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Validamycin and its Derivatives. Discovery, Chemical Synthesis, and Biological Activity [1st Edition]
 9780081011133, 9780081009994

Table of contents :
Content:
Front-matter,CopyrightEntitled to full textChapter 1 - An Introduction to Validamycins and Their Derivatives, Pages 1-8
Chapter 2 - Production of Validamycins, Pages 9-113
Chapter 3 - Bioactivities of Validamycins and Related Natural Compounds, Pages 115-164
Chapter 4 - Chemical Synthesis of Validamycin and Related Natural Compounds, Pages 165-235
Chapter 5 - Voglibose: An Important Drug for Type 2 Diabetes, Pages 237-278
Chapter 6 - N-Octyl-β-Valienamine and N-Octyl-4-epi-β-Valienamine: Two Highly Potent Drug Candidates for Chemical Chaperone Therapy, Pages 279-311
Chapter 7 - Prospects and Concluding Remarks, Pages 313-314
Index, Pages 315-330

Citation preview

Validamycin and Its Derivatives

Validamycin and Its Derivatives Discovery, Chemical Synthesis, and Biological Activity

Xiaolong Chen, Yuele Lu, Yongxian Fan and Yinchu Shen Institute of Fermentation Engineering, Zhejiang University of Technology, Hangzhou, PR China

Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States Copyright r 2017 Elsevier Ltd. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein).

Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-08-100999-4 For Information on all Elsevier publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: John Fedor Acquisition Editor: Anneka Hess Editorial Project Manager: Anneka Hess Production Project Manager: Mohanapriyan Rajendran Designer: Christian J. Bilbow Typeset by MPS Limited, Chennai, India

Chapter 1

An Introduction to Validamycins and Their Derivatives 1.1 IMPORTANCE OF ANTIBIOTICS The term “antibiotics” was first coined by the American microbiologist Selman Waksman and his colleagues to describe chemical substances produced by microorganisms and having antagonistic effects on the growth of other microorganisms. It excluded synthetic antimicrobials (sulfur drugs) and biological products of nonmicrobial origin having antagonistic effects on bacteria. Though antibiotics were introduced into clinical practice only in the middle of the 20th century, the use of microorganisms for the management of microbial infections in ancient Egypt, Greece, China, and some other places in the world is well documented. The modern era of antibiotics started with the serendipitous discovery of penicillin from the culture filtrate of a fungus, Penicillium notatum, by Alexander Fleming in 1928 (Fleming, 1929). In the present scenario, antibiotics available in the market are either produced by microbial fermentation or are derived via semisynthetic route using the existing antibiotic backbone structure. They are classified into different chemically defined groups. Antibiotics target bacterial physiology and biochemistry, causing microbial cell death or the cessation of growth. A significant number of these antibiotics affect cell walls or membranes (e.g., β-lactam and glycopeptides), while several others exert their antibacterial activity by targeting protein synthetic machinery via interaction with ribosomal subunits, and these include antibiotics such as macrolides, chloramphenicol, tetracycline, linezolid, and aminoglycosides. Other “mechanistic” groups include molecules that interfere with the nucleic acid synthesis [e.g., fluoroquinolones (FQ) and rifampin], while some others exert their effects by interfering with the metabolic pathways (e.g., sulfonamides and folic acid analog) or by disruption of the bacterial membrane structure (e.g., polymyxins, daptomycin, and others). The successful use of antibiotics against bacterial diseases of human beings has led to a large-scale screening of antibiotics’ effect for plant disease control in the world.

Validamycin and Its Derivatives. DOI: http://dx.doi.org/10.1016/B978-0-08-100999-4.00001-0 © 2017 Elsevier Ltd. All rights reserved.

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Validamycin and Its Derivatives

1.2 AGRICULTURAL ANTIBIOTICS FOR PLANT PATHOGENS Many antibiotics developed for medical purposes were investigated for activity against plant pathogens. Furthermore, screening of soil organisms for production of antibiotic substances was started with the prime purpose of plant disease control. However, the results obtained with antibiotics and antibiotic-containing culture broth did not fulfill the high expectations. Many of them were too unstable under field conditions or showed toxic side effects on plants. Most antibiotics were rather expensive, even when used as a crude product. In the 1950s, soon after the introduction of antibiotics in human medicine, the potential of these "miracle drugs" to work wonders in controlling plant diseases was explored (Mcmanus and Stockwell, 2000). Nearly 40 antibiotics were screened for plant disease control. To be a viable candidate for disease control, the antibiotic needed to (1) be active on or inside of the plant; (2) tolerate oxidation, UV irradiation, rainfall, and high temperatures; (3) be safe to plants; and (4) select for resistant pathogens only at a low or nondetectable rate. Of the screened compounds, fewer than 10 were used commercially and only streptomycin, tetracycline, cycloheximide, and griseofulvin saw significant use worldwide (Kumar et al., 2005). Streptomycin, the first antibiotic introduced in agriculture, was used in the United States for the control of pear fire blight. This antibiotic and a mixture of streptomycin and tetracycline have been used for the control of bacterial plant diseases, while cycloheximide and griseofulvin have been used for the control of fungal plant diseases. Cycloheximide is a very powerful fungicide, but unfortunately, is highly toxic to plants, which restricts its use against plant diseases. Griseofulvin is a much less phytotoxic systemic fungicide, but its use is also restricted, because the relation of its manufacturing cost to its performance under field conditions is not quite satisfactory. Later, in Japan (Misato, 1976), blasticidin S and kasugamycin have been in practical use for rice blast control instead of mercuric fungicides, and polyoxins and validamycin have been used to control the sheath blight of the rice plant instead of arsenic fungicides. Since then, more and more antibiotics were found to be applied in plant diseases.

1.3 VALIDAMYCINS: MAGIC AGRICULTURAL ANTIBIOTICS Validamycin (Asano et al., 1990; Horii et al., 1972; Iwasa et al., 1970; Kameda et al., 1986), also called as jinggangmycin in China (Agricultural Antibiotic Group, 1975a, 1975b), is a magic agricultural antibiotic produced by fermentation with Streptomyces hygroscopicus var. limoneus or S. hygroscopicus var. jinggangensis, and has been widely used in Asia as the rice and wheat protectant against Rhizoctonia solani since the 1970s. Sheath blight caused by R. solani is a major disease of rice and wheat that greatly reduces yield and grain quality. And validamycin is a nonsystemic antibiotic with fungistatic action and exhibits remarkable therapeutic effects on the disease by inhibiting the extension of R. solani without its growth inhibition (Shibata et al., 1982, 1989; Trinci, 1984).

An Introduction to Validamycins and Their Derivatives Chapter | 1

3

Validamycin can also be used for the control of R. solani in potatoes, vegetables, strawberries, tobacco, ginger, and other crops, and damping-off diseases of cotton, rice and sugar beet, etc. Besides its excellent control effect, low price, low drug-resistance, and low toxicity are the other outstanding merits of validamycin, and it is now one of the most important agricultural antibiotics with the biggest production in China (Shen, 1996). Validamycin is a mixture of aminoglycoside compounds. There have been eight members termed A to H (Fig. 1.1) (Asano et al., 1990; Horii

R5OH2C R6O HO

OH HN

HO

R3 OH

R1 R2OH2C

R4O

Compound Validamycin A

R1 H

R2 H

R3 H

R4 β-D-Glc

R5 H

R6 H

Validamycin B

H

H

OH

β-D-Glc

H

H

Validamycin C

H

H

H

β-D-Glc

α-DH Glc

Validamycin D

H

α-D-Glca

H

Validamycin E

H

H

H

H

H

H

H

H

α-D-Glc(1-4)- βD-Glc Validamycin F

H

H

H

β-D-Glc

H

α-D-Glc

Validamycin G

OH

H

H

β-D-Glc

H

H

Validamycin H

H

H

H

H

H

β-D-Glc(1-6)- βD-Glc aGlc=Glucopyranosyl.

FIGURE 1.1 Chemical structures of validamycins.

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Validamycin and Its Derivatives

OH

CH2OH

CH2OH

CH2 OH OH

NH2

OH

OH

Valienamine

NH2

OH

OH OH

OH

OH

HO

NH2

OH

Hydroxyvalidamine

CH2 OH NH2

Valiolamine

OH

OH

Validamine

CH 2OH OH

OH

OH

OH OH

OH

N H

OH

Voglibose

FIGURE 1.2 Chemical structures of valienamine and its related compounds.

et al., 1972; Iwasa et al., 1970; Kameda et al., 1986) isolated from the broth of S. hygroscopicus var. limoneus, with validamycin A [1L-(1,3,4/2,6)-2,3dihydroxy-6-hydroxymethyl-4-[(1S,4R,5S,6S)-4,5,6-trihydroxy-3-hydroxymethylcyclohex-2-enylamino] cyclohexyl β-D-glucopyranoside] as the main member and the most active. So the content of validamycin A is the most important quality parameter of the commercial products of validamycin. Many researchers also focused on the chemical synthesis, biosynthesis genes, and derivatives of validamycins. Among them, the most interesting work is that validamycins were lysed to produce valienamine or validamine (Fig. 1.2) (Asano et al., 1984; Chen et al., 2005a, 2005b; Kameda et al., 1980, 1981), which could be applied to synthesize valiolamine (Horii et al., 1985; Kameda et al., 1984), and then voglibose (Babu et al., 2005; Chen et al., 2006; Floss et al., 1999; Fukase, 1997; Kameda and Horii, 1986; Lu et al., 2005; Yuan et al., 2005). Voglibose (code number: AO-128, trade name: Basen), is an N-substituted derivative of valiolamine, which is a branched-chain aminocyclitol, or pseudoamino sugar. Voglibose has attracted considerable interest due to its wide range of therapeutic and pharmacological properties, which include its excellent inhibitory activity against α-glucosidases and its action against hyperglycemia and various disorders caused by hyperglycemia. It has shown strong antiobesity and antidiabetic activities, as it is a new, potent glucosidase inhibitor and is a drug used for NIDDM (noninsulin-dependent diabetes mellitus) in Japan, China, and Korea. Therefore, the industry of validamycin has converted from the agricultural antibiotic to pharmaceutical. Valienamine could also be used to synthesize N-octyl-4-epi-β-valienamine (NOEV) (Iwasaki et al., 2006; Ogawa et al., 2004; Suzuki, 2006, 2008, 2013; Suzuki et al., 2007) and N-octyl-β-valienamine (NOV) (Ogawa et al., 1996, 1998; Suzuki, 2013) (Fig. 1.3), two highly potent drug candidates

An Introduction to Validamycins and Their Derivatives Chapter | 1 OH

OH

HO H N

HO

5

HO (CH2 )7 CH3

H N

HO

HO

(CH2 )7 CH3

HO

NEOV

NOV

FIGURE 1.3 Chemical structures of NEOV and NOV.

for chemical chaperone therapy. NOEV and NOV are promising therapeutic agents for human β-galactosidase deficiency disorders (GM1gangliosidosis and Morquio B disease) and β-glucosidase deficiency disorders (phenotypic variations of Gaucher disease), respectively (Suzuki, 2013). Originally NOEV and NOV had been discovered as competitive inhibitors, and then their paradoxical bioactivities as chaperones were confirmed in cultured fibroblasts from patients with these disorders. Subsequently, GM1-gangliosidosis model mice have been used for confirmation of clinical effectiveness, adverse effects, and pharmacokinetic studies. Orally administered NOEV entered the brain through the blood brain barrier enhanced β-galactosidase activity, reduced substrate storage, and improved neurological deterioration clinically. Computational analysis revealed pH-dependent enzyme chaperone interactions. The recent study (Suzuki, 2013) indicated chaperone activity of a new 1-deoxygalactonojirimycin derivative, MTD118, for β-galactosidase complementary to NOEV. NOV also showed the chaperone effect toward several β-glucosidase gene mutants in Gaucher disease. Furthermore a commercial expectorant drug, ambroxol, was found to be a chaperone for β-glucosidase. A few Gaucher patients responded to this drug with remarkable improvement of oculomotor dysfunction and myoclonus. It is hoped that chaperone therapy will become available for some patients with Fabry disease, GM1-gangliosidosis, Gaucher disease, and other lysosomal storage diseases particularly with central nervous system involvement.

1.4 AIMS OF THE WORK Validamycins have nice characteristics, such as high and long efficiency, low toxicity and safe to the environment, and low production cost. Most important of all, there is no report about discovery of validamycinresistant strains of R. solani. Thus, since the production of validamycins in large scale, the yield was more than 30,000 40,000 ton per year (in 5% preparations) for 1 1.3 3 107 hm2 rice field against R. solani and the loss of rice yield was decreased 5000, 000 ton per year at least in China

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Validamycin and Its Derivatives

(Shen, 1996). In recent years, the production of validamycins in other Southeast Asian countries, such as India, Vietnam, Laos, has been booming. In addition, three drugs, voglibose, NEOV, and NOV, were derived from validamycins. These drugs will give a second rise to validamycin production and make products from the agricultural industry to the medical industry. The aims of the work are to review the magic agricultural antibiotics, validamycins, including their discovery, biosynthesis, production, biological activities, chemical synthesis, important derivatives, etc., and to provide references in the research of antibiotics.

REFERENCES Agricultural Antibiotic Group SIOAPS, 1975a. Classification and identification of jinggangmycin producing strains. Weishengwu Xuebao 15 (2), 110 113. Agricultural Antibiotic Group SIOAPS, 1975b. Isolation and identification of jinggangmycins. Weishengwu Xuebao 15 (3), 223 226. Asano, N., Kameda, Y., Matsui, K., Horii, S., Fukase, H., 1990. Validamycin H, a new pseudotetrasaccharide antibiotic. J. Antibiot. 43 (8), 1039 1041. Asano, N., Takeuchi, M., Ninomiya, K., Kameda, Y., Matsui, K., 1984. Microbial degradation of validamycin A by Flavobacterium saccharophilum. Enzymatic cleavage of C N linkage in validoxylamine A. J. Antibiot. (Tokyo) 37 (8), 859 867. Babu J.S., Chavan G.J., Khanduri C.H., Kumar Y., Ray P.C., (Ranbaxy Laboratories Limited, India). assignee. 2005 20050324. Processes for the purification of voglibose and the preparation and purification of its intermediates. Application: WO patent 2005-IB777, 2005092834. Chen, X., Zheng, Y., Shen, Y., 2005a. A new method for production of valienamine with microbial degradation of acarbose. Biotechnol. Prog. 21 (3), 1002 1003. Chen, X., Zheng, Y., Shen, Y., 2005b. Quantitative analysis of valienamine in the microbial degradation of validamycin A after derivatization with p-nitrofluorobenzene by reversed-phase high-performance liquid chromatography. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 824 (1 2), 341 347. Chen, X., Zheng, Y., Shen, Y., 2006. Voglibose (Basen, AO-128), one of the most important alpha-glucosidase inhibitors. Curr. Med. Chem. 13 (1), 109 116. Fleming, A., 1929. Classics in infectious diseases: on the antibacterial action of cultures of a penicillium, with special reference to their use in the isolation of B. influenzae. Br. J. Exp. Pathol. 10, 226 236. Floss H.G., Lee S., Tornus I., (Bayer A.-G., Germany). assignee. 1999 19990329. Preparation of valiolone as synthon for acarbose and voglibose. Application: WO patent 1999-EP2141, 9950217. Fukase, H., 1997. Development of voglibose (Basen), an antidiabetic agent. Yuki Gosei Kagaku Kyokaishi 55 (10), 920 925. Horii, S., Fukase, H., Kameda, Y., 1985. Stereoselective conversion of valienamine and validamine into valiolamine. Carbohydr. Res. 140 (2), 185 200. Horii, S., Kameda, Y., Kawahara, K., 1972. Validamycins, new antibiotics. VIII. Isolation and characterization of validamycins C, D, E, and F. J. Antibiot 25 (1), 48 53.

An Introduction to Validamycins and Their Derivatives Chapter | 1

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Iwasa, T., Yamamoto, H., Shibata, M., 1970. Validamycins, new antibiotics. I. Streptomyces hygroscopicus var. limoneus nov. var., validamycin-producing organism. J. Antibiot. (Tokyo) 23 (12), 595 602. Iwasaki, H., Watanabe, H., Iida, M., Ogawa, S., Tabe, M., Higaki, K., et al., 2006. Fibroblast screening for chaperone therapy in beta-galactosidosis. Brain Dev. 28 (8), 482 486. Kameda, Y., Asano, N., Teranishi, M., Matsui, K., 1980. New cyclitols, degradation of validamycin A by Flavobacterium saccharophilum. J. Antibiot. 33 (12), 1573 1574. Kameda, Y., Asano, N., Teranishi, M., Yoshikawa, M., Matsui, K., 1981. New intermediates, degradation of validamycin A by Flavobacterium saccharophilum. J. Antibiot. 34 (9), 1237 1240. Kameda, Y., Asano, N., Yamaguchi, T., Matsui, K., Horii, S., Fukase, H., 1986. Validamycin G and validoxylamine G, new members of the validamycins. J. Antibiot. 39 (10), 1491 1494. Kameda, Y., Asano, N., Yoshikawa, M., Takeuchi, M., Yamaguchi, T., Matsui, K., et al., 1984. Valiolamine, a new alpha-glucosidase inhibiting aminocyclitol produced by Streptomyces hygroscopicus. J. Antibiot. (Tokyo) 37 (11), 1301 1307. Kameda Y., Horii S.; (Takeda Chemical Industries, Ltd., Japan). assignee. 1986 19860423. Valiolamine derivatives. Application: EP patent 1986-303048, 199591. Kumar, K., Gupta, S.C., Chander, Y., Singh, A.K., 2005. Antibiotic use in agriculture and its impact on the terrestrial environment. Adv. Agron. 87 (05), 1 54. Lu, C., Ye, W., Hu, S., Pan, Y., Yuan, J., 2005. Preparation of voglibose from valiolamine. Zhongguo Yiyao Gongye Zazhi 36 (9), 525. Mcmanus, P., Stockwell, V., 2000. Antibiotics for plant diseases control: silver bullets or rusty sabers. Core Discussion Papers Rp (May), 27 51. Misato, T., 1976. The development of agricultural antibiotics. Environ. Qual. Safety 5 (5), 48 55. Ogawa, S., Ashiura, M., Uchida, C., Watanabe, S., Yamazaki, C., Yamagishi, K., et al., 1996. Synthesis of potent β-D-glucocerebrosidase inhibitors: N-alkyl-β-valienamines. Bioorg. Med. Chem. Lett 6 (8), 929 932. Ogawa, S., Kobayashi, Y., Kabayama, K., Jimbo, M., Inokuchi, J.-I., 1998. Chemical modification of β-glucocerebrosidase inhibitor N-octyl-β-valienamine: synthesis and biological evaluation of N-alkanoyl and N-alkyl derivatives. Bioorg. Med. Chem. 6 (10), 1955 1962. Ogawa, S., Sakata, Y., Ito, N., Watanabe, M., Kabayama, K., Itoh, M., et al., 2004. Convenient synthesis and evaluation of glycosidase inhibitory activity of α- and β-galactose-type valienamines, and some N-alkyl derivatives. Bioorg. Med. Chem. 12 (5), 995 1002. Shen, Y.C., 1996. Research and development on jinggangmycins for 25 years. Zhiwu Baohu (Plant Protect.) 22 (4), 44 45. Shibata, M., Kido, Y., Honda, Y., Shimizu, K., 1989. Hyphal extension inhibitors I and II with similar inhibitory spectra to validamycin, isolated from hyphae of Rhizoctonia solani. Agric. Biol. Chem. 53 (3), 869 873. Shibata, M., Mori, K., Hamashima, M., 1982. Inhibition of hyphal extension factor formation by validamycin in Rhizoctonia solani. J. Antibiot. 35 (10), 1422 1423. Suzuki, Y., 2006. β-Galactosidase deficiency: an approach to chaperone therapy. J. Inherit. Metab. Dis. 29 (2/3), 471 476. Suzuki, Y., 2008. Chemical chaperone therapy for GM1-gangliosidosis. Cell. Mol. Life Sci. 65 (3), 351 353. Suzuki, Y., 2013. Chaperone therapy update: Fabry disease, GM1-gangliosidosis and Gaucher disease. Brain Dev. 35 (6), 515 523.

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Suzuki, Y., Ichinomiya, S., Kurosawa, M., Ohkubo, M., Watanabe, H., Iwasaki, H., et al., 2007. Chemical chaperone therapy: clinical effect in murine G(M1)-gangliosidosis. Ann. Neurol. 62 (6), 671 675. Trinci, A.P.J., 1984. Antifungal agents which affect hyphal extension and hyphal branching. Symp. Br. Mycol. Soc. 9 (Mode Action Antifungal Agents), 113 134. Yuan J., Shao C., Chen D., Ye W.; (Shanghai Laiyi Biopharmaceutical Research and Development Center Co., Ltd., Peop. Rep. China; Xinchang Pharmaceutical Factory, Zhejiang Medicine Co., Ltd.). assignee. 2005 20050303. Preparation of valiolamine as intermediate of voglibose. Application: CN patent 2005-10024194, 1683320.

Chapter 2

Production of Validamycins 2.1 DISCOVERY OF VALIDAMYCINS In the course of screening for new antibiotics effective in the control of sheath blight, a destructive disease of rice plants caused by Pellicularia sasakii (Shirai) S. Ito, validamycins were first discovered in the broth of Streptomyces hygroscopicus var. limoneus T-7545, which was isolated from a soil sample collected in Akashi City, Hyogo Prefecture, Japan in 1970 (Iwasa et al., 1970). Five years later, they were also discovered in the broth of S. hygroscopicus var. jinggangensis Yen. TH82, which was isolated from a soil sample of Jinggang Mountain in Jiangxi, China (Agricultural Antibiotic Group, 1975). Since then, they have been widely used in Asia as the rice and wheat protectant against P. sasakii. Now, eight validamycins have been purified and identified, including A, B, C, D, E, F, G, and H.

2.2 MICROBES FOR PRODUCING VALIDAMYCINS 2.2.1 General Characteristics for Microbes Microbes for producing validamycins belong to S. hygroscopicus, including S. hygroscopicus var. limoneus (Iwasa et al., 1970) and var. jinggangensis (Agricultural Antibiotic Group, 1975). The strains were similar except in some respects, such as the morphology and cultural characteristics. The morphological characteristics of the strains are shown in Fig. 2.1. The aerial mycelium of strain T-7545 was simply branched and terminated in coils of three to five volutions. The spores were oval or cylindrical and measure 1.01.3 3 1.01.5 μm. Their surfaces were smooth. On the other hand, the aerial mycelium of strain TH82 was simply branched and terminated in coils. The spores were oval and not of a uniform size. Their surfaces were smooth. The cultural characteristics of S. hygroscopicus var. limoneus T-7545 and var. jinggangensis Yen. TH82 are listed in Table 2.1. The colors of aerial mycelium are grey to grey and yellow, and black moist areas form in the aerial mycelium on glucose asparagine agar and starch agar. The colors of the vegetative mycelium on most of the media are bright yellow to ochre with, in some cases, a slight greenish tinge. A light yellow to faint yellowish brown diffusible pigment was noted in various media, but because dark Validamycin and Its Derivatives. DOI: http://dx.doi.org/10.1016/B978-0-08-100999-4.00002-2 © 2017 Elsevier Ltd. All rights reserved.

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Validamycin and Its Derivatives

(A)

(B)

(×950)

(×10,000×1/1.5)

(×7500)

(×7500)

FIGURE 2.1 Morphology of S. hygroscopicus var. limoneus T-7545 (A) and S. hygroscopicus var. jinggangensis Yen. TH82 B. (A) Reprinted with permission courtesy of Japan Antibiotics Research Association (JARA).

brown soluble pigment on proteinaceous media was not observed, T-7545 and TH82 are considered to be nonchromogenic. Physiological characteristics of T-7545 and TH82 are shown in Table 2.2. As show in the table, a wide temperature range and rather high optimum temperature for growth are characteristic for these two organisms. Starch hydrolysis and milk peptonization tests are positive, whereas tyrosinase, nitrate reduction, cellulose decomposition, and serum liquefaction are negative for T-7545. On the other hand, for TH82, starch hydrolysis and milk peptonization tests are positive, whereas cellulose decomposition and serum liquefaction are negative. Furthermore, nitrate reduction for TH82 is positive in Czapek’s solution and negative in peptone solution. Gelatin is slowly liquefied for two strains. Products of T-7545 are validamycins A. However, products of TH82 are validamycins and other antibiotics. For T-7545 and TH82, a variety of carbon sources, such as D-xylose, L-arabinose, L-galactose, D-glucose, D-fructose, L-rhamnose, melibiose, maltose, sucrose, lactose, raffinose, inositol, D-mannitol, mannose, and glycerol,

Production of Validamycins Chapter | 2

11

TABLE 2.1 Cultural Characteristics of S. hygroscopicus var. limoneus T-7545 and var. jinggangensis Yen. TH82 Cultural Characteristics Medium Czapek’s agar

S. hygroscopicus var. limoneus T-7545

S. hygroscopicus var. jinggangensis Yen. TH82

Growth (G): Moderate, colorless, folded

R: Yellowish Brown to Maroon

Reverse (R): Raw Sienna (Rda.a, III 17-i) to Sudan Brown (Rdg., III 15-k)

AM: Pink White to Jasmine Buff to Mouse Grey with black moist areas

Aerial mycelium (AM): Tilleul-Buff (Rdg., XL 17’’’-f) to Light Buff (Rdg., XV 17’-f), partially Mouse Grey (Rdg., LI 15’’’’) along the periphery of the colony

SP: Faint Brownish tinge

Soluble pigment (SP): Yellow with a faint Brownish tinge Glucose Czapek’s agar

G: Moderate, colorless to Sulphin Yellow (Rdg., IV 21-i), folded

G: Banana-Buff to Maroon

R: Raw Sienna

AM: Faint Yellow to Dust Grey

AM: Tilleul-Buff to Massicot Yellow (Rdg., XVI 21’-f), partially Light Olive-Grey (Rdg., LI 23’’’’’-d) along the periphery of the colony

SP: Faint Brownish tinge

SP: Yellow with a faint Brownish tinge Glycerol Czapek’s agar

G: Moderate, colorless to Orange-Citrine (Rdg., IV 19-k), folded

G: Sulphin Yellow to Brown

R: Raw Sienna

AM: Faint Grey to Almond Yellow to Mouse Grey

AM: Tilleul-Buff to Massicot Yellow, partially Light Olive-grey

SP: Faint Brownish tinge

SP: Yellow with a faint brownish tinge (Continued )

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Validamycin and Its Derivatives

TABLE 2.1 (Continued) Cultural Characteristics Medium Glucose asparagine agar

S. hygroscopicus var. limoneus T-7545

S. hygroscopicus var. jinggangensis Yen. TH82

G: Colorless

G: Grey Green to Brown

R: Old Gold (Rdg., XVI 19’-i) to Antimony Yellow (Rdg., XV 17’-b) to Cinnamon-Brown (Rdg., XV15’-k)

AM: Mouse Grey with yellow areas to black moist areas

AM: Light Olive-Grey to Mouse Grey, with yellow patches and black moist areas

SP: Faint Yellow

SP: Light brown Calcium malate agar

b

Starch agar

G: Primuline Yellow (Rdg., XVI 19’)

G: Colorless

R: Primuline Yellow. AM: Sparse at first, but later Tilleul-Buff to Light Olive-Grey

AM: Faint Yellow

SP: Pale Yellow

SP: Faint Yellow

G: Colorless to Barium Yellow (Rdg., XVI 23’-d)

G: Pheasant Brown to Tea Brown

R: Deep Colonial Buff (Rdg., XXX 21’’-b) to Snuff Brown (Rdg., XXIX 15’’-k)

AM: Grey White to Mouse Grey with black moist areas

AM: Cartridge Buff (Rdg., XXX 19’’-f) to Mouse Grey, with black moist areas

SP: Faint Yellow

SP: Light Brown. Hydrolysis of starch was observed Gelatin (25 C)

G: Very poor



AM: None SP: None. Liquefaction, slow Nutrient gelatin (25 C)

Same results as with gelatin

 (Continued )

Production of Validamycins Chapter | 2

13

TABLE 2.1 (Continued) Cultural Characteristics Medium Whole egg (37 C)

S. hygroscopicus var. limoneus T-7545

S. hygroscopicus var. jinggangensis Yen. TH82

G: Colorless



AM: None SP: None Tyrosine agar

G: Colorless to Strontium Yellow (Rdg., XVI 23’)

R: Bamboo Shoot Brown to Maroon AM: Ic43’ to Id64’

R: Pale Ochraceous-Buff (Rdg., XV 15’-f) to Light Ochraceous-Buff (Rdg., 15’-d)

SP: Faint Brown

AM: None SP: None Yeast extract agar

G: Colorless, folded

R: Colorless

R: Cream Color (Rdg., XVI 19’-f)

AM: Grey White

AM: White

SP: Colorless

SP: Light brown 

Nutrient agar (37 C)

G: Colorless

R: Colorless

R: Colorless

AM: None

AM: None

SP: Colorless

SP: None Glucose nutrient agar (37 C)

G: Colorless wrinkled

R: Pheasant Brown

R: Cartridge Buff to Pale Ochraceous-Buff

AM: Ic 11’ to Id 42’ with wrinkles

AM: None

SP: Pale brown

SP: None Peptone glucose agar

G: Chartreuse Yellow

R: Yellowish Brown to Brown

R: Honey Yellow (Rdg., XXX 19’’)

AM: Grey White to Jasmine Yellow

AM: Thin, Cream Buff

SP: Pale brown

SP: Yellow with a Brownish tinge (Continued )

14

Validamycin and Its Derivatives

TABLE 2.1 (Continued) Cultural Characteristics Medium Nutrient broth (37 C)

Glucose nutrient broth

S. hygroscopicus var. limoneus T-7545

S. hygroscopicus var. jinggangensis Yen. TH82

G: Surface growth colorless and colorless flocculent growth at bottom of tubes

G: Surface growth colorless and colorless flocculent growth at bottom of tubes

AM: None

AM: None

SP: None

SP: None

G: Surface growth Cartridge Buff, and colorless flocculent growth at bottom of tubes



AM: None SP: None Potato plug

Carrot plug

Cellulose



Litmus milk (37 C)

G: Colorless to Pale Ochraceous-Buff

G: Colorless to White

AM: Tilleul-Buff to Mouse Grey. Color of the plug turned to Sayal Brown (Rdg., XXIX 15’’-i)

AM: Grey White to Green Grey to Pale Brown

G: Colorless

R: Colorless

AM: White to Mouse Grey. Color of the plug turned to CinnamonRufous (Rdg., XIV 11’-i) to Cinnamon-Brown

AM: Grey White to Mouse Grey to Faint Brown

G: Poor growth, Chartreuse Yellow (Rdg., XXXI 25’’-d) to Reed Yellow (Rdg., XXX 23’’-d)

R: Colorless

AM: Mouse Grey

SP: Pale Yellow

G: Surface growth Cream Color to Seashell Pink (Rdg., XIV 11’-f)



SP: Faint Yellow

SP: Pale Yellow

AM: Mouse Grey and grown poorly

AM: None SP: Army Brown (Rdg., XL 13’’’-i). Peptonization (Continued )

Production of Validamycins Chapter | 2

15

TABLE 2.1 (Continued) Cultural Characteristics Medium

S. hygroscopicus var. limoneus T-7545

S. hygroscopicus var. jinggangensis Yen. TH82

with or without weak coagulation. Reaction of the medium, weakly acidic Lo¨effler’s medium (37 C)

G: Naples Yellow (Rdg., XVI 19’-d) becoming Light Buff



AM: None SP: None. No liquefaction a

Rdg.: Ridgway. Soluble starch 1%, potassium monohydrogen phosphate 0.3%, calcium carbonate 0.3%, magnesium sulfate 0.1%, ammonium sulfate 0.2%, sodium chloride 0.05%, agar 2%.

b

TABLE 2.2 Physiological Properties of Strains T-7545 and TH82 Properties T-7545

TH82 

Temperature and pH rangesa

Growth occurs at 1545 C, better growth at 3745 C, no growth at 10 C and 50 C. Growth occurs at pH 510, no or poor growth at pH 4, optimum range pH 67

Growth occurs at 1545. Growth occurs at pH 510, better growth at pH 67

Gelatin

Slow liquefaction

Slow liquefaction

Starch

Hydrolysis. Diameter of hydrolyzed area/diameter of colony 5 33 mm/8 mm

Hydrolysis

Tyrosinase reaction

Negative

Nb

Litmus milk

Peptonization. Coagulation, doubtful. Reaction, weakly acidic

Peptonization. Coagulation, doubtful. Reaction, weakly acidic

Reduction of nitrate to nitrite

Negative (in peptone solution and Czapek’s solution)

Positive in Czapek’s solution. Negative in peptone solution (Continued )

16

Validamycin and Its Derivatives

TABLE 2.2 (Continued) Properties T-7545

TH82

Cellulose decomposition

Negative

Negative

Chromogenicity

Negative

Negative

Liquefaction of serum

Negative

N

Products

Validamycins

Validamycins and other antibiotics

a

On glucose asparagine agar. Not tested.

b

are well utilized for growth. In view of the relatively high optimum temperature for growth of T-7545 and TH82, the cultural characteristics at 45 C were observed and compared with those at 28 C. The characteristics at the higher temperature were found to be almost the same as those at 28 C. A few properties, such as sparse formation of white aerial mycelium on Czapek’s agar, Czapek’s glucose agar, Czapek’s glycerol agar, and filter paper, and a slightly deeper color of the vegetative mycelium on calcium malate agar, were noted (Table 2.3). The characteristics of T-7545 and TH82 are summarized as follows: good growth and abundant aerial mycelia and coiled chains of spores form at 2545 C; on a variety of media, grey to grey and yellow aerial mycelium, bright yellow to ocher vegetative mycelia and faint, brownish yellow diffusible pigment form; on certain media, black moist spots form in the aerial mycelium. Dark brown diffusible pigment is not produced on proteinaceous media, i.e., the strain is nonchromogenic.

2.2.2 Complete Genomes for the Two Microbes Recently, complete genomes of the two strains, S. hygroscopicus var. jinggangensis 5008 (Wu et al., 2012) and var. limoneus KCTC 1717 (Leea et al., 2016), were sequenced.

2.2.2.1 General Features of the S. hygroscopicus var. jinggangensis 5008 Genome Except for a linear chromosome, the strain 5008 also harbors a linear plasmid pSHJG1 and a 73,282-bp large circular plasmid. With a total length of 10,383,684 bp, the genome of strain 5008 is larger than most published

Production of Validamycins Chapter | 2

17

TABLE 2.3 Utilization of Carbon Sources by T-7545 and TH82 Carbon Source

T-7545

TH82

Erythritol

6

6

Adonitol

6

a

Carbon Source

T-7545

TH82

Inositol

11

11

N

D-Mannitol

11

11

Dulcitol

Sorbitol

1

6

D-Xylose

11

11

Trehalose

6

6

11

N

L-Arabinose

11

11

Salicin

6

N

6

1

Esculin

6

6

D-Galactose

11

11

Inulin

11

N

Glucose

11

11

Dextran

1

1

D-Fructose

11

11

Mannose

11

11

L-Rhamnose

11

11

Glycerol

11

11

Melibiose

11

11

Na-Acetate

1

6

Maltose

11

11

Na-Succinate

1

11

Sucrose

11

11

Na-Citrate

1

N

Lactose

11

11

Ca-2-ketogluconate

6

N

Raffinose

11

11

Carbon-free control

6

N

L-Sorbose

Not tested. 1 1 : Good growth. 1 : Fair growth. 6: No or very poor growth.

a

Streptomyces genomes (Table 2.4). The linear chromosome (10,145,833 bp) of strain 5008, with an average G 1 C% mol content of 71.9%, comprises 8849 predicted protein-coding sequences (locus tagged as SHJG), 6 rRNA operons (16S23S5S), and 68 tRNA genes (Table 2.4). The replication origin oriC contains at least 18 DnaA box-like sequences (Jakimowicz et al., 1998) and is shifted 875 kb away from the center to the right (Fig. 2.2A). Intriguingly, it only has 14-bp terminal inverted repeats (TIRs), which is one of the shortest TIRs hitherto found in actinomycetes. Based on a BLASTCLUST analysis, 4607 (41.6%) of predicted protein coding sequences (CDSs) are clustered into 924 families. The linear plasmid pSHJG1 (164,566 bp) (Fig. 2.1B) contains 184 CDSs possibly involved in replication, partitioning, transfer, and other biological functions. It lacks a conserved telomere-associated protein (TAP) and TIRs. However, the rightmost 1.2-kb region of pSHJG1 demonstrates a strong homology to the right arm of the chromosome, implying an evolutionary recombination event occurred between the linear plasmid and the chromosome. Moreover, the left terminus of pSHJG1 is equipped with atypical nucleotide sequences

TABLE 2.4 General Features of Seven Completely Sequenced Streptomyces Chromosomesa Species

Length (bp)

TIR (bp)

GC Content (%)

CDS (No.)

Average CDS size (bp)

Coding (%)

rRNA Operons (No.)

tRNA (No.)

S. hygroscopicus 5008

10,145,833

14

71.9

8849

952

83.2

6

68

S. coelicolor A3(2)

8,667,507

21,653

72.1

7825

991

88.9

6

63

S. avermitilis MA-4680

9,025,608

49

70.7

7582

1027

86.3

6

68

S. griseus IFO13350

8,545,929

132,910

72.2

7138

1055

88.1

6

66

S. scabies 87.22

10,148,695

18,488

71.5

8746

1005

86.2

6

75

S. bingchenggensis BCW-1

11,936,683

40,000

70.8

10,023

1031

86.6

6

ND

S. clavuligerus ATCC 27064

6,760,392

ND

72.0

5710

1031

87.1

6

66

a

Data were obtained from GenBank: S. coelicolor A3(2), NC_003888; S. avermitilis MA-4680, NC_003155; S. griseus IFO13350, NC_010572; S. scabies 87.22, NC_013929; S. bingchenggensis BCW-1, CP002047; S. clavuligerus ATCC 27064, CM000913. ND, not determined.

Production of Validamycins Chapter | 2

19

FIGURE 2.2 Schematic representation of the S. hygroscopicus 5008 chromosome and two plasmids. (A) The chromosome atlas. The outer scale is numbered in megabases from the left to the right ends and indicates the core (red) and noncore (blue) chromosomal regions; Circles 1 and 2 (forward and reverse strands), predicted protein coding sequences colored according to Clusters of Orthologous Groups (COG) function categories; Circles 3 and 4 (forward and reverse strands), distribution of conserved (red) or strain-specific genes (blue) in 5008 compared with other Streptomyces chromosomes; Circle 5, distribution of secondary metabolic gene clusters (red); Circle 6, distribution of tRNA (red) and rRNA operon (blue); Circle 7, GC content; Circle 8, GC bias. Ori, origin of replication. val, validamycin biosynthetic gene cluster. (B) Atlas of linear plasmid pSHJG1. Circles 1 and 2, predicted coding sequences on the plus and minus strands, respectively, colored according to COG functional categories; Circle 3, GC content; Circle 4, GC bias. (C) Atlas of circular plasmid pSHJG2. Circles 1 and 2, predicted coding sequences on the plus and minus strands, respectively, colored according to COG functional categories; Circle 3, GC content; Circle 4, GC bias.

consisting of several packed palindromes with nonconserved loop sequences, thereby forming a different secondary structure from its right end and both chromosome ends. Interestingly, a complete bacterial immune system CRISPR-Cas (Horvath and Barrangou, 2010) was identified in pSHJG1, suggesting a resistance to phages and other invading genetic elements by strain 5008. Genome-wide comparison among completely sequenced Streptomyces chromosomes revealed highly conserved core regions ranging from 5.50 to 7.25 Mb [SCLAV0503-SCLAV5245 (5.50 Mb), SCO1209SCO6774 (6.25 Mb), SGR0954SGR6311 (6.36 Mb), SAV1638SAV7128 (6.48 Mb), SBI25785 SBI889 (7.12 Mb), SCAB12831SCAB78641 (7.25 Mb)], substantially in

20

Validamycin and Its Derivatives

proportion to the corresponding chromosomal length. However, the genome of strain 5008 was predicted to have a relatively small core region (5.56 Mb), with a left arm of 3.16 Mb and a right arm of 1.43 Mb (Fig. 2.2A). Syntenic analyses showed that, except for S. scabies, large continuous or separate inversions centered at oriC were detected in the chromosome of strain 5008, when compared with other Streptomyces species. To further identify commonly conserved or species-specific proteins in strain 5008, orthologs shared among the seven Streptomyces strains were analyzed by Microbial Genome Database (MBGD) (Uchiyama, 2007). The results showed that 2954 SHJG proteins (33.3% of the total CDSs), 2899 SCO proteins (37.3%), 2901 SAV proteins (38.3%), 2879 SGR proteins (40.3%), 2989 SCAB proteins (33.4%), 2989 SBI proteins (29.8%), and 2806 SCLAV proteins (49.1%) could be classified into 2754 clusters. The major conserved proteins are assigned with functions for transcription, translation, energy production, and amino acid and carbohydrate metabolisms. Notably, 1640 strain-specific orthologous clusters including 1749 proteins for strain 5008 could be detected. Surprisingly, the amfABST cluster (Capstick et al., 2007) and the ramR-activated gene (rag) cluster for aerialmycelium formation and sporulation (San Paolo et al., 2006) were not found in strain 5008. In total, 29 gene clusters were determined in the chromosome of strain 5008. Twenty are located in subtelomeric regions with 14 in the left arm and 6 in the right arm. The validamycin A gene cluster (val) is located at a region 350 kb away from the left end of the chromosome (Fig. 2.2A). Among an additional 27 gene clusters putatively for secondary metabolites, 6 were assigned for the biosynthesis of polyketide synthases (PKSs), 8 for non-ribosomal peptide synthases (NRPSs), 5 for hybrid PKSNRPSs, 4 for terpenoids, 1 for lantibiotics, and the other 3 for melanin, norcardamine siderophore, and ochronotic pigment, respectively. Similar to most Streptomyces, the central carbon metabolism of strain 5008 includes complete glycolysis, the pentose phosphate pathway (PPP), the tricarboxylic acid (TCA) cycle, and gluconeogenesis pathway with multiple copies of genes encoding key enzymes for these pathways (Fig. 2.3). Validamycin A synthesis requires sedoheptulose 7-phosphate and UDP-glucose derived from carbohydrate metabolism as precursors. The UDP-glucose synthesis is possibly catalyzed by UDP-glucose-1phosphate uridylyltransferases (UGP), SHJG4652, and SHJG7333 (sharing 77% identity), preceded by the isomerization of glucose-6-phosphate to glucose-1-phosphate catalyzed by phosphoglucomutase (SHJG1995). Unlike strain 5008, only one copy of the ugp gene is present in other sequenced Streptomyces genomes, implicating stronger carbon fluxes from glucose to UDP-glucose for the validamycin A synthesis in strain 5008 (Fig. 2.3).

Production of Validamycins Chapter | 2

21

FIGURE 2.3 Schematic diagram of central carbon and nitrogen metabolisms for validamycin A production in strain 5008. Red arrows and characters represent metabolic pathways and precursors directly related to validamycin A biosynthesis, respectively. Numbers in parentheses indicated copy numbers of annotated proteins or complexes in the metabolic pathways. Abbreviations: ALD, alanine dehydrogenase; DHAP, dihydroxyacetone phosphate; FBP, fructose-1, 6-bisphosphate; GAP, glyceraldehyde-3 phosphate; GDH, glutamate dehydrogenase; Glc, glucose; GlcNAc, N-acetylglucosamine; GOGAT, glutamate synthase; GS, glutamine synthetase; Mal, maltose; PEP, phosphoenolpyruvate.

2.2.2.2 General Features of the S. hygroscopicus subsp. limoneus KCTC 1717 Genome The complete genome of S. hygroscopicus subsp. limoneus KCTC 1717 consists of two linear chromosomes of 10,537,932 bp with 71.96% G 1 C content (Table 2.5). The coding regions that cover 84.62% of the genome (8,916,963 bp) encode 8983 proteins. The genome also encodes 85 pseudogenes, 18 rRNAs (6 operons), 67 tRNAs, 1 tmRNA, and 1 ncRNA, all of which make up 1.14% of the genome (Table 2.4). Signal peptides and transmembrane helices were detected in 576 (6.41%) and 1924 (21.42%) protein coding genes, respectively. The genome encodes a 27-gene cluster (val) located from 337,192 to 374,655 nucleotide positions on chromosome II (locus tags: SHL15 7990 to SHL15 8016), which is responsible

22

Validamycin and Its Derivatives

TABLE 2.5 Genomic Features of S. hygroscopicus subsp. limoneus KCTC 1717 Features

Chromosome I

Chromosome II

Genome size (bp)

8,648,026

1,889,906

DNA coding (bp)

7,424,859

1,492,104

G 1 C content (%)

72.1

71.4

Total genes

7586

1569

Protein coding genes

7447

1536

rRNA (operons)

18(6)

0(0)

tRNA

67

0

tmRNA

1

0

ncRNA

1

0

for producing validamycin. The detection and localization of complete val gene cluster is expected to confer useful genetic information regarding biosynthesis of aminocyclitols (e.g., validamycin and valienamine). Moreover, 111 putative gene clusters responsible for production of diverse secondary metabolites were also detected. Of these, the existence of genes encoding NRPSs, types IIII PKSs, and a type II PKS/NRPS hybrid synthase reveals the potential of KCTC 1717 to produce industrially important natural products.

2.3 PRODUCTION AND ISOLATION OF VALIDAMYCINS AND RELATED NATURAL COMPOUNDS 2.3.1 Fermentation of Validamycins Validamycins are weakly basic, water soluble antibiotics produced by S. hygroscopicus var. limoneus. Cultural conditions for the production of validamycins with S. hygroscopicus var. limoneus No. 7545 were studied by Iwasa et al. (1971b).

2.3.1.1 Temperature of Submerged Culture As the optimum temperature for the growth of T-7545 was found to be fairly high, it was cultivated at 37 C. Since the fermented broth thus obtained was turbid and filtration was very difficult, fermentation at 27 C was compared with that at 37 C. As shown in Table 2.6, the validamycin titer of the filtered broth obtained in 37 C incubation was a little higher than that at 27 C, but

Production of Validamycins Chapter | 2

23

TABLE 2.6 Effect of Temperature on Validamycin Production Incubation Period (days)

27 C

37 C

Validamycin Titera (unit/mL)

Brothb

Validamycin Titera (unit/mL)

Brothb

4

20,000

Clear, easy filtration

35,000

Turbid, difficult filtration

6

20,000

do

35,000

do

a

Assayed by dendroid-test method. The medium used in this experiment consists of 5.0% glucose, 3.6% soybean flour, 0.5% peptone, and 0.6% CaCO3, pH 7.0. 60 mL of the medium in each Erlenmeyer flask of 200 mL capacity was inoculated and cultivated on the rotary shaker (5.0 cm radius) at 220 rpm. b

TABLE 2.7 Effect of Medium Volume on Validamycin Production Volume of the Mediumb

Incubation Period (h)

20 mL

60 mL

pH

Validamycin Titera (unit/mL)

pH

Validamycin Titera (unit/mL)

66

8.0

10,000

7.4

15,000

90

8.0

15,000

7.8

20,000

114

8.2

20,000

8.0

50,000

138

8.6

20,000

8.2

50,000

a

Assayed by dendroid-test method. Composition of the medium used in this experiment was the same as in Table 2.4.

b

filtration was more difficult and essentially equivalent results were obtained in a greenhouse test. So the lower temperature was used to prepare the antibiotic.

2.3.1.2 Effect of Medium Volume on Validamycin Production Erlenmeyer flasks of 200 mL capacity containing 20 mL or 60 mL of the fermentation medium were compared. As shown in Table 2.7, 60 mL of the medium gave superior production. 2.3.1.3 Effect of Medium Composition on Validamycin Production (1) Selection of carbon and nitrogen sources. A variety of media containing glucose, glycerol, or starch as a carbon source and various nitrogen sources

24

Validamycin and Its Derivatives

such as peptone, beef extract, corn steep liquor (CSL), soybean flour (SBF), and corn gluten meal (CGM) were compared by the reversed layer method. The results are shown in Table 2.8. Good growth occurred in the media containing glucose or glycerol as the carbon source regardless of nitrogen sources. As for the yield of validamycin, glucose was notably superior to other carbon sources, and when it was used, no remarkable differences were found among the nitrogen sources. As a result of repeated examinations, CSL and CGM were judged relatively better than SBF and beef extract. When starch was adopted as the carbon source, considerable validamycin production was obtained, although slowly, in the medium containing CSL and CGM as the nitrogen sources. (2) Effects of inorganic salts. Effects of FeSO4, MnSO4, ZnSO4, NaCl, KCl, and NH4Cl on the production of validamycin were investigated in the following two kinds of media: (a) 5.0% glucose, 3.0% SBF, 1.0% CSL, 0.5% CaCO3; (b) 4.0% glucose, 2.0% corn starch, 2.0% CSL, 3.0% CGM, 1.5 CaCO3. Little effect of these inorganic salts was found in the first medium, but in the latter medium, remarkable effects of NaCl and NH4Cl were found as shown in Table 2.9. (3) Selection of medium for high yield of validamycins. From these and other results, a medium composed of glucose 2.0%, corn starch 4.0%, CSL 2.0%, CGM 4.0%, NH4Cl 0.5%, NaCl 1.5%, and CaCO3 1.5% was selected, yielding about 620 μg/mL of validamycins.

2.3.2 Isolation of Validamycins From the Broth Validamycins A and B were first isolated as follows (Iwasa et al., 1971c). The culture broth of S. hygroscopicus var. limoneus was acidified with oxalic acid to pH 34 and the calcium ions in the broth were removed with the mycelium by filtration. To remove cationic and anionic impurities, the broth filtrate was first treated with Amberlite IR-120 (H1) and IR-45 (OH2) and then applied to a column of Dowex-50 X-2. The antibiotics were adsorbed on the resin and then eluted with aqueous ammonia. The elute concentrate was chromatographed on Dowex-50 X-2 with pyridineacetic acid buffer at pH 6.5. Validamycin A was adsorbed on the column and validamycin B passed through. Validamycin A adsorbed on the resin was eluted with pyridineacetic acid, pH 7.5, and the elute was concentrated to give pure crystalline validamycin A. The concentrate of the column effluent was dissolved in water, applied to a column of Dowex-1 X-2 and developed with water to give pure validamycin B. The purity of each antibiotic was confirmed by thin-layer chromatography (TLC) (Fig. 2.4) and its crystalline derivative. Later, the crude validamycins were chromatographed on Dowex 1 3 2 column (OH2 form, 100200 mesh), and the column was developed with water to give the eight components: validoxylamines A and B, and

TABLE 2.8 Production of Validamycins by T-7545 and Its Growth on Various Media Media 1 Component (%)

Glucose

2

3

5

Glycerol

5

5

5

Peptone

1

1

1

Beef extract

0.5

0.5

0.5

CSL 0.5

0.5

10

11

12

5

5

5 5

5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

3

3

3

1

1

1

1

1

1

1

1

1

3

3

3

3

3

3

0.3

0.3

0.3

0.3

0.3

0.3

0.3

0.3

0.3

0.3

0.3

0.3

CaCO3

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

111

111

1 B1 1

111

111

1 1 B1 1 1

111

111

111

111

111

1 B1 1

7.5

7.25

8.0

8.2

7.3

8.3

7.75

7.15

8.0

7.55

8.1

8.6

64

12.5

12.5

140

12.5

74

103

12.5

38

64

28

12.5

Growth pH

b

1 1 1 : Very good growth; 1 1 : Good growth; 1 : Fair growth. Five-day culture was assayed by reversed layer method.

b

9

NaCl

a

a

8

5

CGM

Validamycin titer

7 5

5

0.5

6

5

Starch

SBF

4

26

Validamycin and Its Derivatives

TABLE 2.9 Effects of NH4Cl and NaCl on the Production of Validamycin Salt Added (%)

Growth

pH

Validamycin Titera (μg/mL)

NH4Cl 0.5

111

7.8

345

NaCl 1.5

111

7.45

300

NH4Cl 0.5 1 NaCl 1.5

111

7.1

380

No addition

111

8.4

75

a

Seven-day culture was assayed by reversed layer method.

Rf 1.0

0.8

0.6

0.4

0.2

0

1

2

3

FIGURE 2.4 TLC chromatograph of validamycins A and B. Stationary phase: Silica gel G; Solvent system: n-PrOH/AcOH/H2O 5 4/1/1; Detection: Naphthoresorcin-H2SO4 reagent. (1) Crude powder of validamycins; (2) Validamycin A; (3) Validamycin B. Reprinted with permission courtesy of Japan Antibiotics Research Association (JARA).

validamycins D, A, C, B, F, and E, in order of elution from the column (Horii et al., 1972). The peak of validamycin C appeared just before validamycin B, and these two components were slightly overlapped. On the other hand, validamycin D was eluted just before validamycin A. Validamycin C-rich fraction and fractions mainly containing validamycin D were rechromatographed on a silica gel column (elution with n-PrOH/AcOH/H2O 5 4/1/1) and Dowex 50W 3 2 column (elution with pyridineacetic acid buffer, pH 6.0). Validamycins E and F were strongly retarded on Dowex 1 3 2 column owing to their great affinity for the resin, which showed a rather diffuse elution pattern on a gravity flow. The complete resolution of validamycins E and F was not accomplished on Dowex 1 3 2 column. Validamycin E-rich fractions and validamycin F-rich fractions obtained by Dowex 1 3 2

Production of Validamycins Chapter | 2

27

chromatography were further chromatographed on Dowex 50W 3 2 column (elution with pyridineacetic acid buffer, pH 6.0). In this chromatography, early fractions contained validamycin F. Then, validamycin E was eluted. These chromatographic purifications were repeated once more, if necessary. Finally, chromatographically homogeneous validamycins C, D, E, and F were obtained by rechromatography on Dowex 1 3 2 (OH2 form, developed with water). Validoxylamine A was eluted just before validamycin D on Dowex 1 3 2 column, and crystallization from waterethanol gave the pure validoxylamine A. Although validoxylamine B and validamycin D were almost overlapped on Dowex 1 3 2 chromatography, these components were easily separated on Dowex 50W 3 2 column (eluted with pyridineacetic acid buffer, pH 6.0). These compounds were identical with validoxylamines A and B obtained by the hydrolysis of validamycins A and B, respectively. The chromatograms on Dowex 1 3 2 columns of these components are shown in Fig. 2.5 (validamycins A, B, C, D, E, F, and validoxylamines A) and Fig. 2.6 (validoxylamines A and B). VA-A Refractive index

D

A

C

F B

0

1

2

3

E

4

5

6

7

8

9 hours

FIGURE 2.5 Chromatogram of authentic mixture of validamycins A, B, C, D, E, F and validoxylamines A. Column: Dowex 1 3 2 (OH form, 100200 mesh, 27 cm 3 15 mm I.D.). Carrier: Water (gravity flow rate; 1.1 mL/min). Reprinted with permission courtesy of Japan Antibiotics Research Association (JARA).

Refractive index

VA-A

VA-B

0

10

20

30

40

50

60

min.

FIGURE 2.6 Chromatogram of authentic mixture of validoxylamines A and B. Column: Dowex 1 3 2 (OH2 form, 100200 mesh, 27 cm 3 15 mm I.D.). Carrier: Water (gravity flow rate, 1.1 mL/min). Reprinted with permission courtesy of Japan Antibiotics Research Association (JARA).

28

Validamycin and Its Derivatives

Validamycin G and validoxylamine G were isolated from the broth using a similar method (Kameda et al., 1986). The crude validamycins were chromatographed on a column of Dowex 1-X2 (OH2 form) and eluted with H2O to give 10 components, validoxylamines A, G, and B, and validamycins D, A, G, C, B, F, and E in order of elution from the column. The validoxylamine G and validamycin G fractions were further chromatographed on a Dowex 50W-X8 column (eluted with 0.2 mol/L pyridineacetate buffer, pH 6.0). Finally, each component was rechromatographed on Dowex 1-X2 column (OH2 form) to obtain homogeneous validoxylamine G and validamycin G. The last member of validamycins, validamycin H, was discovered and isolated in 1990 (Asano et al., 1990). The crude validamycin complex, which was prepared in the process described previously, was passed through a column of Amberlite IR-120B (H1) and the column was eluted with 0.5 mol/L ammonium hydroxide. The eluate was concentrated and applied onto a column of Amberlite CG-50 (NH41). The effluent and washings were concentrated and lyophilized to give the validamycin complex. The complex was then chromatographed on a column of Bio-Gel P-2 (200B400 mesh) and the tetrasaccharide fraction was pooled. This fraction was further purified on a column on Dowex 1-X2 (OH2) and eluted with H2O to give validamycins C, H, and a mixture of validamycins E and F in order of elution from the column.

2.4 STRUCTURES, CHARACTERIZATION, AND PROPERTIES OF VALIDAMYCINS AND RELATED NATURAL COMPOUNDS Validamycins and related natural compounds include validamycins A, B, C, D, E, F, G, and H, validoxylamines A, B, and G, validamine, valienamine, valiolamine, and hydroxyvalidamine.

2.4.1 Structures of Validamycins and Related Natural Compounds The structures of validamycins A, B, C, D, E, F, G, and H, validoxylamines A, B, and G are listed in Fig. 2.7 (Asano et al., 1990; Horii and Kameda, 1972; Suami et al., 1980). The structures of validamine (Horii et al., 1971), valienamine (Kameda et al., 1980a, 1980c), valiolamine (Kameda et al., 1986), and hydroxyvalidamine (Horii et al., 1971) are shown in Fig. 2.8.

2.4.2 Characterization of Validamycins and Validoxylamines Validamycin A (Iwasa et al., 1971c) was obtained as colorless hydrophilic powder and did not show sharp melting point; it softened about 100 C and     decomposed about 135 C, ½α23 D 110 6 15 (c 1, H2O), 110 6 15 (c 1, pyri  dine), 92 6 10 (c 1, dimethylformamide), pKa 6.0. The molecular weight

29

Production of Validamycins Chapter | 2 7' 7

CH 2OR 5 R1 R 6O

CH2OR 2

R3

4' 3'

6 1

6' 1'

2' HO

OR 4

5

5'

4 3

2

N H

OH

OH

OH

Components

R1

R2

R3

R4

R5

R6

Validamycin A

H

H

H

β-D-Glc

H

H

Validamycin B

H

H

OH

β-D-Glc

H

H

Validamycin C

H

H

H

β-D-Glc

α-DGlc

H

Validamycin D

H

α-D-Glc

H

H

H

H

Validamycin E

H

H

H

α-D-Glc(1-4)β-D-Glc

H

H

Validamycin F

H

H

H

β-D-Glc

H

α-D-Glc

Validamycin G

OH

H

H

β-D-Glc

H

H

Validamycin H

H

H

H

β-D-Glc(1-6)β-D-Glc

H

H

Validoxylamine A

H

H

H

H

H

H

Validoxylamine B

H

H

OH

H

H

H

Validoxylamine G

OH

H

H

H

H

H

*

*Glc = Glucopyranosyl .

FIGURE 2.7 Chemical structures of validamycins and validoxylamines. CH 2OH

CH 2OH

CH 2 OH HO

HO

HO

HO

HO

NH 2

HO

OH Valienamine

FIGURE 2.8 Chemical hydroxyvalidamine.

CH 2 OH

NH 2

NH 2

Valiolamine

of

valienamine,

OH

HO

NH 2 OH

OH

OH Validamine

structures

HO

HO

validamine,

Hydroxyvalidamine

valiolamine,

and

was found to be 519 6 30 by the titration method. The elemental analysis and the molecular weight of validamycin A supported the molecular formula of C20H3337NO1314. It is soluble in water, methanol, dimethylformamide, and dimethyl sulfoxide, sparingly soluble in ethanol and acetone, and insoluble in ethyl acetate and diethyl ether. It shows a green color with the anthrone reagent, reddish brown color with phenol-sulfuric acid, reddish brown color with orcin sulfuric acid, blue-violet color with GreigLeaback’s reagent, and is positive with benzidineperiodate, but is negative to

30

Validamycin and Its Derivatives 2.5

3

4

5

6

7

8

9 10

20 25 μ

12 14

100 Validamycin A 80 60 40 20 0 4000 2.5 100

3000 3

2000 1800 1600 1400 1200 1000 4

5

6

7

8

9 10

800

600 400 cm–1

12 14

20 25 μ

Validamycin B

80 60 40 20 0 4000

3000

2000

1800 1600 1400 1200 1000 800

600 400 cm–1

FIGURE 2.9 Infrared spectra of validamycins A and B (KBr). Reprinted with permission courtesy of Japan Antibiotics Research Association (JARA).

Sakaguchi’s and ElsonMorgan’s reagents. The ultraviolet spectrum of validamycin A in aqueous solution shows only end absorption, and the infrared spectrum indicates the presence of hydroxyl and ether linkage (10001100 cm21) (Fig. 2.9). The nuclear magnetic resonance (NMR) spectrum (in D2O) of validamycin A indicates the presence of an anomeric proton (δ 6.3 ppm) and many methane protons that are adjacent to hydroxyls (

δ 35 ppm) (Fig. 2.10).

Validamycin B (Iwasa et al., 1971c) was obtained as basic hydrophilic   substance, colorless powder; ½α23 D 102 6 10 (c 1, H2O), pKa 5.0. The molecular weight was found to be 530 6 30 by the titration method. The elemental analysis and the molecular weight of validamycin B indicate the molecular formula of C20H3337NO1415. The solubility and color reaction of validamycin B were similar to those of validamycin A, and the ultraviolet (in H2O) and infrared (KBr) spectra of validamycin B were also similar to those of validamycin A. However, the NMR spectrum (in D2O) of validamycin B was different from that of validamycin A (Fig. 2.10). Validamycins C, D, E, F, G, and H (Asano et al., 1990; Horii et al., 1972; Kameda et al., 1986) are all white amorphous solids that have no definite melting point. A variety of chromatographic procedures have indicated that they are distinguishable from each other and are homogeneous. All are soluble in water, dimethylformamide, and dimethyl sulfoxide, and sparingly

Production of Validamycins Chapter | 2

31

Validamycin A

D2O (100 MC)

TMS

8

7

6

5

4

3

2

1

δ (ppm)

Validamycin B

D2O (100 MC)

TMS

7

6

5

4

3

2

1

δ (ppm)

FIGURE 2.10 NMR spectra of validamycins A and B (100 MC in D2O, TMS as reference). Reprinted with permission courtesy of Japan Antibiotics Research Association (JARA).

soluble or insoluble in ethanol, acetone, ethyl ether, benzene, chloroform, and ethyl acetate. In methanol, validamycin D is easily soluble, but validamycins C, E, and F are less soluble. All are weakly basic substances giving positive reaction with anthrone reagent, orcinolsulfuric acid, GreigLeaback’s reagent and benzidineperiodate, but negative reaction with Sakaguchi’s and ElsonMorgan’s reagents. The ultraviolet absorption spectra of validamycins C, D, E, and F all show only end absorption. The infrared absorption spectra of these are very similar to those of validamycins A and B, and are suggestive of polyhydroxy compounds. The 13C NMR spectral data of validamycins A, G, H, and validoxylamine G are listed in Table 2.10 (Asano et al., 1990; Kameda et al., 1986). And 1H NMR spectral data of validamycins A and H are listed in Table 2.11. Other characterization data on validamycins AH and validoxylamines AB and G are presented in Tables 2.12 and 2.13 (Asano et al., 1990; Horii et al., 1972; Iwasa et al., 1971c; Kameda et al., 1986).

TABLE 2.10

13

C NMR Chemical Shifts of Validamycins A, G, H, and Validoxylamine G

Carbon

Validamycin A

Validamycin G

Validamycin H

Validoxylamine G

C-1

56.53 d

54.5 d

56.60 d

54.4 d

C-2

75.96 d

75.4 d

75.96 d

75.5 d

C-3

75.54 d

72.9 d

75.80 d

74.3 d

C-4

87.10 d

86.0 d

87.75 d

76.6 d

C-5

40.23 d

79.0 s

40.20 d

78.4 s

C-6

29.67 t

31.8 t

29.64 t

31.4 t

C-7

64.60 t

67.1 t

64.71 t

64.8 t

C-1ʹ

55.20 d

56.4 d

55.22 t

56.7 d

C-2ʹ

125.97 d

125.0 d

126.05 d

125.3 d

C-3ʹ

141.98 s

142.1 s

142.05 s

142.2 s

C-4ʹ

74.28 d

75.8 d

74.34 d

75.6 d

C-5ʹ

76.49 d

74.3 d

76.54 d

74.3 d

C-6ʹ

72.23 d

72.5 d

72.29 d

72.4 d

C-7ʹ

64.37 t

64.3 t

64.41 t

64.3 t

C-1ʺ

105.67 d

105.8 d

105.87 d

C-2ʺ

76.22 d

76.2 d

76.17 d

C-3ʺ

78.43 d

78.5 d

78.37 d

C-4ʺ

72.23 d

72.1 d

72.33 d

C-5ʺ

78.80 d

78.7 d

77.58 d

C-6ʺ

63.34 t

63.2 t

71.61 t

C-1ʹʺ

105.51 d

C-2ʹʺ

75.96 d

C-3ʹʺ

78.45 d

C-4ʹʺ

72.44 d

C-5ʹʺ

78.84 d

C-6ʹʺ

63.58 t

34

Validamycin and Its Derivatives

TABLE 2.11 1H NMR Data of Validamycins A and H (δ in ppm, J in Hz) Proton

Validamycin A

Validamycin H

1-H

3.287 (br q)

3.290 (br q)

2-H

3.633 (dd, J 5 9.5, 4.0)

3.639 (dd, J 5 10.1, 4.0)

3-H

3.748 (t, J 5 9.5)

3.757 (dd, J 5 10.1, 9.3)

4-H

3.515 (dd, J 5 10.0, 9.5)

3.515 (dd, J 5 10.5, 9.3)

5-H

2.097 (m)

2.103 (m)

6-Hax

1.366 (ddd, J 5 14.5, 13.0, 2.8)

1.364 (ddd, J 5 14.8, 13.2, 2.8)

6-Heq

1.960 (dt, J 5 14.5, 3.2)

1.967 (dt, J 5 14.8, 3.2)

7-Ha,b

3.788 (d, J 5 4.3)

3.786 (d, J 5 4.6)

1’-H

3.382 (Br t, J 5 4.9)

3.367 (br s)

2’-H

6.046 (dq, J 5 4.9, 1.5)

6.048 (dq, J 5 5.0, 1.5)

4’-H

4.095 (br d, J 5 5.7)

4.097 (br d, J 5 5.9)

5’-H

3.633 (dd, J 5 9.0, 5.7)

3.636 (dd, J 5 9.5, 5.9)

6’-H

3.635 (dd, J 5 9.0, 4.0)

3.636 (dd, J 5 9.5, 3.9)

7’-Ha

4.138 (br d, J 5 13.9)

4.139 (br d, J 5 13.8)

7’-Hb

4.251 (dq, J 5 13.9, 1.0)

4.253 (dq, J 5 13.8, 1.0)

1”-H

4.528 (d, J 5 8.0)

4.532 (d, J 5 8.0)

2”-H

3.347 (dd, J 5 9.1, 8.0)

3.367 (dd, J 5 9.3, 8.0)

3”-H

3.524 (t, J 5 9.1)

3.513 (t, J 5 9.3)

4”-H

3.435 (dd, J 5 9.5, 9.1)

3.497 (dd, J 5 9.8, 9.3)

5”-H

3.497 (m)

3.675 (m)

6”-Ha

3.744 (dd, J 5 12.3, 5.8)

3.874 (dd, J 5 12.4, 6.3)

6”-Hb

3.916 (dd, J 5 12.3, 2.1)

4.224 (dd, J 5 12.4, 2.1)

1’“-H

4.512 (d, J 5 8.0)

2’“-H

3.332 (dd, J 5 9.3, 8.0)

3’“-H

3.513 (t, J 5 9.3)

4’“-H

3.402 (dd, J 5 9.8, 9.3)

5’“-H

3.463 (m)

6’“-Ha

3.675 (dd, J 5 12.4, 5.6)

6’“-Hb

3.992 (dd, J 5 12.4, 2.1)

TABLE 2.12 Properties of Validamycins AF and Validoxylamines AB (1) Formula

Elemental Analysis Found

Calcd.

C%

H%

N%

C%

H%

N%

Validamycin A

C20H35NO13  H2O

46.64

7.15

2.95

46.60

7.24

2.72

Validamycin B

C20H45NO18  H2O

45.58

6.92

2.71

45.20

7.02

2.64

Validamycin C

C26H35NO13  H2O

46.48

7.22

2.15

46.08

6.99

2.07

Validamycin D

C20H35NO13  H2O

46.97

7.23

2.58

46.60

7.24

2.72

Validamycin E

C26H45NO18  H2O

46.22

7.19

1.94

46.08

6.99

2.07

Validamycin F

C26H45NO18  H2O

46.00

6.90

1.95

46.08

6.99

2.07

Validamycin G

C20H35NO14  H2O

45.19

7.01

2.63

45.31

7.19

2.47

Validamycin H

C26H45NO18  H2O







46.08

6.99

2.07

Validoxylamine A

C14H25NO8  H2O

47.86

7.86

3.89

47.58

7.70

3.96

Validoxylamine B

C14H25NO9  H2O

46.01

7.28

3.59

45.52

7.37

3.79

Validoxylamine G

C14H25NO9  H2O

45.52

7.36

3.79

45.83

7.45

3.62

36

Validamycin and Its Derivatives

TABLE 2.13 Properties of Validamycins AF and Validoxylamines AB (2) ½α23 D (H2O)

pKa Valuea (neut. equiv.)

TLCb Rf Value

TLCc Rf Value

NMR (D2O)d Anomeric Proton

Validamycin A

1 112.5

6.0 6 0.2 (520 6 30)

0.20

0.29

δ 4.75 (J 5 7.5 Hz)

Validamycin B

1 102.3

5.0 6 0.2 (530 6 30)

0.29

0.23

δ 4.75 (J 5 7.5 Hz)

Validamycin C

1 132.9

6.0 6 0.2 (680 6 30)

0.14

0.14

δ 4.73 (J 5 7.3 Hz) δ 5.12 (J 5 3.3 Hz)

Validamycin D

1 169.3

6.0 6 0.2 (520 6 30)

0.20

0.21

5.12 (J 5 3.3 Hz)

Validamycin E

1 148.2

6.1 6 0.2 (680 6 30)

0.14

0.21

δ 4.75 (J 5 7.3 Hz) δ 5.59 (J 5 3.3 Hz)

Validamycin F

1 130.7

6.1 6 0.2 (680 6 30)

0.14

0.21

δ 4.75 (J 5 7.3 Hz) δ 5.61 (J 5 3.3 Hz)

Validamycin G

1 52.8



0.18

0.14



Validamycin H

1 74.9





0.14

0.21



Validoxylamine A

1 170.0

6.2 6 0.2 (355 6 30)

0.30

0.40



Validoxylamine B

1 130.7

5.0 6 0.2 (370 6 30)

0.37

0.32



Validoxylamine G

1 118.6



0.23

0.24



a

In water. Solvent: n-PrOH/CH3COOH/H2O 5 4/1/1; Silica gel: Kieselgel 60F254 (Merck). Solvent: BuOH/MeOH/CHCl3/condensed NH4OH 5 4/5/2/5; Silica gel: Kieselgel 60F254 (Merck). d At 100 MHz with TMS standard. b c

Another characteristic for validamycins is that the products of their acid hydrolysis are validoxylamines. Validamycins A, C, D, E, and F can be degraded to validoxylamine A. And validamycins B and G can be degraded to validoxylamines B and G, respectively. Validamycin A is harmless to mammals and fish; the 50% lethal dose (LD50) for rats is 20 g/kg (Ishikawa et al., 2005). Validamycin A is degraded rapidly in soil, with a half-life of ,5 h. The United States Environmental Protection Agency (EPA) has classified validamycin A into the toxicity class IV, or practically nontoxic. In Asia and Europe, more than 300 tons of validamycins are used annually. There has been no report of the occurrence of validamycin-resistant R. solani although validamycin A has been used in Japan for the control of rice sheath blight for more than 30 years (Ishikawa et al., 2005; Tomlin and Editor, 1997).

Production of Validamycins Chapter | 2

37

2.4.3 Characterization of Valienamine, Validamine, Valiolamine, and Hydroxyvalidamine Valienamine [(1S, 2S, 3S, 4R)-1-amino-5-(hydroxymethyl)-cyclohex-5-ene2, 3, 4-triol] and validamine (1S)-(1, 2, 4/3, 5)-1-amino-5-hydroxymethyl-2, 3, 4-cyclohexanetriol] were isolated from the microbial degradation of validoxylamine A with Pseudomonas denitrificans (Kameda and Horii, 1972; Kameda et al., 1975) and Flavobacterium saccharophilum (Asano et al., 1984; Kameda et al., 1980a, 1981). Later, valienamine, validamine, valiolamine [(1S)-(1(OH), 2, 4, 5/1, 3)-5-amino-1-hydroxymethyl-1, 2, 3, 4-cyclohexanetetrol], and hydroxyvalidamine [1-amino-5-hydroxymethyl-2, 3, 4, 6-cyclohexanetetrol] were isolated from the broth of S. hygroscopicus (Kameda et al., 1984). The absolute configurations of valienamine and its related pseudosugars are similar to that of α-D-glucose. Thus, they demonstrate strong glucosidase inhibitory activity (Kameda et al., 1980c, 1984; Takeuchi et al., 1990a, 1990b). There has been increasing interest in the chemistry and biochemistry of glycosidase inhibitors because of their potent use as chemotherapeutic agents. Glycosidases are enzymes for the cleavage of glycosidic bonds and are responsible for glycoprotein processing on the surface of the cell wall and for carbohydrate digestion in animals. Inhibition of these enzymes has significant implications for both antiviral and antidiabetic chemotherapy (Fleet, 1989). Plasma levels of D-glucose and insulin are usually high in diabetics, especially after food ingestion. Limiting intestinal digestion of dietary carbohydrates by inhibition of intestinal α-glucosidases has been suggested as a possible means of controlling diabetes mellitus and obesity. Thus, α-D-glucosidase inhibitors are thought to be valuable aids in the treatment of diabetes. They act by delaying the absorption of carbohydrates, thereby inhibiting postprandial hyperglycemia and hyperinsulinemia. Furthermore, several studies have confirmed the value of the inhibitors of the processing enzyme glucosidase I in the treatment of cancer (Nishimura et al., 1997) and in inhibiting the human immunodeficiency virus (HIV) replication etiologic agent for acquired immune deficiency syndrome (AIDS) and AIDS-related complex (Fleet et al., 1988). It has also been demonstrated that inhibition of the glycoprotein-processing enzyme mannosidase I may provide leads for the treatment of AIDS (Montefoiori et al., 1988). In the pseudoaminosugars, valienamine also showed antibiotic activity against Bacillus species (Kameda et al., 1980c). Valienamine has been found to be a key component for biological activities in pseudoaminosugars and pseudooligosaccharides such as validamycins (Horii and Kameda, 1972), acarbose (Wehmeier and Piepersberg, 2004), amylostatins (Chen et al., 2003), adiposins (Chen et al., 2003), acarviosin (Kato et al., 2016), and trestatins (Chen et al., 2003) (Fig. 2.11).

38

Validamycin and Its Derivatives

HOH2 C

H3 C

HOH 2 C

OH H

O OH

OH

OH

N H

OH

O

O

OH

m

O

O

OH

OH

O

HOH 2 C

HOH 2 C O

OH

OH

OH

n

Acarbose: m = 0, n = 1 Amylostatins HOH 2C OH

O OH

OH

N H

OH

HOH2 C

HOH 2 C

OH OH

N H

OH

OH

Acarviosin

Voglibose

HOH2 C

HOH2 C

HOH 2 C

OH O OH

N H

OH

OH

OH

O

O

OH

m

O

O

OH

OH

O

HOH 2 C

HOH 2 C O

H

OCH 3 OH

OH

OH

OH

OH

n

Adiposins

H

OH

OH

O

OH

HOH2C

HOH2C

H3C

HOH2C N H

O

OH

O

OH O

O OH

OH

OH CH2OH

HOH2C O

OH

O

OH

n

OH

O

O

HO

OH

Trestatins HOH2 C

HOH 2 C

H3 C

OH

OH H

O

O OH

m

N H OH

OH

OH

OH

O

O OH

O

O

OH

OH

HOH 2 C

HOH 2 C O

OH

n

OH

Oligostatins

FIGURE 2.11 Chemical structures of various glucosidase inhibitors.

These pseudooligosaccharides exhibit stronger enzyme inhibitory activities than valienamine itself. Valienamine and its related pseudosugars could also synthesize their N-substituted branch-chain derivatives, such as acarbose, acarviosin, voglibose (1,N-(2-hydroxy-1-(hydroxymethyl)ethyl) valiolamine, AO-128, Basen), etc., some of which have very strong enzyme inhibitory activity for different glucoside hydrolases. Therefore, valienamine and its related pseudoaminosugars suppress blood sugar elevation and are useful in treating symptoms of hyperglycemia and various disorders caused by hyperglycemia, such as obesity, adiposity, hyperlipemia (arteriosclerosis), diabetes, and prediabetes, as well as diseases attributable to sugar metabolism by microorganisms in the oral cavity, such as prophylaxis of dental caries. They also have value as inhibitors of the

Production of Validamycins Chapter | 2

39

processing enzyme glucosidase I in the treatment of cancer and as inhibitors of the HIV replication etiologic agent for AIDS and AIDS-related complex. They are also very important chemical intermediates in the synthesis of other strong α-glucosidase inhibitors, such as acarbose, adiposins, acarviosin, trestatins, voglibose, etc. Valienamine has the molecular formula C7H15NO4 and contains one primary NH2 (Van Slyke), one C 5 C, one CH2OH, and three secondary  OH groups: monohydrochloride (C7H13NO4,HCl), ½α23 D 1 68.6 (1N-HCl); 23   pentaacetate, mp 95 C, C17H23NO9, ½αD 1 30.2 (CHCl3), pKa0 8.4; positive to ninhydrin and Lemieux tests, negative to anthrone and Fehling tests. Validamine has the molecular formula C7H15NO4 and contains one primary NH2 (Van Slyke), one CH2OH, and three secondary OH groups:  monohydrochloride (C7H15NO4,HCl), mp 229232 C dec, ½α23 D 1 57.4 21  (1N-HCl), ½αD 1 60.6 (H2O), pKa0 8.2; positive to ninhydrin and Lemieux tests, negative to anthrone and Fehling tests. The physicochemical properties of valiolamine and epi-valiolamine are described in Table 2.14 (Chen et al., 2003). Hydroxyvalidamine has the molecular formula C7H15NO5 and contains one primary NH2 (Van Slyke), one CH2OH, and four secondary OH groups: crystal, mp 164165 C, ½αD 1 80:73 (H2O), pKa0 7.0; positive to ninhydrin and Lemieux tests, negative to anthrone and Fehling tests.

2.5 BIOSYNTHESIS OF VALIDAMYCINS Filamentous gram-positive Streptomyces strains produce myriads of secondary metabolites with diversified structures and biological activities, including antibacterial, antifungal, antitumor, immunosuppressant, pesticidal, and herbicidal effects (Demain, 2000). S. hygroscopicus subsp. jinggangensis, isolated from the Jinggang Mountain area of China in 1974 (Agricultural Antibiotic Group, 1975), produces at least two antibiotics of agricultural importance. Jingangmycin, a weakly basic water-soluble aminocyclitol antibiotic, which was later proven to be identical to validamycin A produced by S. hygroscopicus var. limoneus (Iwasa et al., 1970), has been widely used as a prime control reagent against sheath blight disease of rice plants and dumping-off of cucumber seedlings in China and many other eastern Asian countries. The other antibiotic, jingsimycin, is an acidic polypeptide similar to saramycetin and has activity against various fungi. A structural comparison between validamycin A and acarbose (Fig. 2.9), a compound used for the treatment of type II insulin-independent diabetes, revealed that they contain an identical C7N aminocyclitol moiety (Mahmud, 2003), valienamine, whose structure and stereochemistry resemble that of D-glucose and which is responsible for the strong inhibitory activity of acarbose against α-glucosidases. Although the valienamine moiety in both acarbose and validamycin A is derived from the same precursors,

40

Validamycin and Its Derivatives

TABLE 2.14 Physicochemical Properties of Valiolamine and epi-Valiolamine Valiolamine

epi-Valiolamine

Appearance

Both hygroscopic, white powder

Element analysis

Calcd. for C7H15NO5  H2O: C 39.80; H 8.11; N 6.63. Found: C 39.35; H 7.82; N 6.59

Calcd. for C7H15NO5  H2O: C 39.80; H 8.11; N 6.63. Found: C 39.55; H 8.32; N 6.50

Specific rotation ½α20 D

18.8 (c1, H2O)

18.2 (c1, H2O)

UV absorption spectruma

No absorption maximum

IR absorption spectrumb

3340, 2920, 1575, 15001300, 1090, 1045, 910, and 815 cm21

3340, 2920, 1570, 15001300, 1090, and 1045 cm21

1

δ 1.65 (1H, dd, J 5 3.8, 15.2), 1.90 (1H, dd, J 5 0.9, 15.2), 3.33 (1H, m), 3.40 (1H, d, J 5 9.5), 3.48 (2H), 3.56 (1H, dd, J 5 3.9, 9.5), 3.83 (1H, t, J 5 9.5)

δ 1.72 (1H, dd, J 5 4.6, 14.3), 1.96 (1H, dd, J 5 6.6, 14.3), 3.42 (1H, m), 3.58 (1H, d, J 5 6.6), 3.60 (2H, d, J 5 3.1), 3.76 (1H, dd, J 5 4.0, 6.6), 4.00 (1H, t, J 5 6.6)

13

δ 35.06 (t), 52.94 (d), 68.24 (t), 73.88 (d), 76.30 (d), 76.47 (d), 78.71 (s)

δ 37.16 (t), 48.79 (d), 67.93 (t), 74.27 (d), 75.44 (d), 77.03 (d), 77.85 (s)

Solubility

Both are soluble in water, dimethyl sulfoxide, and methanol; both are sparingly soluble or insoluble in ethanol, ethyl acetate, chloroform, and acetone

Color reactions

Both are positive to ninhydrin reaction and GreigLeaback reagent and negative to naphthoresorcinolsulfuric acid

H NMRc

C NMRd

a For both amino sugars, the aqueous solution does not exhibit any characteristic absorption maximum in the region 200360 nm except end absorption. b The IR absorption spectrum was measured by the KBr method. The wavenumbers of the main absorption peaks are given. c The 1H NMR spectrum was measured in D2O at 100 MHz. The chemical shift δ values and coupling constants J (hertz) are given. d The 13C NMR spectrum was measured in D2O at 100 MHz under decoupling conditions. The chemical shift δ values, as well as the splitting patterns as measured under the off-resonance conditions, are given.

2-epi-5-epi-valiolone (Dong et al., 2001; Mahmud et al., 1999; Stratmann et al., 1999), further downstream their biosynthetic pathways appear to be different (Mahmud et al., 2001). Feeding experiments with isotopically labeled precursors to the validamycin producing S. hygroscopicus var. limoneus revealed the incorporation of 2-epi-5-epi-valiolone, 5-epi-valiolone, valienone, and validone into validamycin A (Fig. 2.12) (Dong et al., 2001).

Production of Validamycins Chapter | 2

OH

OH

OH

HO

41

OH

HO

HO

HO

HO

O HO OH

2-epi-5-epi-Valiolone

HO

O OH

5-epi-Valiolone

HO

O

HO

O

HO

OH

Valienone

OH

Validone

FIGURE 2.12 Important intermediates in the biosynthesis of validamycin A.

Based on the results of these feeding experiments, it was proposed that in validamycin A biosynthesis 2-epi-5-epi-valiolone is epimerized at the C-2 stereocenter followed by dehydration between C-5 and C-6 to give valienone. The latter compound was found to be a common precursor for both the unsaturated and saturated cyclitol moieties of validamycin A (Dong et al., 2001). Identification and functional analysis of the biosynthetic gene cluster of validamycin in S. hygroscopicus var. jinggangensis 5008 has been reported by Deng and coworkers, revealing 16 structural genes; 2 regulatory genes; 5 genes related to transport, transposition/integration, and tellurium resistance; and another 4 genes with no obvious identity (Bai et al., 2006). The involvement of the gene cluster in validamycin biosynthesis was confirmed by deletion of a 30-kb DNA fragment from this cluster in the chromosome that resulted in the loss of validamycin production. In addition, valA, a homolog of the acbC gene of acarbose biosynthesis, was disrupted in the validamycin producer and found to be essential for the production of validamycin A. Biochemical studies using the ValA protein heterologously expressed in E. coli revealed the function of this protein as a 2-epi-5-epi-valiolone synthase. Among 16 structural genes, only eight were found to be essential for the synthesis of validamycin A in a heterologous host, S. lividans 1326. Those are valA (encoding a 2-epi-5-epi-valiolone synthase), valB (encoding a nucleotidyltransferase), valC (encoding a cyclitol kinase), valG (encoding a glycosyltransferase), valK (encoding an epimerase/dehydratase), valL (encoding a validoxylamine A 7-phosphate synthase), valM (encoding a aminotransferase), and valN (encoding a cyclitol reductase) (Fig. 2.13 and Table 2.15) (Bai et al., 2006). In vivo inactivation of the putative glycosyltransferase gene (valG) abolished the final attachment of glucose for validamycin production, and resulted in the accumulation of the precursor, validoxylamine A (Scheme 2.1) (Bai et al., 2006). Complementation with valG restored the normal production of validamycin A, and in vitro enzymatic assays using the recombinant ValG protein demonstrated the glycosylation of validoxylamine A to validamycin A. Based on these genetic and biochemical data as well as the previously reported feeding experiments,

42

Validamycin and Its Derivatives S. hygroscopicus var. jinggangensis 5008

valJ

vall

valH

valG

valF valE

valD valC valB valA valK valL valM valN valO orf3

orf2 orf1

S. hygroscopicus var. limoneus KCCM 11405

vldS vldU vldW vldX vldC vldB vldA vldT vldV Validamycin biosynthetic genes Transporter/efflux pump Possible tailoring genes

vldD vldE vldF vldG vldH

vldl

vldJ

vldK

Unknown/unrelated open reading frames Transposase gene

FIGURE 2.13 Genetic organization of the validamycin biosynthetic gene clusters from S. hygroscopicus var. jinggangensis 5008 and S. hygroscopicus var. limoneus KCCM 11405. Blue arrows are genes proposed to be involved in the biosynthesis of validamycin A. Dashed lines show the inverted locations of three almost identical genes in strains 5008 and KCCM 11405.

TABLE 2.15 Comparison of Related Genes Present in the val, vld, and acb Clusters and Their Putative Function Val Protein

Vld Protein (% sim.)a

Acb Protein (sim.)a

Putative Function

ValA

VldA (100)

Acb (72)

2-epi-5-epi-Valiolone synthase

ValB

VldB (99)

Acb (72)

Nucleotidylyltransferase

ValC

VldC (99)

Acb (41)

Cyclitol kinase

ValD

VldX (99)



Glyoxalase

ValE





Oxidoreductase

ValF





Oxidoreductase

ValG

VldK (98)



Glycosyltransferase

ValH

VldJ (99)



Transport protein

ValI

VldI (99)



Glycosylhydrolase

ValJ

VldW (97)



Oxidoreductase

ValK

VldD (100)



Epimerase/dehydratase

ValL

VldE (98)



Validoxylamine A 7’-phosphate synthase

ValM

VldF (99)



Cyclitol aminotransferase

ValN

VldG (99)



Cyclitol reductase (Continued )

43

Production of Validamycins Chapter | 2

TABLE 2.15 (Continued) Val Protein

Vld Protein (% sim.)a

Acb Protein (sim.)a

Putative Function

ValO

VldH (96)



Phosphatase/ phosphohexomutase

ValP





Regulatory protein

ValQ





Regulatory protein

Orf4

VldV (100)



Unknown

a

% similarity to the corresponding Val protein at the amino acid level.

OH

HO

Sedoheptulose 7-P

HO

ValA

ValK

HO

O

HO

OP

OH

OH HO

HO

HO

ValC

ValK HO

O

HO

OH 2-epi-5-epi-Valiolone

O

HO

O OH Valienone 7-P

OH Valienone

OH 5-epi-Valiolone

ValN? OP

OP

OH(P)

HO

HO

HO

?

HO

OP

HO

OH OH

OH 1-epi-Valienol 1-P

1-epiValienol 7-P

HO

O OH Validone 7-P

ValB OH(P) HO

OP

OP

OH(P) OH

HO

HO

ValL HO

N OH H OH OH Validoxylamine A 7'-phosphate

HO

ONDP

OH NDP-1-epi-Valienol

NH2

HO

OH Validamin 7-P

Phosphatase ValG Validoxylamine A

UDP-glucose

Validamycin A

SCHEME 2.1 Biosynthesis pathway to validamycin A proposed by Deng and coworkers.

Deng and coworkers then proposed the biosynthetic pathway to validamycin A as shown in Scheme 2.1 (Bai et al., 2006). Recently, two other validamycin biosynthetic gene clusters from related strains of bacteria have been reported (Jian et al., 2006; Singh et al., 2006).

44

Validamycin and Its Derivatives

Deng and coworkers identified the biosynthetic gene cluster of jinggangmycin in S. hygroscopicus 1022 (Jian et al., 2006). The chemical structure of jinggangmycin was recently proposed to be identical to validamycin A, based on comparisons of its high-performance liquid chromatography (HPLC) retention time, tandem mass spectrometry (MS/MS) fragmentation pattern, and bioactivity, as well as the sequence similarity of its biosynthetic gene clusters. While only part of the cluster (ca. 6 kb) has been sequenced, three open reading frames identified in the clusters, orf1orf3, showed 99% identity at the nucleotide level and 99% similarity at the amino acid level to valA, valB, and valC, respectively. In addition, Suh and coworkers independently sequenced a 37-kb DNA fragment harboring a complete set of validamycin biosynthetic genes from S. hygroscopicus var. limoneus KCCM 11405 (IFO 12704) (Singh et al., 2006). Direct comparison of the validamycin cluster from S. hygroscopicus var. jinganggensis 5008 (the val cluster) and that from S. hygroscopicus var. limoneus KCCM 11405 (the vld cluster) has shown that both clusters contain an almost identical set of genes necessary for the biosynthesis of validamycin A, or at least for that of validoxylamine A (Fig. 2.14). The similarity of the genes involved in validamycin biosynthesis in both clusters ranges from 96% to 100% (see Table 2.15). Combined with the high similarity in the genetic organization of the gene clusters, these data indicate that both producers are very closely related.

(A)

(B)

SAV-DEGs P2 422

611

SHJG-DEGs

SHJG-DEGs

SHJG-DEGs

P2

P2

P4

701

841

667

738

122

237

Gene number of COG categories

30 Down-regulated at 37°C Up-regulated at 37°C 20

10

A C min ar o b a C ohy cid el d m l e ra n t e C ve e m tab oe lo e o n p ta lis E In n D zym e b bo m or e ef i ga rg en e oge lism y ni p se me ne c ro io d m lab si n u tra ct DN ech oli s ns ion A an sm po an re is rt d pli ms an co ca Se P co os N L d m nve tion nd ttr uc ipi et rs l Si ary ans eot d m abo ion gn m ta id et lis al et tio e m ab m tra ab na e oli ns oli l m lab sm du tes od ol ct b ifi ism io io ca n sy ti m n on ec th Tr ha esis an nis sc m Tr rip s an tio st n at io n

0

FIGURE 2.14 Transcriptomic analysis of strain 5008 (SHJG) and S. avermitilis NRRL8165 (SAV) response to elevated temperature. (A) Differentially expressed genes (DEGs) at 37 C and 30 C; (B) COG category distributions of DEGs in 5008 by DNA microarray (P , 0.01, n 5 4). .4-fold up-regulated (red); 4-fold down-regulated (green).

Production of Validamycins Chapter | 2

45

The reported validamycin gene clusters generally fit well with the proposed scheme of validamycin A biosynthesis derived from the feeding experiments (Scheme 2.2). In vivo inactivation of valA and vldA and in vitro characterization of the gene products confirmed the initiation step from sedoheptulose 7-phosphate to the cyclic product, 2-epi-5-epi-valiolone. Subsequently, epimerization at C-2 and dehydration at C-5/C-6 of 2-epi-5epi-valiolone by the proposed bifunctional ValK would give valienone. In acarbose biosynthesis, 2-epi-5-epi-valiolone is phosphorylated by AcbM (the cyclitol kinase) to give 2-epi-5-epi-valiolone 7-phosphate. Further reactions in the acarbose pathway were proposed to involve phosphorylated intermediates (Zhang et al., 2002). The presence of valC or vcdC, a homolog of acbM, in the val and vld gene clusters suggests the involvement of phosphorylated intermediates in validamycin A biosynthesis as well. However, because 2-epi-5-epi-valiolone, 5-epi-valiolone, valienone, and validone were efficiently incorporated into validamycin A, the kinase (ValC) might have broad substrate specificity, activating all these cyclitols and making it possible for them to be incorporated into the biosynthetic pathway. Alternatively, ValC may only utilize valienone and/or validone as substrate. Suh and coworkers (Singh et al., 2006) proposed a rather intriguing hypothesis regarding the function of vldC (Scheme 2.2). They speculated that VldC is a 5-epi-valiolone 7-kinase that phosphorylates 5-epi-valiolone to its 5-epi-valiolone 7-phosphate. The latter compound could be converted to 5-epi-valiolone 5-phosphate by the action of a phosphomutase (VldH), setting the stage for a dehydration reaction leading to valienone. However, inactivation of the vldH homolog, valO, in S. hygroscopicus 5008 did not abolish the production of validamycin A (Bai et al., 2006), which raises questions whether VldH is really involved in the biosynthesis of validamycin A. Alternatively, VldC was also proposed to function as a glucose kinase, the product of which is converted to nucleotidyldiphosphate (NDP)-glucose by the action of VldB. Our recent investigation of the function of ValC has shown that the kinase phosphorylates valienone and validone to their corresponding 7-phosphorylated derivatives, but not 2-epi-5-epi-valiolone, 5-epi-valiolone, or glucose. Valienone 7-phosphate may serve as a branching point in the pathway: a portion of it is reduced to validone 7-phosphate and the rest is reduced to valienol 7-phosphate or 1-epi-valienol 7-phosphate by the reductase ValN (VldG) (Scheme 2.1) and/or other reductases/dehydrogenases, e.g., ValE, ValF, or ValJ. However, the heterologous expression experiments in S. lividans suggest that ValE, ValF, and ValJ are not necessary for the biosynthesis of validamycin A (Bai et al., 2006). Similarly, inactivation of vldW, which corresponds to valJ, did not abolish the production of validamycin A in S. hygroscopicus var. limoneus (Singh et al., 2006), which leaves this part of the pathway unclear. It was proposed that these oxidoreductases might be involved in the biosynthesis of the hydroxylated analogs of

OH

HO

Sedoheptulose 7-P

OH

HO HO

VldA

HO HO

HO

HO

VldC

HO

O

OH 2-epi-5-epi-Valiolone

HO

OH

OP

O OH 5-epi-Valiolone

O HO

O

Glucose 1-P

HO

O

O OH Valienone 7-P

OH Valienone

VldF reductive coupling

VldB

NDP-glucose

Reduction

HO

OH 5-epi-Valiolone 5-P

VldC

HO

HO

Dehydration

OH 5-epi-Valiolone 7-P

Glucose

PO

VldH

HO

OP

OH

OH

OH

HO

OH

Validamycin A N H

HO OH

OH OH

Validoxylamine A

SCHEME 2.2 Biosynthetic pathway to validamycin proposed by Suh and coworkers.

Production of Validamycins Chapter | 2

47

validamycin A, validamycin B, and/or validamycin G. Transamination of the keto group of validone 7-phosphate catalyzed by the aminotransferase ValM (or VldF) would give validamine 7-phosphate. In the other branch of the pathway, (1-epi-) valienol 7-phosphate is either phosphorylated at C-1 by an unidentified kinase to generate the 1,7-diphosphate derivative as proposed in acarbose biosynthesis or transformed into valienol 1-phosphate through catalysis by a phosphoglucomutase. The product is subsequently converted to an NDP-valienol derivative by the nucleotidyl transferase ValB (or VldB). Condensation of NDP-(1-epi-) valienol with validamine 7-phosphate is predicted to give phosphorylated validoxylamine A. However, an enzyme required for the dephosphorylation of validoxylamine A 70 -phosphate to validoxylamine A has not been found within the cluster. A nonspecific sugar phosphatase may be able to carry out this hydrolytic reaction. Finally, validoxylamine A is converted to validamycin A through catalysis by the glucosyltransferase ValG using UDP-glucose as the sugar donor. Interestingly, the location of the glycosyltransferase genes is different in the two clusters (Fig. 2.13). In the 5008 strain, ValG is located upstream of the main cluster, and in the KCCM 11405 strain the homologous gene (vldK) is located downstream of the main cluster (Singh et al., 2006). Although Suh and coworkers (Singh et al., 2006) excluded vldK from the proposed structural genes for validamycin biosynthesis, the high similarity (98%) between VldK and ValG suggest that vldK is also involved in the biosynthesis of validamycin A. The role of vldI in validamycin biosynthesis is substantiated with the observation that validamycin A productivity decreased significantly in a vldI knockout mutant (Singh et al., 2007). The gene valC, which encodes an enzyme homologous to the 2-epi-5-epivaliolone kinase (AcbM) of the acarbose biosynthetic pathway, was identified in the validamycin A biosynthetic gene cluster (Minagawa et al., 2007). Inactivation of ValC resulted in mutants that lack the ability to produce validamycin A. Complementation experiments with a replicating plasmid harboring full-length ValC restored the production of validamycin A, thus suggesting a critical function of ValC in validamycin biosynthesis. The biochemical analysis of VldB, VldE, and VldH and the establishment of their roles in validoxylamine A biosynthesis was investigated by Mahmud and coworkers (Asamizu et al., 2011; Yang et al., 2011). Particularly, the involvement of VldE and VldH, which resemble OtsA and OtsB, in validoxylamine A formation is evolutionarily and mechanistically intriguing. VldE might have evolved from an ancestral trehalose 6-phosphate synthase to the extent that it no longer recognizes activated sugars as substrates, but still retains some of the catalytic properties, including the net retention of the anomeric configuration of the product. In addition, the use of the aminocyclitol unit as pseudosugaracceptor by VldE to form N-pseudoglycosyl linkage is a significant departure from the OtsA catalyzed reaction. In comparison to the O-glycosyltransferases such as OtsA, glycogen synthase, cellulose

48

Validamycin and Its Derivatives

synthase, and others, N-glycosyltransferase enzymes are less abundant, but play significant roles, in biological systems. More recently, the thermoregulated biosynthesis of validamycin in S. hygroscopicus 5008 was studied with genomic analysis (Wu et al., 2012). S. hygroscopicus 5008 has been used for the production of the antifungal validamycin/jinggangmycin for more than 40 years. A high yield of validamycin is achieved by culturing the strain at 37 C, rather than at 30 C for normal growth and sporulation. The mechanism(s) of its thermoregulated biosynthesis was largely unknown. To explore the molecular mechanism of the positive thermoregulation on validamycin A biosynthesis, the transcriptomes of strain 5008 cultured at 30 C or 37 C in liquid medium were compared by microarray analysis. Given more shared orthologs (4845) between strain 5008 and S. avermitilis NRRL 8165, they chose S. avermitilis cultivated under the same conditions as a filter. Using the statistical criteria of .2-fold change and p , 0.05, a total of 1542 differentially expressed genes (DEGs) were identified at 37 C in strain 5008 (Fig. 2.14A). Likewise, 1033 genes were differentially transcribed by NRRL8165 under the same cultivation condition (Fig. 2.14A). Filtered with the DNA microarray dataset from NRRL8165, the number of DEGs in 5008 was reduced to 1405, and subsequently to 359 using more stringent criteria of .4-fold change and P , 0.01 (Fig. 2.14A). The markedly down-regulated DEGs at 37 C are largely assigned with functions for amino acid transport and metabolism, inorganic ion transport and metabolism, and cell envelope biogenesis (Fig. 2.14B). Consistent with previous observation (Liao et al., 2009), numerous ribosomal protein genes were moderately up-regulated at 37 C. As expected, the transcriptional levels of most of the validamycin A biosynthetic genes were markedly enhanced at 37 C, except for glucosyltransferase gene valG, transporter gene valH, and the two-component regulatory genes valP and valQ. Furthermore, three other gene clusters of PKS NPRSs and type-III PKS were also up-regulated by the strain at 37 C (Table 2.16). Although the genes involved in PPP were not dramatically overexpressed at 37 C, key enzymes for glycolysis were moderately down-regulated, including the 6-phosphofructokinase (Pfk), glyceraldehyde-3-phosphate dehydrogenase (Gap), and pyruvate kinase (Pyk). Moreover, gluconokinase (GntK) for phosphorylation of gluconate to generate 6-phosphogluconate and citrate lyase (CitE), catalyzing the cleavage of citrate to yield oxaloacetate and acetyl-CoA, had notably enhanced transcription, which could increase the carbon flux to the PPP and decrease the TCA cycle (see Table 2.12). A glutamine synthetase gene glnA and its positive regulatory gene glnR (Wray et al., 1991) were also down-regulated by strain 5008 at 37 C, suggesting a low concentration of glutamine and a high concentration of ammonium accumulated in bacterial cells. On the other hand, the gene of glutamate dehydrogenase GdhA (SHJG7666) for converting 2-oxoglutarate

Production of Validamycins Chapter | 2

49

TABLE 2.16 Selected Differentially Expressed Genes in S. hygroscopicus 5008 at 37 C Compared with at 30 C Description

Gene ID or Name

Fold Change

Up-regulated genes at 37 C Validamycin biosynthesis

valABCD, ValKLMN, valEF, valIJ

4.123.8

PKS-NRPS biosynthesis

SHJG0303SHJG0325

6.8707.3

PKS-NRPS biosynthesis

SHJG1907SHJG1918, SHJG1921SHJG1929

2.111.8

Type III PKS biosynthesis

SHJG8479

12.9

Central carbon metabolisms

SHJG3123 (gntK), SHJG3510 (citE)

4.65.2

Nitrogen metabolism

SHJG7666 (gdhA), SHJG7685 (glnA3)

2.73.0

Ribosomal proteins

SHJG0705SHJG0706, SHJG0835SHJG0836, SHJG2929, SHJG5163SHJG5164, SHJG5778, SHJG5809SHJG5816

2.13.6

Heat shock proteins

SHJG4359 (hspX), SHJG5369SHJG5372 (dnaK-grpE-dnaJ-hspR), SHJG7073, SHJG8393

3.35.3

Markedly expressed regulators

SHJG0301, SHJG0319, SHJG0322, SHJG0477, SHJG6588, SHJG6678, SHJG6961, SHJG7352

4.3128.2

Down-regulated genes at 37 C Glycolysis

SHJG2653 (pfk), SHJG3403 (gap), SHJG3488 (pyk)

2.63.5

Nitrogen metabolism

SHJG2665 (ureD), SHJG2667 (ureE), SHJG2668SHJG2670 (ureCBA), SHJG3687 (glnA), SHJG3702 (glnII), SHJG3730 (glnA2), SHJG3958 (nirB), SHJG4429 (narK), SHJG4923 (glnR), SHJG6704 (amtB), SHJG6705 (glnB), SHJG6706 (glnD)

4.116.7

Phosphate starvation response

SHJG0329 (phoA), SHJG2705, SHJG4290 (ppe), SHJG4865 (phoU), SHJG4934 (ppk), SHJG4942SHJG4945 (pstBACS), SHJG5998SHJG5999 (neuAB), SHJG8008 (glpQ), SHJG8009

3.9144.3

(Continued )

50

Validamycin and Its Derivatives

TABLE 2.16 (Continued) Description

Gene ID or Name

Fold Change

Sulfate assimilation

SHJG1828SHJG1830, SHJG4909 (cysA), SHJG7116, SHJG7181SHJG7183 (ssuABC), SHJG7184SHJG7187 (cysCNDH), SHJG7188, SHJG7189 (cycI)

4.716.8

Markedly expressed relators

SHJG0180, SHJG1204, SHJG1557, SHJG4402, SHJG4865, SHJG5108, SHJG5332, SHJG6822, SHJG7752, SHJG8626, HJG8650, SHJG8654, SHJG8656, SHJG8657

4.331.3

into L-glutamate was moderately up-regulated at 37 C, implying a mechanism for generating more amino group for validamycin A biosynthesis. Accordingly, SHJG7666 was deleted in strain 5008, and a desired mutant JG33 was obtained (Figs. 2.15AB). HPLC analysis of the extracts from the mutant JG33 displayed obvious reduction of validamycin A production (Fig. 2.15C). Also, the intracellular concentration of glutamate in strain 5008 and its mutant XH3, with val gene cluster deleted, was quantified after 24- and 48-h cultivation using an amino acid analyzer. In 48-h cultured mutant XH3 with validamycin productivity abolished, the intracellular glutamate concentration at 37 C (5123 ng/mg dry weight) was higher than that at 30 C (4201 ng/mg dry weight), indicating an efficient synthesis of glutamate in S. hygroscopicus 5008 and its derivatives at both temperatures. Moreover, when validamycin was overproduced in strain 5008 at 37 C for 48 h, the intracellular glutamate concentration dropped to 1203 ng/mg dry weight, less than a fourth of mutant XH3 and a fifth of strain 5008 cultivated at 30 C (6933 ng/mg dry weight) (Fig. 2.15D). Therefore, the dramatic decrease of intracellular glutamate concentration and the synchronic accumulation of validamycin A in strain 5008 indicated most of the glutamate was consumed for validamycin A biosynthesis at the higher temperature. Among the 22 markedly expressed regulators by strain 5008 at 37 C (Table 2.17), a SARP-family regulatory gene (SHJG0322) was most highly expressed, with a maximum enhancement of 128-fold. SHJG0322 was inactivated by replacing a 610-bp internal sequence with the apramycin resistance gene aac(3)IV in strain 5008, generating a thiostrepton-sensitive, apramycinresistant (ThioSAprR) mutant (JG27) (Fig. 2.16AB). The wild-type 5008 and the SHJG0322 mutant JG27 were cultivated at 30 C or 37 C for 2 days, and the extracts of these cultures were analyzed by HPLC. At 30 C, the mutant produced 0.07 g/L validamycin A, similar to the amount produced by the wild-type 5008 (0.09 g/L). At 37 C, however, the yield of validamycin A in the mutant JG27 was 0.49 g/L, which was less

Production of Validamycins Chapter | 2

51

FIGURE 2.15 Inactivation of the glutamate dehydrogenase gene SHJG7666, and measurement of intracellular glutamate (Glu) in strain 5008 and its mutant XH3 with validamycin gene cluster deleted. (A) Schematic deletion of SHJG7666 in strain 5008. (B) Polymerase chain reaction (PCR) analysis of strain 5008 and SHJG7666 mutant JG33. (C) Validamycin A production in strain 5008 and mutant JG33 cultivated at 30 C or 37 C. Mean values of three independent experiments with SD are indicated by error bars. (D) Concentration of intracellular Glu in strain 5008 and mutant XH3. Samples were extracted in strain 5008 and mutant XH3 for one or two days at 30 C or 37 C. Each value is the average of two measures using an amino acid analyzer. XH3 is a mutant with val gene cluster deleted.

than 20% of the wild-type productivity (2.52 g/L) (Fig. 2.16C). Detected by quantitative RT-PCR, the relative transcription of validamycin A biosynthetic genes valA and valK of the wild-type 5008 were increased by 100-fold and 26-fold at 37 C than at 30 C, respectively (Fig. 2.16D). However, the transcription of valA and valK in the mutant JG27 at 37 C were both dropped to only 6-fold than at 30 C (Fig. 2.16D). Furthermore, the mutant JG27 was complemented with a cloned SHJG0322 under the control of the PermE constitutive promoter (pJTU5287) or its native promoter (pJTU5288). A similar amount of validamycin A was produced in both complemented derivatives JG27/ pJTU5287 (2.23 g/L) and JG27/pJTU5288 (2.06 g/L) at 37 C, which accounted respectively for 88.6% and 81.6% of the wild-type yield (Fig. 2.16C). At 30 C,

52

Validamycin and Its Derivatives

TABLE 2.17 Optimization of Corn Powder and Soybean Powder Concentrations Using Single-Factor Experiment Factors

Concentration (g/L)

Validamycin A (g/L)

Corn powder

90

9.5 6 0.9

70

7.0 6 0.8

Soybean powder

(A)

50

5.1 6 0.5

40

9.5 6 0.9

30

12.3 6 2.5

20

12.0 6 1.1

0322-C-F 0322-C-R 0.66 kb SHJG0321

SHJG0322

SHJG0323

5008

SHJG0324

0.61 kb Deleted 4775 bp

3863 bp aac(3)IV

pJTU5271

oriT ori pIJ101

tsr

Double crossover Truncated SHJG0322 aac(3)IV

SHJG0321 oriT 0322-C-F

(D) 4

120 va/A va/K

3 2 1

1.40 kb

Relative quantity

Validamycin A (g/L)

08

JG 36

50

1k

1.65 kb

JG27

SHJG0324

0322-C-R

(C) b

(B)

SHJG0323

1.40 kb

90

60

30

08

27 JG

50

27 JG

08

0 50

0.66 kb

50 08 JG 30° 27 C 50 30° 08 C JG 27 JG 37° / JG pJT 27- C 27 U5 37 ° 2 / JG pJT 87 C 27 U5 -30 ° /p 2 JG JT 88 C 27 U5 -30 ° 2 /p JT 87 C -3 U 52 7° 88 C -3 7° C

0 0.65 kb

FIGURE 2.16 Inactivation of the regulatory gene SHJG0322. (A) Schematic replacement of an internal 610-bp fragment of SHJG0322 with aac(3)IV-oriT cassette. (B) PCR analysis of strain 5008 and SHJG0322 mutant JG27. (C) Validamycin A production in strain 5008, mutant JG27, JG27/ pJTU5287 (with PermE promoter), and JG27/pJTU5288 (with native promoter) cultivated at 30 C or 37 C. Mean values of three independent experiments with SD are indicated by error bars. (D) Relative transcriptional levels of valA and valK in strain 5008 or JG27 at 37 C against 30 C.

Production of Validamycins Chapter | 2

53

both strains produced comparable amounts of validamycin A to strain 5008 and mutant JG27 (Fig. 2.16C). These results suggested that the SARP gene SHJG0322 was necessary but not adequate for the thermoregulated validamycin biosynthesis in strain 5008. Probably some other regulatory factors are recruited as well. The function of valL was identified later (Zheng et al., 2012). In order to genetically prove the involvement of valL in validamycin A biosynthesis, a 1.18-kb internal region of valL was replaced by an aac(3)IV cassette in strain 5008. This was achieved by using a pHZ1358-derived plasmid pJTU685, in which valL had been replaced by aac(3)IV between a 2.98-kb left-flanking and a 2.12-kb right-flanking sequence of valL (Fig. 2.17A). The plasmid

FIGURE 2.17 Inactivation of valL and complementation of the mutant. (A) Schematic representation of the replacement of an 1.18-kb internal fragment of valL with the 1.4-kb aac(3)IV. In shuttle plasmid pJTU685, aac(3)IV was inserted between the 2.98-kb and 2.12-kb genomic fragments originally flanking the deleted 1.18-kb region. While wild-type S. hygroscopicus 5008 should give a 1.30-kb PCR-amplified product, mutant 5008ΔvalL should yield a 1.60-kb product by using a pair of primers, valL-Det-F and valL-Det-F. (B) PCR analysis of wild-type S. hygroscopicus 5008 and valL mutant. (C) HPLC profiles of the standards, 5008, 5008ΔvalL, and 5008ΔvalL::pJTU640. Plasmid pJTU640 is an integrative plasmid cloned with intact valL under the control of PermE promoter. (D) Bioassay comparison between the 5008 and 5008ΔvalL. One mL of fermentation supernatant was mixed with 14 mL of melted 0.8% agar. An agar plug with the fungal indicator P. sasakii was transferred to the center of the agar plate. After 24 h incubation at 30 C, the diameter of the colony was measured, which is inversely related to the inhibitory potency.

54

Validamycin and Its Derivatives

pJTU685 was introduced into strain 5008 through conjugation, and thiostrepton-sensitive and apramycin-resistant exconjugants were selected. The mutants 5008ΔvalL were further confirmed by PCR amplification. The valL mutant gave an expected 1.60-kb PCR product, whereas the wildtype strain 5008 gave a 1.30-kb product (Fig. 2.17B). Fermentation broths of the mutants were analyzed by HPLC and bioassay. No peak corresponding to validamycin A and validoxylamine A was detected by HPLC analysis (Fig. 2.17C), and inhibition of the fungus P. sasakii could not be observed in the bioassay (Fig. 2.17D), indicating a complete loss of production of both compounds in the valL mutants. When a pPM927-derived integrative plasmid pJTU640, with an intact valL under the control of PermE promoter, was introduced into 5008ΔvalL, the culture broth of the thiostrepton-resistant exconjugant was found to regain the productivity of validamycin A and validoxylamine A by HPLC analysis (Fig. 2.17C). Both inactivation and complementation experiments clearly demonstrated that valL is essential for the biosynthesis of validamycin A and its intermediate validoxylamine A. ValA is involved in the biosynthetic pathway of validamycin A. The crystal structure of valA from S. hygroscopicus 5008 was described by Kean et al. (2014). The large majority of the main chain as well as an active site NAD1 and Zn21 are well ordered with strong and clear density, and an absorption scan and anomalous difference map clearly confirm the presence and placement of the active site Zn21 (Fig. 2.18). The final structure includes

FIGURE 2.18 Electron density map quality and active site structure. Stereoview of the valA active site residues (purple carbons) and a water (red sphere) that are near the NAD1 (gray carbons) and the Zn21 (silver sphere) cofactors. Coordination bonds (black lines) and select H-bonds (black dashes) are shown along with the 2Fo2Fc electron density (orange, contoured at 1ρrms) and an anomalous difference map (green, contoured at 12ρrms).

Production of Validamycins Chapter | 2

55

360 of the 414 expected residues, 188 waters, one PEG, one Zn21, and one NAD1. The missing residues (125, 46, 47, 5862, 244249, and 399414) are not modeled because of weak or unclear electron density. Additionally, three sections, including the residues just N-terminal to residue 26, a β-hairpin turn at residues 32 and 33, and a weakly ordered helix at residues 4650, lay on or near crystallographic twofold axes and had weak, ill-formed density, making them challenging to model. A crystallographic twofold axis brings two valA chains together to form a dimer that, according ˚ 2 of surface area (i.e., 2110 A ˚ 2 per to the PISA server, buries 4220 A monomer). This dimer (Fig. 2.19A) is equivalent to those observed for the homologous enzymes DHQS and DOIS, and the dimer interface is well conserved, implying that it is the physiological form of valA. Each chain of valA encompasses the expected N-terminal yielded a model for the one chain in the asymmetric unit with final R and R-free ˚ NAD1-binding domain values of 17.9% and 26.2%, respectively, to 2.1 A and C-terminal metal-binding domain common to the DHQS-like superfamily. The domain topologies (Fig. 2.19B) using a secondary structure nomenclature that takes into account which elements are conserved among the SPCs were described (Fig. 2.20). The NAD1-binding domain has a core seven-strand β-sheet (with a 1296534 strand order) surrounded by five α-helices, one β-hairpin (β7 and β8), and two short 310-helices. The metal-binding domain is mainly α-helical and includes eight α-helices, one 310-helix, and one β-hairpin. This domain contains not only the Zn21-binding residues but also, on the basis of what has been seen in DHQS and DOIS, the majority of the residues involved in substrate recognition, and so has also been called the substrate-binding domain. However, the sugar phosphate substrate actually binds in a cleft between the two domains, and its recognition involves residues from both domains. GlnR was proved to act simultaneously as an activator and a repressor in validamycin biosynthesis by binding to different loci within a promoter region of the gene cluster (Qu et al., 2015). The involvement of glnR in validamycin A production of 5008 was investigated by the deletion of an internal 632-bp fragment of glnR (SHJG4923), which resulted in a mutant named as JG38 (Fig. 2.21A). The internal deletion of glnR in JG38 was confirmed by PCR amplification with primers glnR-C-F/R. The PCR product of JG38 is expected to be 435 bp, whereas the expected PCR product from wild-type 5008 is 1067 bp (Fig. 2.21B). Strains 5008 and JG38 were individually cultivated with fermentation medium at 30 C or 37 C, and the validamycin A production was measured after 48 h by HPLC. At 30 C, the mutant JG38 produced 0.15 g/L validamycin A, which was less than 50% of the wild-type yield (0.32 g/L). At 37 C, the yield of validamycin A in the mutant JG38 was 0.81 g/L, which was about 23% of the wild-type yield (3.50 g/L) (Fig. 2.21C). The results suggested that glnR overall is positively involved in validamycin A biosynthesis.

FIGURE 2.19 Overall structure and topology of valA. (A) Ribbon diagrams of the two chains of the valA dimer are shown in purple and green tones, respectively, with the N-terminal NAD1-binding domains in light hues and the C-terminal metal-binding domains in dark hues. Dashed lines indicate internal unmodeled backbone segments. The NAD1 and the Zn21 with its coordinating ligands are shown. Secondary structural elements in each domain of one monomer are labeled. (B) Topology diagram showing α-helices (cylinders), β-stands (arrows), 310-helices (triangular prisms), and π-helices (wider cylinder) with their first and last residues given. The minimal length α- and 310-helices (five and three residues, respectively) are left out of the family secondary structure nomenclature. The domains are colored light and dark purple as indicated, and helices (H) and strands (β) common to the sugar phosphate cyclases (SPCs) are named sequentially within each domain. Dashed lines denote unmodeled backbone segments. The three Zn21-binding residues (red asterisks) and the glycine-rich turn and acidic residues (green asterisks) important for NAD1 binding are indicated.

FIGURE 2.20 Sequence alignment of valA with representative related enzymes. The sequence of valA is listed first, and its secondary structure elements are schematically shown above the sequence. Other sequences in descending order are AvDDGS (A. variabilis DDGS, Ava_3858), AmEVS (Ac. mirum EVS, Amir_2000), PDB entry 1DQS (As. nidulans DHQS), PDB entry 2D2X (B. circulans DOIS), and PDB entry 1JQ5 (B. stearothermophilus glycerol dehydrogenase). For the structurally known proteins, the residues in β-strands (yellow), α-helices (teal), 310-helices (blue), and π-helices (orange) are highlighted. Residues involved in metal binding (m), NAD1 binding (n), and substrate binding and/or catalysis (T) are denoted below the sequences, and active site residues with notable variation (k) are denoted above the sequences.

58

Validamycin and Its Derivatives (B) SHJG4922

38

glnR-C-R

1067 bp

JG

glnR-C-F

SHJG4923

50 08

(A) 5008

SHJG4924

Deleted 632 bp

1102 bp

1472 bp pLQ282

tsr

1.65 kb

ori plJ101

Double crossover Truncated SHJG4923 SHJG4922 435 bp

435 bp

glnR-C-R

(D)

JG

50

38

0

8 6 4 2 0 –2

38

1

10

JG

2

va/A va/K

08

3

(E) 12

50

30°C 37°C

Relative quantity (Log2)

4

08

Yield of validamycin A (g/L)

(C)

1067 bp

0.50 kb JG38

SHJG4924

glnR-C-F

1.00 kb

glnR

mutant

5008

FIGURE 2.21 Inactivation of the regulatory gene glnR in 5008. (A) Schematic deletion of an internal 632-bp fragment of glnR in 5008. (B) PCR analysis of 5008 and JG38 (glnR mutant). (C) Validamycin A production in 5008 and JG38 cultivated at 30 C or 37 C. Mean values of three independent experiments with SD are indicated by error bars. (D) Transcriptional levels of valA and valK in 5008 and JG38 at 37 C. (E) Effect of glnR disruption on morphological differentiation.

Additionally, the glnR mutant hardly formed aerial hyphae and was defective in sporulation on serum-free medium (SFM) (Fig. 2.21E). Both validamycin A production and morphological defect were restored by a complementation with plasmid pPM927 carrying an intact glnR and its own promoter region. These results implied that, in addition to its involvement in validamycin A biosynthesis, GlnR is also involved in morphological differentiation, as reported for S. coelicolor (Yang et al., 2009). Subsequently, transcriptional levels of valA and valK between S. hygroscopicus 5008 and the glnR mutant JG38 were compared by qPCR. It was found that the expression of valA and valK were dramatically increased by 250-fold in JG38 compared to the wild-type 5008 at 37 C (Fig. 2.21D). Thus, GlnR exerted a negative effect on the expression of structural genes involved in validamycin A biosynthesis, albeit the overall productivity was decreased. Therefore, it was speculated that GlnR might exert the positive effect on validamycin A production by affecting primary metabolic pathways and its negative regulation on the transcriptional level of structural val genes. Finally, a regulatory model of GlnR involved in validamycin A biosynthesis is proposed (Fig. 2.22). Based on the different affinities of GlnR for sites I and II, it is speculated that GlnR first binds to site II and then site I. The binding of GlnR to site II positively enhances the expression of val genes and validamycin

Production of Validamycins Chapter | 2

PvaIK

AdpA Site

AdpA Site

59

PvaIA

100 bp

6.63 g/L (JG45) Δ GInR Site I

GInR Site II

Enhancement

PvaIK

AdpA Site

AdpA Site

PvaIA

2.26 g/L (JG60) Δ GInR Site I

Δ GInR Site II

Inhibition AdpA Site

AdpA Site

PvaIK

PvaIA

1.48 g/L (JG646) GInR Site I

Δ GInR Site II

Enhancement AdpA Site

AdpA Site

PvaIK

PvaIA

3.29 g/L (5008) GInR Site I

GInR Site II

FIGURE 2.22 Regulatory model of GlnR involved in validamycin A biosynthesis in S. hygroscopicus 5008. Two GlnR binding sites are marked by boxes. The white and black ellipsoids represent AdpA and GlnR molecules, respectively. The arrows show the transcription start points of valK and valA.

A production, without any noticeable interference on the interaction between bound AdpA proteins, as shown in the wild-type 5008 and mutant JG45 (Fig. 2.22). When the concentration of GlnR reaches a critical level, it binds to site I. As shown in Fig. 2.22 (5008 and JG46), the interaction between the two bound AdpA dimers is blocked, causing a decrease of val gene expression and subsequent validamycin A production. When the GlnR binding site I is mutated, as shown in Fig. 2.22 (JG60 and JG45), the failure of GlnR binding results in the interaction between the two bound AdpA dimers, which leads to an increase of val gene expression and subsequent validamycin A production. Mahmud and coworkers’ study demonstrated that validamycin B is derived from validamycin A by the action of VldW, an α-ketoglutarate/Fe(II)dependent dioxygenase that regioselectively hydroxylates the C-60 position of validamycin A (Almabruk Khaled et al., 2012). Inactivation of the vldW gene in the producing strains may abolish the production of validamycin B and validoxylamine B, which in turn may lead to an increased overall production of the important crop protectant validamycin A.

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Validamycin and Its Derivatives

2.6 DETECTION OF VALIDAMYCIN A Validamycin A has been widely used in Asia as rice protectant against R. solani since the 1970s. It can also be used for the control of R. solani in potatoes, vegetables, strawberries, tobacco, ginger, and other crops, and damping-off diseases of cotton, rice, and sugar beet, etc. (He et al., 2003). Besides its excellent control effect, low price, low drug resistance, and low toxicity are the other outstanding merits of validamycin A, and it is now one of the most important agricultural antibiotics, with the biggest production in China. However, in recent years, because of widespread use on large agricultural areas and kinds of crops, consumer health and safety risks may be of concern. The Japanese positive list system tentatively lists validamycin A residue limit at 60 μg/kg in rice, 50 μg/kg in dry soybean, and 50 μg/kg in vegetables and fruit (http://www.m5.ws001.squarestart.ne.jp/foundation/ agrdtl.php?a_inq 5 51100). Therefore, detection of validamycin A is crucial for proper assessment of human exposure from foods. There have been several methods developed for validamycin A analysis, including reversed layer method (Iwasa et al., 1971a), colorimetric method (Zheng et al., 2004), gas chromatography (GC) (Horii et al., 1972; Xu et al., 2008b), highperformance liquid chromatography with ultraviolet detector (HPLC-UV) (reverse phase chromatography) (Yu et al., 2011) (GB/T, 95531993; National Standards of China), liquid chromatography (ion exchange chromatography) (Fredenhagen, 2004; Horii et al., 1972), capillary zone electrophoresis (CZE) (He et al., 2003, 2005; Hsiao and Lo 1999), and liquid chromatographyatmospheric pressure chemical ionizationtandem mass spectrometry (LCAPCIMS/MS) (Wang et al., 2015). Howeve, validamycin A has no characteristic ultraviolet absorption and its maximum absorption wavelength is 210 nm; whether HPLC or CZE, the detection wavelengths were all set at or near 210 nm. At such wavelength, the spectra interference, which may come from other coexistence compounds and even the mobile phase or the running buffer, would be a serious problem for the accuracy and sensitivity, as well. In fact, considering that validamycin A is coexisting with other analogue validamycins, it is very difficult to obtain satisfactory resolution using only GC, HPLC, or CZE, while poor sensitivity might be another issue since the residue concentration is so low and the matrix of real food samples is complex just as the Japanese positive list system requested. LCAPCIMS/MS was a more powerful and sensitive analytical method to overcome these possible drawbacks for both qualitative and quantitative analysis of residues.

2.6.1 Reversed Layer Method The reversed layer method was the first and earliest official method used for the analysis of validamycin A in commercial formulations, although it

Production of Validamycins Chapter | 2

61

is complex and time consuming, and could not distinguish the actual antibiotic from false products (Hsiao and Lo, 1999). The reversed layer method was a diffusion method, different from the dendroid-test method, an agar dilution method.

2.6.1.1 Procedure of Reversed Layer Method 1. Test organism: P. sasakii IFO-9253. 2. Preparation of hyphal suspension (inoculum): The agar disk cut from a culture on modified Pfeiffer’s agar plate is inoculated to 50 mL of the sterilized medium in a 300-mL flask containing 3.0% sucrose, 0.2% L-asparagine, 0.3% NH4NO3, 0.1% KH2PO4, and 0.1% MgSO4, and incubated at 27 C for 4 days on a reciprocal shaker (10 cm, 120 spm). The whole culture obtained was homogenized with a blender, and used as a hyphal suspension of the test organism. 3. Assay media: Bottom layer medium: 0.1% sucrose, 1.0% beef extract, 1.0% peptone, 0.8% agar. Upper layer medium: 0.25% sucrose, 0.45% peptone, 0.1% NaCl, 1.2% agar. 4. Procedure: Melted bottom layer medium is kept at 45 C and 2.5% of the hyphal suspension of the test organism is added. After mixing, 5 mL of this medium is poured into a 9-cm Petri dish. The plate is incubated at 27 C for 4045 h, and overlaid with 10 mL of the upper layer medium melted and kept at 55 C. After solidification, the paper disk dipped in a sample solution is placed on the surface. The inhibition zone is measured after incubation for 2025 h at 27 C. The doseresponse curve for purified validamycin A in reversed layer method was made. The response was observed to be linear over a range of 62.51000 μg/mL.

2.6.2 Colorimetric Method The concentration of validamycins could be measured with the colorimetric method (Zheng et al., 2004). Validamycins belong to the family of aminocyclitol antibiotics, all of which include at least a molecule of glucose. If the glucose is reacted with vitriol oil, it is dehydrolyzed and forms a hydroxyglycuronate, which can be condensed with phenol to produce a red compound. The concentration of the red compound can be determined by colorimeter.

2.6.3 Gas Chromatography The gas chromatography (GC) method was established by Horii et al. (1972) (Figs. 2.23 and 2.24). Apparatus and chromatographic conditions: A Hitachi Model 063 gas chromatograph equipped with flame ionization detector was used.

62

Validamycin and Its Derivatives D

B A

E

5

10

15

20

F

25

C

30 min.

FIGURE 2.23 Gas chromatogram of TMS-validamycins A, B, C, D, E, and F. Reprinted with permission courtesy of Japan Antibiotics Research Association (JARA).

VA- B

VA- A

0

5

10

15 min.

FIGURE 2.24 Gas chromatogram of TMS-validoxylamine A (VA-A) and B (VA-B). Reprinted with permission courtesy of Japan Antibiotics Research Association (JARA).

The conditions for the chromatography of trimethylsilyl (TMS) validamycins AF are: Column: 1% silicone OV-1 chromosorb W AW DMCS (glass column 2 m 3 3 mm I.D.) Column temperature: 280 C Injection temperature: 300 C Carrier gas: He, 60 mL/min

Production of Validamycins Chapter | 2 D

63

A

Absorbancy at 440 mμ

B E

0

1

2

3

4

5

6h

FIGURE 2.25 Liquid chromatogram of validamycins A, B, D, and E with ion exchange chromatography (Conditions: system II). Reprinted with permission courtesy of Japan Antibiotics Research Association (JARA).

The conditions for the separation of TMS-validoxylamines A and B are: Column: 3% silicone OV-17 on chromosorb W AW DMCS (glass column 2 m 3 3 mm I.D.) Column temperature: initial 150 C; final 280 C (program rate 10 C/min); Injection temperature 300 C Carrier gas: He, 45 mL/min Typical chromatograms are shown in Figs. 2.24 and 2.25. Silylation procedure: Approximately 1 mg of sample was weighed into a rubber-capped small tube and dissolved into 100 μL pyridine. Bis(trimethylsilyl) acetamide 100 μL and trimethylchlorolilane 50 μL were added. The tube was heated for 30 min at 7080 C.

2.6.4 HPLC with Reverse Phase Chromatography HPLC with reverse phase chromatography for detection of validamycin A was established by Agricultural Antibiotic Group, Shanghai Institute of Agricultural Pesticides (Shanghai), which was adopted as the National Standard of China. Chromatographic separations were performed by HPLC in an ODS column of 250 mm 3 4.6 mm. The mobile phase was composed of 0.01 mol/L disodium hydrogen phosphate buffer solution (pH 5 7.0) added with 2.5% (v/v) methanol. The flow rate was 1.0 mL/min. The elutant was monitored at 210 nm.

2.6.5 Liquid Chromatography with Ion Exchange Chromatography Liquid chromatography with ion exchange chromatography was first employed for the detection of validamycins by Horii et al. (1972). The Jeol JLC-3BC2

64

Validamycin and Its Derivatives

Absorbancy at 440 mμ

E

F

0

1

2

3

4

5

6h

FIGURE 2.26 Liquid chromatogram of validamycins E and F with ion exchange chromatography (Conditions: system II). Reprinted with permission courtesy of Japan Antibiotics Research Association (JARA).

amino acid analyzer was used as the chromatographic system. In the detection of validamycins AF, a wave length of 440 nm was used to detect orcinolsulfuric acid reaction products, using a visible ray detector. The samples were analyzed with the following two column chromatographic systems. System I: The column (15 cm 3 8 mm I.D.) was packed with Jeol Resin LC-R-3 strongly basic ion exchange resin, 8% cross linkage; borate form) and was jacketed to maintain the temperature at 50 C. The column was developed with 0.11 mol/L borate buffer, pH 7.5 (first buffer) and 0.25 mol/L borate buffer, pH 9.0 (second buffer; buffer change time 100 min), at a flow rate of 0.49 mL/min. System II: The column (70 cm 3 8 mm I.D.) was packed with Bio-Rad resin AG 1 3 2 (200400 mesh, OH form) and developed with water at a flow rate of 0.37 mL/min. Some typical chromatograms are shown in Figs. 2.252.27. Recently, a HPLC with ion exchange chromatography using radiochemical detector was used for validamycin analysis (Fredenhagen, 2004). The liquid chromatograph consisted of an HP 1100 quaternary pump, an HP1100 autosampler, an HP 1100 thermostatted column compartment, an HP1100 diode array detector (Hewlett-Packard, Little Falls, PA, USA), and an LB 506 C1 (EG & G Berthold, Bad Wildbad, Germany) radioactivity monitor. Alternatively a Shimadzu HPLC consisting of two LC-10AD pumps, an autoinjector SIL-10A, an SPD-M10A diode array detector (Shimadzu, Kyoto, Japan), a column oven Merck-Hitachi 655A-52 (Merck, Darmstadt, Germany), and a Berthold LB 506 B were used. In both cases the radiodetector was equipped with an LB 5035 (EG & G Berthold) pump adding liquid scintillation solution (optiflow safe 1, EG & G Berthold) at a flow rate of 2 mL/min to a flow detection cell with a volume of 500 μL. Parameters: nuclide 14C, 3H-resp.; cell Model Z; Ratem. units: min.; H-backgnd: 0 cpm; eff. correct.: no; H-range 500 K cpm; peak-FWHM: 8 s; H-time C 1.5 FWHM. The column was packed with the Mitsubishi MCI-GEL CK 10F (strong acid cation exchanger, H1-form). The result is shown in Fig. 2.28.

Absorybancy at 440 mμ (orcinol - H2SO4)

C

A

B

Buffer change

0

1

2

3

4h

FIGURE 2.27 Liquid chromatogram of validamycins A, B, and C with ion exchange chromatography (Conditions: system I).

(B)

81.2%

4.3%

12.7%

Counts or mAU

100

1.1% 0.3%

120 100

(A)

3 5

1

80 60 2

40

4

20 0 10

20

30 40 Time (min)

50

60

FIGURE 2.28 HPLC of validamycin mixture and radiochemical purity of crude [3H] material. 1: unknown impurity, 2: validamycin B or G, 3: validamycin A, 4: validoxylamine B or G, 5: validoxylamine A. Experimental conditions: Mitsubishi MCI-GEL CK 10F (counter ion: H1) 150 3 4.0 mm2; eluent: 50 mmol/L TFA; 0.6 mL/min; 65 C. (A) [3H] radiosignal: 10 μL of crude [3H] validamycin A corresponding to 300 kBq in H2O injected; (B) UV at 205 nm: 10 μL of a mixture validamycin and validoxylamine 5 mg/mL each in H2O injected.

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Validamycin and Its Derivatives

2.6.6 Capillary Zone Electrophoresis CZE is an efficient separation technique in which charged solutes are differentially transported through open capillaries under the influence of an applied field (Jorgenson and Lukacs, 1981). However, this simple method could not be used directly for detection of validamycin A. An alternative approach to detect these compounds is by the addition of surfactant ions to the mobile phase at concentrations above their critical micelle concentration (Terabe et al., 1984). This technique was designated micellar electrokinetic capillary chromatography (MEKC) (Burton et al., 1986). MEKC was also used to determine validamycin A content in commercial products (Hsiao and Lo, 1999). The MEKC method was performed using a Biofocus 3000 automated capillary electrophoretic apparatus. A Biofocus cartridge capillary 1483040 (50 cm 3 50 μm I.D., uncoated) was employed with a length of 45.4 cm from the point of sample introduction to the point of detection. The column temperature was 20 C. A regulated dc power supply delivering 18 kV was used to provide high voltage between the ends of the column filled with running buffer. The sample was introduced into the capillary vessel using pressure injection mode at 5 psi 3 s. The elution of a solute was monitored by an on-column UVvis detector (195 nm) at the negative pole (Fig. 2.29). This method was capable of analyzing the validamycin A content in formulated products with an instrument detection limit of 0.94 μg/mL and a method detection limit of 1.70 μg/mL. Relative standard deviation (RSD) values of MEKC determination of validamycin A in formulated products ranged from 0.61% to 2.09%. Recoveries of validamycin A in formulated products were in the region of 99.5105.1%. Nevertheless, analyses were somehow interfered by at least one kind of impurity in commercial formulations. Later, determination of validamycin A by CZE with indirect UV detection was investigated by He et al. (2003). Electrophoresis was performed on a P/ACE MDQ CE system (Beckman Coulter, Fullerton, CA), which was equipped with a P/ACE UV detector module, an autosampler, and a temperature-controlled fluid-cooled capillary cartridge. A computer and MDQ software (2.3 version) were used for instrument control and for data collection and processing. An uncoated fusedsilica capillary (total 60 cm, effective length 50 cm 3 75 μm I.D.) from Yongnian Chromatography (Yongnian, Hebei, China) was employed. UV detection was set at 200 nm. Samples were introduced into the capillary by pressure mode (0.5 psi for 10 s). The temperature of the capillary was controlled at 25 C. Before each injection, a 3-min purge of capillary with carrier electrolyte was programmed. The pH of carrier electrolyte was adjusted by acetic acid solution. All carrier electrolyte solutions were filtered through a 0.22-μm syringe membrane filter (Shanghai Yadong Hitech, Shanghai,

Production of Validamycins Chapter | 2 mAU 4.01

mAU

Buffer

4.00

2.84

2.68

1.67

1.37

0.50

0.05

–0.67 0.00

mAU 4.02

3.00

6.00

9.00 12.00 15.00 Min

Samples A and B (3% SL)

7.78

3.00 6.00

9.00 12.00 15.00 Min

Samples C, D, and E (5% SL)

2.75

2.91

1.79

7.79

7.76

1.49

0.23

0.68

–0.43 0.00

Standard

–1.26 0.00

mAU 4.02

67

3.00

6.00

9.00 12.00 15.00 Min

–1.04 0.00

3.00

6.00

9.00 12.00 15.00 Min

FIGURE 2.29 Typical electropherograms of dilution buffer, validamycin A standard, and commercial products. Samples A and B were 3% solution, and samples CE were 5% solution.

China), and all samples were centrifuged (10,000 g for 10 min) before they were introduced into the system. The method of CZE with indirect UV-absorbing detection was used to analyze validamycin compounds, because of their lack of chromophores. First in the optimization of CZE with indirect UV detection is the selection of a background electrolyte (BGE) with large molar absorptivity and effective mobility similar to that of the analyst cation. Being an aromatic amine compound, animopyrine has strong UV absorption. It is suitable for the analysis of validamycin A as indicated by symmetrical peak (Fig. 2.30). The CZE and HPLC yielded similar values for each sample (Fig. 2.31). Nevertheless, the abnormal results obtained from the MEKC method indicated the presence of one or more coeluates in these samples (see the biggest peak in Fig. 2.32). It is due to its low separation efficiency (,30,000 plates/m). The results implied that the MEKC method is not suitable for the

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Validamycin and Its Derivatives

FIGURE 2.30 Electropherogram of sample I. Experimental conditions: uncoated fused-silica capillary, 50 cm (effective length) 3 75 μm (I.D.); carrier electrolyte, 10 mmol/L aminopyrine 2 mmol/L EDTA (pH 5.2); separation voltage, 15 kV; temperature, 25 C; indirect UV detection, 254 nm; pressure injection, 0.5 psi for 10 s. The content of validamycin A (peak 1) is 94.6 μg/mL.

determination of validamycin A in commercial formulations, at least in these samples. By using 10 mmol/L aminopyrine2 mmol/L ethylenediaminetetraacetic acid at pH 5.2 as the carrier electrolyte, high-efficiency separation of validamycin A was achieved with the number of theoretical plates up to 350,000 plates/m. The linear range was across 3 orders of magnitude. The RSDs for migration times and peak areas were less than 0.5% and 3.0%, respectively. The limit of detection for validamycin A was 1.0 μg/mL. The average recoveries ranged from 103.0% to 104.8%. This method has many advantages as compared with HPLC and MEKC in the determination of commercial formulations.

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FIGURE 2.31 Chromatogram of sample I. Experimental conditions: Waters 4.6 mm 3 75 mm symmetry C18 3.5 μm column; mobile phase, 5 mmol/L phosphate (pH 7.0) 1 3% methanol; injection volume, 5 μL; detection wavelength, 210 nm. The content of validamycin A (peak 1) is 93.3 μg/mL.

Nevertheless, the detection sensitivity of MEKC with indirect UV detection was not so satisfactory. Furthermore, there was potential interference from components in samples with complex matrixes. More recently, to overcome these disadvantages, capillary electrophoretic determination of validamycin A in formulations with direct UV detection was studied by He et al. (2005). Electrophoresis was performed on a P/ACE MDQ capillary electrophoresis system (Beckman Coulter, Fullerton, CA, USA) that was equipped with P/ACE UV detector or diode array detector, an autosampler, and a temperature-controlled fluid-cooled capillary cartridge. A computer with MDQ software (2.3 version) was used for instrument control, data collection,

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Validamycin and Its Derivatives

FIGURE 2.32 Electropherogram of sample I. Experimental conditions: carrier electrolyte, 100 mmol/L SDS and 50 mmol/L borate (pH 9.0); separation voltage, 20 kV; temperature, 20 C; UV detection, 200 nm; other experimental conditions as in Fig. 2.31.

and processing. The capillary was a 60 cm 3 75 μm I.D. uncoated fusedsilica capillary (Reafine Chromatography, Yongnian, Hebei, China) with a UV detection window placed on-column 50 cm from the injection end. The detection wavelength was set at 200 nm. The capillary temperature was controlled at 25 C. Samples were introduced from the anodic end of the capillary by pressure mode (0.5 psi for 5 s unless indicated otherwise). The detection for validamycin A by CZE with UV detector is a challenging task because validamycin A has no strong UV chromophore. Validamycin A shows only end absorption in aqueous solution; therefore direct UV detection can only be done at a low wavelength. With the diode array detector, detection sensitivity was found optimal at the maximum absorbance wavelength of 193 nm in acetate buffer (Fig. 2.33A). In this experiment, the detection wavelength was fixed at 200 nm with the UV detector. However, this led to a 24% reduction in sensitivity.

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FIGURE 2.33 Electropherograms of sample 1. Experimental conditions: uncoated fused-silica capillary: 60 cm (effective length 50 cm) 3 75 μm (I.D.); separation voltage: 15 kV; temperature: 25 C; pressure injection: 0.5 psi for 10 s. (A) Running buffer: 10 mmol/L aminopyrine 2 mmol/L EDTA (pH 5.2); indirect UV detection: 254 nm. (B) Running buffer: 100 mmol/L acetate (pH 4.7); UV detection: 200 nm.

The detection was achieved by using direct UV mode at 200 nm and the detection limit was 0.2 μg/mL. Linearity in the concentration range of 5500 μg/mL was excellent (R2 . 0.999). The run-to-run repeatability (n 5 3), as expressed by the RSD for migration times and peak areas, was less than 0.5% and 3.0% respectively. The mean recovery ranged from 97.2% to 101.4%.

2.6.7 Liquid ChromatographyAtmospheric Pressure Chemical IonizationTandem Mass Spectrometry A rapid, sensitive, and accurate liquid chromatographyatmospheric pressure chemical ionizationtandem mass spectrometry (LCAPCIMS/MS) method was developed for determination of validamycin A in agricultural

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food samples (rice, agaric, almond, cabbage, green onion, carrot, tomato, cucumber, and spinach) by Wang et al. (2015). An Agilent 1200 Series LC system (Agilent Technologies, Wald-bronn, Germany) consisting of a degassing unit, a quaternary pump, an autosampler, and a thermostatted column compartment was used in the LCAPCIMS/MS system. Separation of the analyte was achieved on a Waters Atlantis HILIC column (250 mm 3 4.6 mm I.D., 5 μm film thickness, Waters Corp., Milford, MA, USA) with a column oven temperature of 30 C. The mobile phase was composed of acetonitrile (A) and 5 mmol/L ammonium acetate (B), and the flow rate was set at 1.0 mL/min. Gradient elution employed with the ratio of A:B varied as follows: 0 min, 80:20; 4 min, 55:45; 10 min, 55:45; 10.1 min, 80:20; 16 min, 80:20. The elution was diverted to waste during 07.0 and 9.016 min, respectively. An Applied Biosystems-Sciex API 4000 (Applied Biosystems, Concord, ON, Canada) triple-quadrupole mass spectrometer equipped with a Turbo Ion Spray interface was used. Ionization was achieved using atmospheric pressure chemical ionization (APCI) in the positive ion mode at 500 C with N2 as nebulizer. Detection was performed by multiple reaction monitoring (MRM) mode of selected ions at the first (Q1) and third quadrupoles (Q3). To choose the fragmentation patterns of m/z (Q1) and m/z (Q3) for the analyte in the MRM mode, direct infusion of standard solution into the MS was performed and the production scan mass spectra were recorded. Once the fragment ions were chosen, the MRM conditions were further optimized to obtain maximum sensitivity for the compound of interest. The APCIMS/MS product scan spectrum of validamycin A (Fig. 2.34) was acquired, and three fragments at m/z 498.3, 336.2, and 178.2 were observed

FIGURE 2.34 APCIMS/MS product scan spectrum of validamycin A.

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and revealed two clear and obvious transitions (498.3-336.2 and 498.3-178.2). Both transitions were chosen for identification of validamycin A in real samples, and the more sensitive 498.3-178.2 transition was selected for quantitation. A minimum of three identification points are required to meet the identification performance criteria defined by the EU Commission for quantitative mass spectrometric detection. Using LCAPCIMS/MS to monitor one precursor ion and two daughter ions “earns” four identification points (1 for the parent ion and 1.5 for each daughter ion) and therefore fulfills these criteria. LCMS/MS MRM chromatogram of a 10.0 μg/kg spiked rice sample solution is shown in Fig. 2.35. A clean background was obtained and revealed the absence of chromatographic interferences. Since there were no MS-related reports on validamycin A, the most plausible interpretation of MS/MS fragmentation was proposed and given in Fig. 2.36 primarily based on the spectral information in Fig. 2.35. The ion observed at m/z 498.3 derives from [M 1 H]1, the ion observed at m/z 336.2 corresponds to the neutral loss of one glucopyranosyl (Glc) residue (C6H10O5), and the ion observed at m/z 178.2 corresponds to the loss of one glucopyranosyl (Glc) residue and C7H10O4. The average recoveries, measured at three concentration levels (10.0, 50.0, 100.0 μg/kg) were in the range 83.5109.6%. The method offers the best sensitivity and specificity for the routine analysis of validamycin A in agricultural food samples.

FIGURE 2.35 LCAPCIMS/MS MRM chromatograms of a rice sample spiked with 10 μg/kg of validamycin A.

FIGURE 2.36 Proposed fragmentation pattern with structures for the productions of validamycin A. Glc, Glucopyranosyl (C6H10O5).

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A new approach to determine validamycin A by spectrophotometer was developed by Li et al. (2016). In the method, a pigment secretion was found along with the validamycin A biosynthesis during the fermentation of S. hygroscopicus 5008. There was a stable relationship between the concentration of validamycin A and spectral absorption value of this pigment at 450 nm, even in different fermentation cultures or conditions. Therefore, using spectral absorption value as interior label, a rapid spectrophotometric method for determining validamycin A production was established. The method was stable and accurate as standard HPLC method. It could be applied successfully to finding positive strains with high validamycin A productivity and short fermentation time.

2.7 MICROBIAL DEGRADATION OF VALIDAMYCIN A 2.7.1 Degradation of Validamycin A by Pseudomonas denitrificans The microbial hydrolysis by P. denitrificans (Chen et al., 2003; Kameda and Horii, 1972; Kameda et al., 1975) is nonspecific for α- and β-glucosidic linkages, and validamycin A is hydrolyzed to D-glucose and validoxylamine A in the first step. The further decomposition of validoxylamine A proceeds via valienamine and validamine, which can be isolated as the intermediate products. The degradation procedure was carried out with resting cells. The resting cells were obtained as follows. A cell suspension of P. denitrificans from the agar slant was inoculated in 20 mL of a medium that contained glucose 2%, yeast extract 0.1%, peptone 1%, K2HPO4 0.5%, KH2PO4 0.1%, NaCl 0.2%, MgSO4  7H2O 0.02%, pH 7.2, in a 100-mL Erlenmeyer flask and cultured on a rotary shaker at 28 C for 24 h. The broth was transferred to a 1-L Erlenmeyer flask containing 300 mL of the medium and incubated on a rotary shaker at 28 C for 72 h. The cells were harvested by centrifugation and washed several times with water. Validamycin A (2.03 g) was then dissolved in water (2 L), and the resting cells harvested from the culture solution (4 L) of P. denitrificans were suspended in the reaction solution (pH 7.1). The incubation was carried out at 28 C for 8 h under shaking conditions, and then the mixture was centrifuged. The cells were discarded, and the supernatant solution was passed through a column of Amberlite IRC-50 (H1 form, 300 mL) to adsorb the basic degradation products. The column was eluted with 0.5 N ammonia water, and the eluate was concentrated to dryness. The residue was separated by Dowex 1 3 2 (OH2 form) ion-exchange resin chromatography, using water as the developing solvent, into three components: valienamine, validamine, and validoxylamine A.

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2.7.2 Degradation of Validamycin A by Flavobacterium saccharophilum F. saccharophilum, which was isolated from the rice fields of Kanazawa City, Japan, was found to decompose validamycin A efficiently. For the preparation of valienamine and validamine, F. saccharophilum was cultured at 27 C for 4 days on a shaker in a medium consisting of validamycin A 1%, (NH4)2SO4 1%, K2HPO4 0.7%, KH2PO4 0.3%, and MgSO4  7H2O 0.01%, pH 7.1. Two liters of the broth was passed through a column of Amberlite IRC-50 (NH41 form, 500 mL), which was eluted with 0.5 N aqueous ammonia. The concentrate of the eluate was chromatographed on a column of Dowex 1 3 2 (OH2 form, 500 mL) column and developed with water to give valienamine and validamine. The resting cells of F. saccharophilum could also be used to degrade validamycin A. At first, F. saccharophilum was cultured in nutrient broth with shaking at 27 C for 24 h. The cells were harvested by centrifugation at 20,000 g. The washed cells (50 g, wet weight) were suspended in 1000 mL of 0.05 mol/L phosphate buffer, pH 7.0, containing 10 g of validamycin A, and the suspension was incubated at 27 C for 48 h under shaking conditions. The isolation method was as described previously. The proposed degradation pathway of validamycin A by F. saccharophilum (Scheme 2.3) was studied by Asano et al. (1984) and Chen et al. (2003). Validamycin A is first hydrolyzed to D-glucose and validoxylamine A. Validoxylamine A undergoes oxidation at the C-3 position of the validamine or valienamine moiety by 3-dehydrogenase to form two ketoenol compounds, in addition to validamine and valienamine. Validamine and valienamine are also deaminated to form the ketoenol compounds. The resulting ketoenol compounds could be degraded by the same hydrolase into open-chain compounds and further into low-molecular-weight compounds.

2.8 CLONING, EXPRESSION, AND DEFICIENCY OF GENES IN THE VALIDAMYCIN BIOSYNTHESIS AND THEIR APPLICATIONS 2.8.1 Cloning and Expression of valG Gene ValG is a glycosyltransferase. Glycosyltransferases catalyze the transfer of a sugar moiety from an NDP-sugar to an acceptor, which could be a growing oligosaccharide, a lipid, a protein, or a small molecule. On the basis of tertiary structure analysis, glycosyltransferases have been divided into two superfamilies, known as glycosyltransferase-A and glycosyltransferase-B (Bourne and Henrissat, 2001). Members of the glycosyltransferase-A superfamily contain

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HOH2C OH

OGlc NH

OH

HOH2C OH

OH

Validamycin A

OH

HOH2C OH

OH NH

OH

HOH2C OH

OH

D-glucose

Validoxylamine A OH

HOH2C

HOH2C

OH

OH OH O

NH OH

OH NH

OH

HOH2C OH

HOH2C OH

OH

O

OH

HOH2C

OH

OH OH O

NH2 OH OH

HOH2C

HOH2C

HOH2C

OH

Valienamine

NH2 OH

OH

O

OH

OH

Validamine

SCHEME 2.3 Proposed degradation pathway of validamycin A by F. saccharophilum.

two dissimilar domains, one involved in the recognition of the NDP-sugar and the other in the recognition of the acceptor molecule. Most of the glycosyltransferases in the Leloir pathway that reside in the Golgi apparatus and the endoplasmic reticulum belong to this family. The glycosyltransferase-B superfamily is remarkably diverse and contains members that are extremely promiscuous to their NDP-sugar donors (Barton et al., 2002; Jiang et al., 2001). The

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OH

HO

OH OH

ValG HO

N H OH

OH OH

Validoxylamine A

UDP-galactose

OH

HO

O

CH2 OH

O

4''

HO HO

N H OH

OH

OH OH

OH 4''-epi-Validamycin A

SCHEME 2.4 Bioconversion of validoxylamine A to 400 -epi-validamycin A using ValG.

glycosyltransferase-B family consists mostly of prokaryotic enzymes that glycosylate secondary metabolites to produce active natural products, as well as some glycosyltransferases from the primary pathways. For S. hygroscopicus subsp. jinggangensis 5008, the complete biosynthetic gene clusters have been identified (Bai et al., 2006). Among the proteins believed to be directly involved in the biosynthesis, validamycin glycosyltransferase has been characterized both in vivo and in vitro as a glycosyltransferase that catalyzes the conversion of validoxylamine A to validamycin A using UDP-glucose as the sugar donor (Bai et al., 2006). Interestingly, validamycin glycosyltransferase belongs to the glycosyltransferase-A family of glycosyltransferases, even though it functions in secondary metabolism. Later, valG was cloned from S. hygroscopicus subsp. jinggangensis 5008 and expressed to produce validamycin glycosyltransferase (Xu et al., 2008a). To explore the possibility of employing ValG as a tool for generating analogues of validamycin, a number of commercially available NDP-sugars were tested as sugar donors. The recombinant histidine-tagged protein was prepared heterologously in E. coli BL21Gold(DE3)pLysS and purified on a BD TALON affinity column. The enzyme was incubated with validoxylamine A as a sugar acceptor and either UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, UDP-glucuronic acid, or GDP-mannose as a sugar donor. The reactions were carried out in the presence of Mg21 for 16 h and monitored by TLC. UDP-glucose is the natural sugar donor of ValG, which converts validoxylamine A to validamycin A, and was used in this study as a positive control to validate the activity of the enzyme. The results revealed that, in addition to UDP-glucose, ValG also utilizes UDP-galactose as substrate to produce a new validamycin analogue, 400 -epi-validamycin A (Scheme 2.4), whereas incubations with UDP-N-acetylglucosamine, UDP-glucuronic acid, or GDP-mannose did not give any products at a detectable level. However, in the biotransformation process, the enzyme was used as the catalyst and the UDP-glucose as the sugar donor. It is not suitable to be applied in the industrial production of validamycin A because of the production cost, including the purification of the enzyme and purchase of UDPglucose. If the resting cells could be used directly instead of the enzyme, the cost would be decreased definitely. Another sugar donor, D-cellobiose, was

Production of Validamycins Chapter | 2 OH

OH

HO

OH OH

ValG N H

HO OH

OH

UDP-glucose

OH

O

O

CH2OH OH

HO N H OH

Validoxylamine A

OH

HO

HO

79

OH

OH

OH Validamycin A

D-cellobiose

SCHEME 2.5 Bioconversion of validoxylamine A to validamycin A using ValG.

reported to be used in β-glucosidation of validoxylamine A (Kameda et al., 1980b). If D-cellobiose could be utilized as the sugar donor instead of UDPglucose, the cost of the validamycin A production would be decreased again because D-cellobiose is much cheaper than UDP-glucose. Recently, another study focused on cloning and expression of validamycin glycosyltransferase in E. coli and its application in the biotransformation of validoxylamine A to validamycin A with D-cellobiose as the sugar donor using the resting cells (Fan et al., 2013) (Scheme 2.5). The conditions for the expression of valG were optimized to improve the effectiveness of biocatalysis. Validamycins were produced in large scale in China with many product specifications, such as products of 40% (w/w) validamycin A and 60% (w/w) validamycin A. However, it is hard to improve the content of validamycin A more than 70% because of the purifying cost. In validamycin mixture, there is about 60% validamycin A, 2030% validoxylamine A, 58% other members of validamycins, such as validamycin BH, and 23% other members of validoxylamines, such as B and G, reported that 3 g/L validamycin A and 0.5 g/L validoxylamine A accumulated at the end of the fermentation during the fermentation of wild-type S. hygroscopicus var. jinggangensis 5008 (Zhou et al., 2011). A similar phenomenon was also observed in the high-yielding validamycin A producer TL01, with 18 g/L validamycin A and 4 g/L validoxylamine A produced. Furthermore, the molar ratio of validoxylamine A/validamycin A was even higher in the high-yielding strains than that in low-yielding strains. Although the validamycin A content could be increased by adding UDP-glucose to decrease the validoxylamine A accumulation (Zhou et al., 2011), the cost of UDP-glucose is too high to be used in the industrial scale. The other way to solve the problem is to biotransform validoxylamine A to validamycin A using validamycin glycosyltransferase. In this respect, cloning and expression of ValG are very important for validamycin production.

2.8.2 Cloning and Expression of ugp Gene UDP-glucose pyrophosphorylase (or glucose-1-phosphate uridylyltransferase) (Ugp, EC 2.7.7.9) catalyzes the reversible formation of UDP-glucose and

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FIGURE 2.37 (A) HPLC chromatogram of validamycin A fermentation broth. VAL, validamycin A; VO, validoxylamine A. (B) Biosynthesis of UDP-glucose and the glycosylation of validoxylamine A. Ugp: UDP-glucose pyrophosphatase; ValG: glucosyltransferase.

inorganic pyrophosphate from UTP and glucose-1-phosphate (Sheu and Frey, 1978). The activity of Ugp has been detected in S. griseus ATCC 13273 and Streptomyces sp. strain MRS202 (Liu and Rosazza, 1998). But few of the UDP-glucose pyrophosphorylase genes in Streptomyces have been cloned and functionally studied, except in Bai and coworkers’ study (Fig. 2.37) (Zhou et al., 2011). During the fermentation of wild-type 5008, 3 g/L validamycin A and 0.5 g/L validoxylamine A accumulated at the end of the fermentation. A similar phenomenon was also observed in the high-yielding validamycin A producer TL01, with 18 g/L validamycin A and 4 g/L validoxylamine A produced (Fig. 2.38). Additionally, the molar ratio of validamycin A/validoxylamine A was even lower in the high-yielding strain than that in wild-type 5008. It seems quite straightforward that the validamycin titer could be increased by efficient conversion of the accumulated validoxylamine A to validamycin A. The reasons for the accumulation of validoxylamine A perhaps lie in the efficiency of glycosyltransferase ValG and the supply and flux of the precursor UDP-glucose. The supply of UDP-glucose was proved to be the rate-limiting factor, and overexpression of Ugp in strain TL01 could successfully increase the validamycin A titer and reduce the accumulation of validoxylamine A (Zhou et al., 2011). At first, glycosylation activity of the glucosyltransferase ValG was confirmed not to be responsible for the accumulation of validoxylamine A.

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FIGURE 2.38 Expression and enzymatic activity of ValG and production of validamycin A and validoxylamine A in TL01 and its derivatives with an extra copy of valG. (A) Quantitative real-time RT-PCR analysis of valG transcription in TL01, TL01 (PermE 1 valG), and TL01 (PvalA 1 valG). (B) ValG activities in TL01 and TL01 (PvalA 1 valG) using 2-day or 5-day cell-free extracts. (C) Production profiles of validamycin A in TL01 and TL01 (PvalA 1 valG). (D) Molar ratios of validamycin A/validoxylamine A in TL01 and TL01 (PvalA 1 valG) at different fermentation times.

Since no degradation of validamycin was detected during the fermentation process, a low efficiency of glycosylation by the glucosyltransferase ValG was thought to be a possible reason for validoxylamine A accumulation. If the low efficiency was caused by low transcription of valG, usually overexpression of valG in TL01 could be expected to increase the proportion of validamycin A and decrease the proportion of validoxylamine A. The valG gene was cloned to a pSAM2-derived integrative vector downstream of the constitutive PermE promoter (Bibb et al., 1985), or the PvalA promoter from the valABC operon of the validamycin biosynthetic gene cluster (Yu et al., 2005). The two valG constructs were introduced by conjugation from E. coli into TL01. Real-time analysis indicated that the expression of valG was increased in both strains carrying a different extra copy of valG.

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FIGURE 2.39 (A) HPLC chromatograms of validamycin production with exogenous UDPglucose and validoxylamine A (VO) added to 2-day cell-free extracts of TL01. a: VO standard; b: 5 mmol/L VO added; c: 5 mmol/L VO and 5 mmol/L UDP-glucose added; d: 5 mmol/L VO and 10 mmol/L UDP-glucose added; e: 5 mmol/L VO and 50 mmol/L UDP-glucose added. (B) HPLC chromatograms of validamycin A production with exogenous UDP-glucose added to the cell cultures of TL01. a: TL01; b: TL01 supplemented with 10 mg UDP-glucose; c: TL01 supplemented with 50 mg UDP-glucose; d: TL01 supplemented with 100 mg UDP-glucose.

Additionally, the PvalA promoter worked more effectively than PermE in the fermentation medium (Fig. 2.39A). Furthermore, the enzymatic activity of ValG was enhanced from 234 in TL01 to 459 pkat/mg in TL01 (PvalA 1 valG) at 48 h (Fig. 2.38B). However, the TL01(PvalA 1 valG) produced similar proportions of validamycin A as the parent strain. Both TL01 and TL01(PvalA 1 ugp) reached the highest yield at 96 h during the fermentation, and the titer of validamycin A was 17.9 and 17.6 g/L, respectively (Fig. 2.38C). The molar ratios of validamycin A/validoxylamine A were nearly identical throughout the 5-day fermentation process for TL01 and TL01 (PvalA 1 valG; Fig. 2.38D). Therefore, overexpression of glucosyltransferase ValG did not improve validamycin A productivity, reflecting abundant glycosylation activity in the high-yield producer TL01. Secondly, supplementation of UDP-glucose was proved to increase validamycin A but decrease validoxylamine A production. Increased validamycin A production could be detected when 5 mmol/L validoxylamine A and different concentrations of UDP-glucose (5, 10, or 50 mmol/L) were added into a cell-free extract of TL01 (Fig. 2.39A). When a high concentration of UDP-glucose (50 mmol/L) was added into the reaction, almost all of the validoxylamine A was converted into validamycin A, which proved that the glycosylation step could be enhanced and more validamycin A could be

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produced when UDP-glucose was not limiting. UDP-glucose was also added into the fermentation broth at a 5-mL scale. In 5-day small-scale fermentation, the validamycin A and validoxylamine A production was 1.77 g/L and 0.76 g/L, respectively. The glycosylation was enhanced when 10, 50, or 100 mg UDP-glucose was added during fermentation. Validamycin A production reached 2.66 g/L and less than 0.1 g/L of validoxylamine A accumulated when 100 mg of UDP-glucose was added into the small-scale fermentation broth (Fig. 2.39B). Finally, overexpression of the ugp gene SHJG4652 was identified to increase validamycin A but decrease validoxylamine A production. The gene SHJG4652 was cloned into the pSAM2-derived integrative vector pPM927 under the control of PermE or the PvalA promoter and introduced into TL01 through conjugation. Quantitative real-time RT-PCR confirmed that ugp was overexpressed in the recombinant strains, and its transcription reached the highest level to 2.5-fold of that of TL01 at 48 h. Again, the PvalA promoter was shown to be stronger than PermE in TL01 (Fig. 2.40A). The UDP-glucose pyrophosphorylase activity in the cell-free extract of the derivative TL01 (PvalA 1 ugp) was compared with that of TL01. Fig. 2.40B shows that Ugp activity increased to two-fold from 26.4 6 3.5 to 56.2 6 5.2 pkat/mg in TL01 (PvalA 1 ugp) at midfermentation (48 h). These results clearly demonstrated that Ugp was overexpressed in the engineered TL01 (PvalA 1 ugp). UDP-glucose, as the metabolic product, was also detected. In an attempt to increase the accumulation of UDPglucose, a glucosyltransferase valG mutant of TL01 (XZ1) was constructed (unpublished data), and pJTU3391 containing PvalA and ugp was introduced into XZ1. Both the fermentation broth and the cell-free extracts of XZ1 (PvalA 1 ugp) and XZ1 were collected, and the amount of UDP-glucose was measured. However, the intracellular or extracellular accumulation of UDPglucose could not be detected. The productivity of validamycin A and validoxylamine A of TL01 (PvalA 1 ugp) was compared with TL01 by fermentation and HPLC analysis. Both TL01 and TL01 (PvalA 1 ugp) reached the highest yield at 96 h during the fermentation, and the titer of validamycin A was increased by 22% from 18 g/L in TL01 to 22 g/L in TL01 (PvalA 1 ugp). Moreover, the productivity of validoxylamine A was decreased from 4 g/L in TL01 to 2.5 g/L in TL01 (PvalA 1 ugp; Fig. 2.40C), resulting in an increased molar ratio of validamycin A/validoxylamine A from 3.15 to 5.75 on average (Fig. 2.40D). It could be concluded that the availability of UDP-glucose is one of the limiting factors for validamycin production in the high-yield producer TL01, and increasing the UDP-glucose supply is a promising strategy for validamycin A overproduction. Therefore, limiting UDP-glucose supply was confirmed to play an essential role in the accumulation of validoxylamine A during the fermentation of the high-yielding producer S. hygroscopicus TL01 for validamycin production. The nonenzymatic and enzymatic deglycosylation of validamycin A

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FIGURE 2.40 Expression and enzymatic activity of Ugp and production of validamycin A and validoxylamine A in TL01 and its derivatives with an extra copy of ugp. (A) Quantitative realtime RT-PCR analysis of ugp transcription in TL01, TL01 (PermE 1 ugp) and TL01 (PvalA 1 ugp). (B) UDP-glucose pyrophosphorylase activities in TL01 and TL01 (PvalA 1 ugp) using 2-day or 5-day cell-free extracts. (C) Validamycin A produced by TL01 and L01 (PvalA 1 ugp). (D) Molar ratios of validamycin A/validoxylamine A in TL01 and TL01 (PvalA 1 ugp) at different fermentation times.

with exogenous validamycin A added during the fermentation was also ruled out. Furthermore, the expression level of the glucosyltransferase ValG was proved not to be the cause for validoxylamine A accumulation because its overexpression did not improve the validamycin A productivity.

2.8.3 Knocking Out valG Gene Validamycin glycosyltransferase has been characterized both in vivo and in vitro as a glycosyltransferase that catalyzes the last step in the biosynthesis of validamycin A, the conversion of validoxylamine A to validamycin A, using UDP-glucose as the sugar donor (Bai et al., 2006). Knocking out valG

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FIGURE 2.41 Inactivation and complementation of valG. (A) Schematic representation of the replacement of an 806-bp internal fragment of valG with the 1.4-kb aac(3)IV. In shuttle plasmid pJTU609, aac(3)IV was inserted between the 2.4-kb and 1.0-kb genomic fragments originally flanking the deleted 806-bp region. While wild-type S. hygroscopicus should give a 2.1-kb PCRamplified product, mutant LL-1 should yield a 1.5-kb product by using a pair of primers, ValG2-F and ValG2-R. (B) PCR analysis of wild-type S. hygroscopicus and mutant LL-1. (C) Bioassay comparison between the wild-type (left), LL-1 (middle), and LL-101 (right). LL-101 is the derivative of LL-1 harboring shuttle plasmid pJTU612 with valG. (D) HPLC chromatograms of the standards, wild-type, LL-1, and LL-101. The retention time of validamycin A is 9.7 min, and that of validoxylamine A is 6.5 min.

would abolish the biosynthesis of validamycin A and accumulate validoxylamine A (Bai et al., 2006; Deng et al., 2009; Li et al., 2008). LL-1 (Fig. 2.41B) was one of the confirmed mutants, with an internal 806-bp region of valG (from nt number 112 to 918) replaced by an aac(3)IV from the strain 5008 (Fig. 2.41A). Bioassay indicated that the LL-1 fermentation broth had retained inhibitory activity, albeit at a reduced capacity (Fig. 2.41C), but the presence of validamycin A (retention time of 9.7 min) could not be detected by HPLC. Despite the fact that, under normal conditions, the wild-type strains also produced validoxylamine A (retention time of 6.5 min), a significantly increased accumulation of validoxylamine A was observed in LL-1 (Fig. 2.41D). The abolished production of validamycin A and the increased accumulation of validoxylamine A in LL-1 strongly suggest that ValG catalyzes the glycosylation of validoxylamine A to yield validamycin A. The observed weak inhibitory activity agrees with the previous finding that validoxylamine A has much lower in vivo inhibitory activity than validamycin A (Asano et al., 1991).

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Validamycin and Its Derivatives

When pJTU612, a pIJ101-derived plasmid with an intact valG, was introduced into mutant LL-1, the culture broth of the thiostrepton-resistant exconjugant (LL-101) was found to have regained the inhibitory activity (Fig. 2.41C) in bioassay. HPLC analysis unambiguously demonstrated the presence of validamycin A with the retention time of 9.7 min. However, the presence of valG under the strong constitutively expressed PermE promoter in LL-101 was not sufficient for the conversion of all validoxylamine A to validamycin A, as a peak with the retention time of 6.5 min corresponding to validoxylamine A was also observed (Fig. 2.41D). Therefore, the mutant knocking out valG could be employed to produce validoxylamine A.

2.8.4 Inactivation of valN Gene One of the essential genes for validamycin A biosynthesis is valN, which encodes protein homologous with the zinc-dependent sorbitol dehydrogenase from Geobacillus thermodenitrificans NG80-2 (28% identity, 45% similarity) and the alcohol dehydrogenase from Prosthecochloris vibrioformis DSM 265 (29% identity, 42% similarity). Both sorbitol and alcohol dehydrogenases are NAD1-dependent enzymes that catalyze the oxidation of an alcohol to a ketone. ValN also shares conserved domains with other dehydrogenase proteins, such as the shikimate 5-dehydrogenases and the ketopantoate reductases, which catalyze the conversions of shikimate to 5-dehydroshikimate and (R)-pantoate to 2-dehydropantoate, respectively. To investigate the function of valN in validamycin A biosynthesis, the valN gene in S. hygroscopicus 5008 was inactivated. The gene in the genome was replaced by an aac(3)IV-oriT cassette (Fig. 2.42A) (Gust et al., 2003; Xu et al., 2009). A pHZ1358-derived plasmid (pJTU753) containing an aac(3)IV-oriT cassette flanked with sequences of 3924-bp upstream and 2244-bp downstream of valN was obtained by ReDirect Technology in E. coli BW25113 (pIJ790). The plasmid pJTU753 was introduced into strain 5008 by conjugation from E. coli ET12567 (pUZ8002), and the apramycin-resistant phenotype was screened to get the valN-inactivated mutant, XH-2. Total DNA was extracted from the mutant, and the wild-type of S. hygroscopicus 5008 used as template for PCR amplification. The mutant gave a 2.30-kb PCR product, and the wild-type gave a 1.86-kb PCR product (Fig. 2.42B), which confirmed that a 918-bp DNA fragment of valN has been replaced by the 1371-bp aac(3)IV-oriT cassette. The mutant strain lacks the ability to produce validamycin A, but instead produces two new secondary metabolites, 1,10 -bis-valienamine and validienamycin (Fig. 2.43), which can be used as alternative sources of valienamine. 1,10 -bis-Valienamine contains two identical unsaturated cyclitol units, and its efficient conversion to valienamine would provide an alternative avenue to

Production of Validamycins Chapter | 2 (A) BamHI

ValN-det-R

ORF2

valO

ORF3

ValN-det-F BamHI

1860 bp

valM

valN

87

valL

5008

Replaced 918 bp

958

39

3924 bp

2244 bp

pJTU753

aac(3)IV-oriT

ori pIJ101

tsr

Double crossover

Truncated valN ORF2

ORF3

aac(3)IV-oriT

valO

2313 bp

valM

valL

XH-2

ValN-det-F

XH -2

1

kb

(B)

50 08

La dd er

ValN-det-R

4.0 kb 3.0 kb 2.30 kb 2.0 kb

1.86 kb

1.6 kb

1.0 kb

FIGURE 2.42 Inactivation of valN in S. hygroscopicus 5008. (A) Schematic representation of the replacement of a 918-bp fragment of valN with the 1371-bp aac(3)IV-oriT cassette. In shuttle plasmid pJTU753, aac(3)IV-oriT was inserted between the 3924- and 2244-bp genomic fragments originally flanking the 918-bp region. While wild-type 5008 should give a 1860-bp PCRamplified product, mutant XH-2 should yield a 2313-bp product using primers ValN-det-F and ValN-det-R. (B) PCR analysis of wild-type 5008 and mutant XH-2. PCR products were run on an agarose gel.

FIGURE 2.43 Chemical structures of 1,1’-bis-valienamine and validienamycin.

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Validamycin and Its Derivatives OH

OH

HO

OH

N H

HO OH

OH OH

1,1'-bis-Valienamine

OH

OH

NBS

HO

HO

NH3

HO OH Valienamine

HO

O OH Valienone

SCHEME 2.6 Chemical degradation of 1,10 -bis-valienamine.

this important precursor of antidiabetic drugs (Scheme 2.6). The glucoside validienamycin can be hydrolyzed to 1,10 -bis-valienamine by refluxing the compound in 1 N H2SO4/AcOH (1:1) for 48 h. Treatment of 1,10 -bis-valienamine with NBS produced valienamine and its corresponding ketone.

2.8.5 Deletion of γ-Butyrolactone Receptor Genes γ-Butyrolactone (GBL) regulatory systems are important in regulation of secondary metabolism in Streptomyces (Du et al., 2011; Horinouchi and Beppu, 2007; Takano, 2006; Willey and Gaskell, 2011). γ-Butyrolactones are a family of hormone-like signal molecules, and at least 60% of Streptomyces species appear to produce γ-butyrolactones (Takano et al., 2000). A-factor, i.e., 2-isocapryloyl-3R-hydroxymethyl-butyrolactone, produced by S. griseus, is a prototypic γ-butyrolactone and its regulatory mechanism has been well studied. Three key proteins—AfsA, ArpA, and AdpA—are involved in the A-factor regulatory cascade. The biosynthesis of A-factor is catalyzed by AfsA (Kato et al., 2007). A-factor above a critical level could bind to a receptor protein, ArpA, and then trigger antibiotic biosynthesis, resistance, and sporulation by derepressing the transcription of adpA, which encodes a central transcriptional activator (Horinouchi and Beppu, 2007). The genome sequence of S. hygroscopicus 5008 was recently revealed (Wu et al., 2012), and three pairs of afsAarpA and adpA homologs were found in the genome (Tan et al., 2013). ShbR3, one of the γ-butyrolactone receptor homologs, could directly repress the transcription of the adpA ortholog (adpA-H). AdpA-H in turn controls the transcription of valABC and valKLMN by directly binding to the intergenic region between these two operons. By tandem deletion of γ-butyrolactone receptor genes, the roles of the three pairs of afsA-arpA homologs in validamycin biosynthesis were investigated Zhong and coworkers (Tan et al., 2015). The afsA (ΔshbA1, ΔshbA2, and ΔshbA3) and arpA (ΔshbR1 and ΔshbR3) homologs were individually deleted. As shown in Fig. 2.44A, ΔshbA1 inactivation led to more than 90% decrease of the validamycin production; in ΔshbA2 and ΔshbA3, the production also decreased by 77% and 61%, respectively. On the other hand, by arpA homolog deletion, the production of validamycin was increased. As shown in Fig. 2.44B, compared with the wild-type, the validamycin production in ΔshbR1 and

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89

FIGURE 2.44 Effects of afsA homologs (A) and arpA homologs (B) deletion on the production of validamycin.  P , 0.05.

FIGURE 2.45 ShbR1 negatively regulated the transcription of adpA and positively regulated the transcription of ΔshbR3. (A) Quantitative real-time RT-PCR analysis of adpA-H (mean fold of gene transcriptional level over wild-type) after ΔshbR1 deletion; (B) electrophoretic mobilityshift assay (EMSA) with the adpA-H promoter DNA fragments and ShbR1; (C) quantitative real-time RT-PCR analysis of ΔshbR3 transcription (mean fold of gene transcriptional level over wild-type) after ΔshbR1 deletion; (D) EMSA with the ΔshbR3 promoter DNA fragments and ShbR1. (E) EMSA with the adpA-H promoter DNA fragment and ShbR2;  P , 0.05.

ΔshbR3 was significantly increased, by 26% (P , 0.05) and 20% (P , 0.05), respectively. However, ΔshbR2 deletion had no significant effects on validamycin production (P . 0.05). These results suggested that deletion of arpA (ΔshbR1 and ΔshbR3) homologs could facilitate the validamycin biosynthesis, and deletion of afsA homologs could repress the validamycin biosynthesis. Furthermore, by deletion of ΔshbR1, the transcripts of adpA-H increased. Although the transcription of ΔshbR3 was significantly reduced in the ΔsbhR1 mutant, its transcripts could still be detected by qRT-PCR and might therefore contribute to adpA-H repression, and the ΔshbR1/R3 double mutant was constructed. The time courses of validamycin fermentation in the wild-type and a series of ΔshbR mutant strains are shown in Fig. 2.45A.

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Validamycin and Its Derivatives

At the end of fermentation, the validamycin production and productivity in ΔshbR1/R3 double mutant increased by 55% (P , 0.05) and 95% (P , 0.05), respectively, compared with the wild-type. The final production of validamycin in the ΔshbR1/R3 double mutant was also significantly higher than in the ΔshbR1 (P , 0.05) or ΔshbR3 (P , 0.05) single mutant. Surprisingly, in ΔshbR1 mutant and ΔshbR1/R3 double mutant, cell growth was decreased after 48 h of fermentation compared to the wild-type (Fig. 2.45B). Meanwhile, in the ΔshbR1/R3 double mutant, the transcription of adpA-H and valABC was at a high level compared with the wild-type or ΔshbR1 single mutant (Fig. 2.45C and D). It is also worth noting that these ShbRs proteins have no direct binding affinity to the promoter region of valABC and valKLMN. These results suggested that double deletion of ΔshbR1/R3 could almost completely derepress the adpA-H transcription, and then increase the gene cluster transcription and validamycin production.

2.9 FERMENTATION PROCESS FOR PRODUCTION OF VALIDAMYCINS Due to its high efficiency, safety to both animal and human health, and the possibility of economic production from agroindustrial byproducts or feedstock (e.g., soybean meal, peanut cake, corn powder, and rice powder), validamycin A has been widely used in East Asia, especially China, for controlling sheath blight disease of crops and dumping-off disease in vegetable seedlings (Fan et al., 2013; Wei et al., 2011; Zhou et al., 2014a, 2012). Moreover, validamycin A can be converted to valienamine, which is an important precursor for the production of voglibose, an alpha-glucosidase inhibitor in the treatment of diabetes (Fan et al., 2013; Yu et al., 2005; Zhou et al., 2014a). Therefore, many scholars and engineers (An and Yu, 2008; Feng et al., 2013; Guo et al., 2006; He and Zhang, 1998; Hu, 1984; Li et al., 2009, 2013; Liao et al., 2008, 2009; Luan et al., 2015; Qiu and Chang, 2011; Ren et al., 2003; Shi et al., 1988; Tan et al., 2013, 2014; Wang et al., 2001; Wei et al., 2011, 2012a, 2012b; Wei and Zhong, 2013; Yan et al., 2014; Yan and Pu, 1980; Yu and An, 2007; Zheng et al., 2000, 2004; Zhong and Liao, 2008; Zhong et al., 2008, 2009, 2010; Zhou and Zhong, 2010, 2015; Zhou et al., 2012, 2014a, 2014b; Zhu et al., 2008) have been focusing on the fermentation process for the production of validamycins for more than 40 years.

2.9.1 Screening and Breeding High-Yield Strains For industrial-scale production, strain improvement is essential to obtain a relatively high amount of products. Since the discovery of validamycins in China, many methods for screening and breeding high-yield strains have been employed, such as UV (Shen, 1981), UV 1 LiCl (Shen, 1981), NTG

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91

(Shen, 1981), Co60 (Shen, 1981), N1 (Yu and An, 2007), and Ti1 (An and Yu, 2008; Yu and An, 2007). The titer of validamycin A was improved from hundreds to more than 20,000 μg/mL fermentation broth. Because of its high yield, the production had been stable for several decades, 30,00050,000 t (5% agent) (Shen, 1996), and it has been the largest antibiotic in China. Compared to random mutagenesis, metabolic engineering to specific pathways and/or genes is a more effective and targeted strategy for strain improvement (Hwang et al., 2014), resulting in an increase of precursor supply, manipulation of regulatory networks, enhancement of antibiotic resistance, and engineering of biosynthetic structural genes. In contrast, amplification of the biosynthetic gene clusters was believed to be a common and generally applicable method to enhance the production of secondary metabolites (Jung et al., 2013; Liao et al., 2010; Murakami et al., 2011). The amplification of multiple gene clusters was proved to be important for validamycin A overproduction by Zhou et al. (2014a). The schematic diagram for the construction of the recombinant strain with multiple val gene clusters is shown in Fig. 2.46A. As the size of the single amplified unit from RsA to RsB is about 40 kb, the gene cluster copy number of the recombinant strains can be determined by Southern blot analysis. After separation by pulsed-field gel electrophoresis (PFGE), the genomic DNA was hybridized with a 2-kb probe from val gene cluster. As shown in Fig. 2.46B, all five recombinant strains (TC01TC05) had at least three bands with the size ranging from 162 to 243 kb, indicating that the recombinant strains contained multiple copies (mainly three to five copies) of the val gene cluster. In contrast, the WT strain containing a single copy of the val cluster (Wu et al., 2012) only had a 70-kb band. Therefore, the analysis demonstrated that a recombinant strain containing multiple val gene clusters was successfully obtained using the zouA system. In order to investigate the effect of multiple gene clusters on validamycin A production, one recombinant strain TC03 was chosen to study the kinetic profiles of cell growth and production of validoxylamine A and validamycin A compared to the WT strain. As shown in Fig. 2.47A, multiple gene clusters slightly affected the cell growth (represented by total intracellular protein). The maximum total cellular protein in the WT strain and TC03 was 4.6 6 0.20 and 4.9 6 0.29 g/L, respectively. The time course of validamycin A biosynthesis in Fig. 2.47B showed that the validamycin A production of TC03 started to increase more rapidly after 12 h. Both strains reached their highest level of production after 96 h of fermentation. Ultimately, the maximum validamycin A production of TC03 was 21.0 g/L (0.22 g/L h) at 96 h, 34% higher than that of the WT strain. On the other hand, the production of validoxylamine A in TC03 was 3.0 g/L at 96 h, which was only half of that in the WT strain (Fig. 2.47C). Consequently, higher validamycin A production with lower validoxylamine A (intermediate) production resulted in the enhanced molar ratio of validamycin A/validoxylamine A in TC03 compared

92

Validamycin and Its Derivatives (A) One copy Spe1

BstZ171 val gene cluster aac(3)IV-RsB zouA-RsA 81,9 kb

probe

Three copies Spe1

40 kb

BstZ171

val gene cluster val gene cluster val gene cluster RsB-RsA zouA-RsA RsB-RsA 161,9 kb

(B)

M 1 2 3 4 5 6 1 2 3 4 5 Cluster copy number 291.0 kb 242.5 kb 194.0 kb 170.0 kb 145.5 kb 121.0 kb 97.0 kb 73.0 kb

6 5 4 3 2 1 1*

48.5 kb 24.5 kb

FIGURE 2.46 Amplification of the val gene cluster in S. hygroscopicus 5008 and genetic stability analysis. (A) Schematic representation of the single copy (top) and three copies “head-totail” val gene clusters (bottom). DNA fragments of zouA-RsA and aac(3)IV-RsB were inserted to the left and right boundaries of val gene cluster, respectively. Positions of SpeI, BstZ17I, and probe were indicated with expected fragment sizes. (B) Stained PFGE (left) and Southern blot hybridization (right) results of S. hygroscopicus 5008 WT strain and five randomly selected recombinant strains. Genomic DNA digested by SpeI and BstZ17I from WT strain, recombinant strains TC01, TC02, TC03, TC04, and TC05 are shown in lanes 16, respectively. M MidRange I PFG Marker (New England Biolabs). The val gene cluster copy number of recombinant strains is indicated by arrow on the right. Asterisk, a 70.7-kb DNA fragment of WT strain that only contained a single copy of val gene cluster and without the zouA system.

to WT, i.e., 8 versus 1.8, suggesting that more validoxylamine A was glycosylated owing to the multiple copies, which might be the rate-limiting step for validamycin A production. Meanwhile, the total molar concentration of validoxylamine A and validamycin A was about 10% higher in TC03 as well, which reached 53.4 mmol/L at 96 h compared with 48.4 mmol/L in the WT strain (Fig. 2.47D). Other genes were enhanced or knocked out as described in Section 2.7 in S. hygroscopicus and the engineered strains were applied in the production of validamycin A or other derivatives.

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FIGURE 2.47 Fermentation profiles of S. hygroscopicus 5008 wild-type (WT) strain and recombinant strain TC03. (A) Cell growth (represented by total intracellular protein); (B) validamycin A (VALA) production; (C) validoxylamine A (VO) production; (D) total molar concentration of validamycin A and validoxylamine A.

2.9.2 Optimization of Cultural Conditions Due to a large market for validamycin A, a great deal of research and development has been undertaken to improve its production titer and to reduce its production cost as low as possible. These investigations range from process improvement such as medium optimization with agroindustrial residues (Guo et al., 2006; Hu, 1984; Liao et al., 2008; Ren et al., 2003; Wei et al., 2012a; Zhong et al., 2008; Zhou et al., 2014b), application of fermentation strategies (He and Zhang, 1998; Liao et al., 2009; Qiu and Chang, 2011; Wei et al., 2012b; Zheng et al., 2000, 2004; Zhong and Liao, 2008; Zhong et al., 2010; Zhou and Zhong, 2015; Zhu et al., 2008), stimulator addition (Tan et al., 2013; Wei et al., 2011; Zhou et al., 2012), and precursor supply (Feng et al., 2013; Wei and Zhong, 2013; Zhou et al., 2011).

2.9.2.1 Optimization of Fermentation Medium Antibiotic production is influenced by various environmental factors including nutrients, oxygen supply, temperature, etc. (Lin et al., 2011). The effects of medium components are traditionally investigated as an important step to

94

Validamycin and Its Derivatives

improve antibiotic production. Statistical methods, such as PlackettBurman design and response surface methodology (RSM), have been widely used in medium optimization processes. For validamycin A production, complex media using agroindustrial residues such as sweet potato meal, rice starch, corn powder, soybean meal, cotton seed powder, and peanut powder as carbon sources and nitrogen sources were developed to reduce the cost of the medium and enhance validamycin A production by experience (Liao et al., 2009). Statistical methods, such as PlackettBurman design and RSM, are used in medium optimization during validamycin A fermentation (Wei et al., 2012a). Corn powder and soybean powder were used as a carbon source and nitrogen source in validamycin A fermentation. The effects of their concentrations on validamycin A production were studied by single-factor experiment. As the results of Table 2.17 indicate, they had significant effects on validamycin A production. The effects of other components, such as yeast extract, NaCl, KH2PO4, MgSO4, NH4Cl, and pH on validamycin A were analyzed in PlackettBurman design. The effects of six variables on validamycin A production are summarized in Table 2.18. The P-value is the probability that the magnitude of a contrast coefficient is due to random process variability, and is used as a tool to check the significance of each tested variable. A low P-value indicates a significant effect. As shown in Table 2.17, among the variables, yeast extract had the most significant effect on validamycin A production (P , 0.05). From the results of the single-factor experiment and PlackettBurman design, yeast extract, corn powder, and soybean powder were therefore selected for further optimization by RSM. Each variable had three levels. 100 g/L corn powder, 25 g/L of soybean powder, and 5 g/L yeast extract were selected as the levels corresponding to

TABLE 2.18 Statistical Data for the Determination of Variable Significance in the PlackettBurman Design Experiment Code

Factors

Low Level

High Level

ANOVA Analysis

(21) (g/L)

(21) (g/L)

t-value

P

Sequence

D

Yeast extract

5

15

211.3

0.001

1

E

NaCl

0.5

1.5

4.52

0.020

2

G

KH2PO4

1

2

0.50

0.652

6

H

pH

6

7

23.47

0.040

4

J

MgSO4

0

0.5

4.28

0.023

3

K

NH4Cl

0

0.5

23.12

0.052

5

Production of Validamycins Chapter | 2

95

the center point. Regression analysis of the experimental data was performed using the STATISTICA program and the following quadratic polynomial model was produced: Y 52216:3013:62X1 12:98X2 13:05X3 0:0175X1 2 0:0520X2 2 0:122X3 2

where Y is the validamycin A production in g/L, and X1, X2, and X3 represent the coded values of corn powder, soybean powder, and yeast extract. The parameters in this equation and their significance levels are shown in Table 2.19. ANOVA analysis showed that R2 was found to be 0.935, adjusted R2 was 0.851, F-value was 11.15, and P-value was 0.002. The three-dimensional response surfaces were generated to study the interaction among the three tested factors and to visualize the combined effects of factors on validamycin A production (Fig. 2.48). The response surfaces were studied in detail to determine the optimum medium composition. The effects of interaction of the medium components on validamycin A production were tested by contour plots for three possible combinations of factors, keeping one factor as a constant at one time. It was observed from contour plots that when corn powder and soybean powder were varied from lower to higher limit, an optimum point was found. The optimum value for corn powder and soybean powder was identified to be 101 and 26 g/L, respectively. Similarly, the predicted optimal value for yeast extract was found to be 6 g/L. The new optimized medium caused 70% enhancement of validamycin A production compared to the original medium. And low nitrogen source level facilitated structural gene transcription and validamycin A production, but did not affect cell growth and enzyme activity related to precursor metabolism.

TABLE 2.19 Regression Coefficients and Their Significance for Response Surface Model Factors

Coefficient

t

P

Constant

2216.30

24.48

0.003

X1

3.6225

4.14

0.004

X2

2.980

2.45

0.044

X3

3.05

3.09

0.017

X12

20.0175

24.14

0.004

X22

20.052

23.08

0.018

X32

20.122

27.22

0.000

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Validamycin and Its Derivatives

FIGURE 2.48 Response surface and contour plot of validamycin A production. (A) Effect of soybean powder and corn powder; (B) Effect of corn powder and yeast extract; (C) Effect of soybean powder and yeast extract.

2.9.2.2 Application of Fermentation Strategies Reducing the validamycin A production cost is critical to its large-scale production due to its rather low price. The medium in industrial-scale validamycin A production contains more than 100 g/L of starch (like rice and corn powder) as carbon source, which takes about 70% of the total medium cost. Therefore, developing a cheap culture medium by using biomass feedstock as substrates could save the industrial production cost, reduce the competition of the starch-based food consumption, and also contribute to the sustainable growth of society and economy. Hemicellulosic biomass, which is the second most available renewable resource in nature, has great potential as a substrate for microbial fermentation. For the validamycin production strain, S. hygroscopicus 5008, it was proved to have the ability to utilize the D-xylulose (Wu, 2012). Corncob hydrolysate (also called corncob molasses, xylose mother liquid) is a typical kind of lignocellulosic biomass and is widely available in China. It is an acid hydrolysate waste that is generated during the industrial-scale production of D-xylose from corncobs. Corncob hydrolysatecontains a high concentration of mixed sugars (including D-xylose, L-arabinose, and D-glucose),two-thirds of which is D-xylose. This low-cost

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and high-carbohydrate content feedstock has attracted increasing attention for the production of industrially important chemicals, such as acetone, butanol and ethanol propionic acid, L-lactic acid, xylitol, and validamycin A. Zhou et al. (2011) investigated the possibility of validamycin A production from corncob hydrolysate and the contribution of each sugar in the corncob hydrolysate to validamycin A biosynthesis by S. hygroscopicus 5008. Singleand mixed-sugar fermentation experiments indicated that D-xylose in the corncob hydrolysate had the major contribution to the validamycin A biosynthesis. In addition, 17.4 g/L validamycin A was produced with fed-batch culture from the medium containing 13% (v/v) corncob hydrolysate by an engineered strain, which was 41% higher than that of the wild-type strain. In order to lower the cost of validamycin fermentation, an external-loop airlift bioreactor with low height-to-diameter ratio of 2.9 was employed and evaluated in the validamycin production (Zheng et al., 2000, 2004). Airlift bioreactors are a class of bioreactors where a region of gassed liquid is connected to a region of ungassed liquid, the difference in hydrostatic pressure between the two regions resulting in circulation of the liquid phase. External-loop airlift bioreactors are those loop reactors in which the injection air into the bottom of one of the risers aerates the broth and causes liquid circulation between riser and down-comer. The geometry gives several advantages over the traditional mechanically stirred tank bioreactor, including (1) absence of high-shear regions such as near the impeller; (2) simple construction; (3) low energy consumption; (4) few chances of media contamination; (5) ease of operation; and (6) versatility. Therefore, the structures, properties, and applications of the airlift bioreactor were widely investigated. However, most airlift bioreactors had high height-to-diameter ratios, usually more than 3.0, even .20. In contrast, the height-to-diameter ratios of mechanically stirred tank bioreactors are mostly less than 3.0, especially in China. So an external-loop airlift bioreactor with low height-to-diameter ratio of 2.9 was developed and investigated to evaluate its suitability for validamycin production from crude substrates and the possibility of fitting a mechanically stirred tank bioreactor with external loops. The highest concentration of validamycins, 19,975 μg/mL, was obtained in the external-loop airlift bioreactor with sparger hole diameter of 1.5 mm under the optimized condition in which gas flow rate was 1.10 vvm. The fermentation cycle in the external-loop airlift bioreactor was almost the same as that in the mechanically stirred tank bioreactor, and much shorter than that in the shaking flasks under the same operation conditions including fermentation medium composition, inoculum ratio, and culture temperature. Although under the optimal fermentation condition, gas flow rate in the external-loop airlift bioreactor needed to be 37.5% higher than that in the mechanically stirred tank bioreactor, there was no agitation system and power was not required for agitation. Taking these factors into account, the total consumed energy of the external-loop airlift bioreactor was about 25% less than that of the mechanically stirred tank

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bioreactor. Furthermore, the manufacture cost of the external-loop airlift bioreactor was about two-thirds that of the mechanically stirred tank bioreactor without agitation system. So it is possible to retrofit the existing mechanically stirred tank bioreactor with the external-loop airlift bioreactor to produce validamycins from crude substrates economically.

2.9.2.3 Stimulator Addition In recent decades, considerable effort has been focused on enhancing validamycin A production, including strain screening, medium optimization, and culture condition optimization (e.g., temperature). Stimulators, such as external ethanol (Wei et al., 2011), H2O2 (Zhou et al., 2012), and 1,4-butyrolactone (Tan et al., 2013), were also investigated to enhance validamycin A production. 2.9.2.3.1 Addition of External Ethanol The effects of external ethanol addition on validamycin A biosynthesis by S. hygroscopicus 5008 were studied by Zhou et al. (2012). Different addition concentrations and addition times of ethanol brought a change to the antibiotic production. Validamycin A production titer was greatly enhanced under an optimal ethanol addition condition. Pure ethanol was added into the fermentation medium after inoculation with the final concentrations of 1200 mmol/L in the medium. Various concentrations of ethanol were tested for their effect on fermentation performance. The results in Fig. 2.49A showed there was no significant difference in cell growth as measured by intracellular protein with less than 200 mmol/L ethanol added. As shown in Fig. 2.49B, validamycin A production was increased significantly with addition of ethanol in concentrations (2, 10 mmol/L). When the ethanol level was above 10 mmol/L, validamycin A production was reduced in proportion to the increase of ethanol concentration in the medium (100, 200 mmol/L). The optimal concentration of ethanol was 10 mmol/L, which resulted in a 50% validamycin A enhancement on the 4th day. From the results, ethanol treatment led to more pigments being secreted into the fermentation broth from the 3rd to the 5th day compared with the control. These results suggest that ethanol may be involved in regulation of secondary metabolism in S. hygroscopicus. Pure ethanol was added at 0, 10, and 20 h separately during validamycin A fermentation with a final concentration of 10 mmol/L in the medium to study the effect of its addition time on the validamycin A production. As shown in Fig. 2.50C and D, the treatments had no evident effect on cell growth when ethanol was added at different stages of the fermentation. However, the addition of ethanol in the early phase of growth (0, 10 h) was beneficial to validamycin A production (Fig. 2.49D). The later supply at 20 h resulted in less enhancement of the production, approximating that of the control. The addition of ethanol at 10 h almost never improved cell

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FIGURE 2.49 Effect of ethanol addition concentration and time on S. hygroscopicus cell growth and validamycin A production. (A) Cell growth when different concentrations of ethanol were added at 0 h; (B) Validamycin A production when different concentrations of ethanol were added at 0 h; (C) cell growth when 10 mmol/L ethanol was added at different times; (D) Validamycin A production when 10 mmol/L ethanol was added at different times.  Indicates statistical significance (P , 0.05) compared to control (culture without ethanol addition).

growth and greatly increased the production of validamycin A (Figs. 2.49C and D). This finding indicated that the response of validamycin A biosynthesis to the induction time was closely related to the cellular physiological state of S. hygroscopicus. The optimal ethanol addition condition in this research was determined as 10 h (inoculation time) with a final concentration of 10 mmol/L in the medium, in which the production of validamycin A increased by 60%. In the following experiments, this addition condition was applied to an investigation of the positive effect of ethanol on validamycin A production compared with that of the control sample without the addition of ethanol. The addition of ethanol was accompanied by the multiple responses of intracellular signal, gene transcription, and enzyme activity levels. On the basis of the results, a model was presented for the effect of ethanol on validamycin A production in S. hygroscopicus (Fig. 2.50). Upon cellular perception of low ethanol in the medium, intracellular ROS is induced, leading to the subsequent activation of global regulators afsR and glnR, which regulate the transcription of structural genes valABC, valKLMN, and valG. Meanwhile, ethanol addition is accompanied by a higher PPP metabolic reaction rate, which provides the validamycin A biosynthetic pathway with plenty of precursor S7P. Finally, accumulation of precursors and

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FIGURE 2.50 A proposed signal transduction cascade of S. hygroscopicus cells induced by ethanol at low concentration (10 mmol/L).

transcriptional activation of validamycin A biosynthetic genes lead to an enhanced production of validamycin A. 2.9.2.3.2 Addition of External H2O2 Reactive oxygen species (ROS) play multiple roles in many organisms. ROS can regulate secondary metabolism on gene transcription levels in bacteria, such as in xanthan gum production by Xanthomonas campestris. Furthermore, ROS may be expected to play a significant role in determining bioreactor productivity (Rao and Sureshkumar, 2001). In order to verify whether ROS induction could be useful for improving antibiotics production (important secondary metabolites) in Streptomyces, which occupy a large group of popular drugs and pesticides having been used worldwide, the external addition of H2O2 into the fermentation medium of S. hygroscopicus 5008 was attempted (Wei et al., 2011). 50 μmol/L H2O2 was added to the medium at 0 h (the beginning of fermentation), 8 h (early exponential phase), and 24 h (late exponential phase) during validamycin A fermentation.

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FIGURE 2.51 Effect of H2O2 amount and addition time on S. hygroscopicus 5008 cell growth and validamycin A production. (A) Concentrations of total intracellular protein (as cell growth index) when a different concentration of H2O2 was added at 8 h; (B) concentrations of total intracellular protein when 50 mol/L H2O2 was added at different time; (C) validamycin A production when a different concentration of H2O2 was added at 8 h; (D) validamycin A production when 50 mol/L H2O2 was added at different time. Each data point represents the mean 6 SD from three independent samples,  P , 0.05.

Fig. 2.51B and D show the effects of H2O2 addition time on cell growth and validamycin A production. The results showed no significant difference in cell growth when H2O2 was added. However, validamycin A production was enhanced when H2O2 was added at three different phases. The optimal H2O2 addition time was at 8 h, which led to a 30% enhancement of validamycin A production titer on the 4th day of fermentation. The effect of the amount of H2O2 on cell growth and validamycin A production is shown in Fig. 2.51A and C. Cell growth was not significantly affected when H2O2 was added, but validamycin A production was enhanced under H2O2 addition. The optimal amount was 25 μmol/L, which had a 40% validamycin A enhancement on the 4th day. ROS induction time was reported to be critical in the manipulation of metabolite accumulation (Rao and Sureshkumar, 2001). So H2O2 was added at different stages to investigate the effects of H2O2 addition time on cell growth and validamycin A production. The treatments had no significant effect on validamycin A production when H2O2 was added at the beginning of fermentation and late log phase. However, validamycin A production was enhanced by more than 30% at 96 h when H2O2 was added

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at the early log phase. This indicated that the response of validamycin A biosynthesis to ROS is closely related to the growth stage of S. hygroscopicus 5008. The optimal amount of H2O2 for validamycin A production was 25 μmol/L; lower amounts of H2O2 were not sufficient to enhance validamycin A production, while higher amounts of H2O2 had an inhibitory effect on cell growth. The effect of ROS on gene transcription and enzyme activity was investigated. The results indicated that ROS induction is a novel and powerful strategy for improving the important antibiotic biosynthesis. 2.9.2.3.3

Addition of Exogenous 1,4-Butyrolactone

γ-Butyrolactones, such as A-factor, are one type of signaling molecule produced by Streptomyces species and have been reported to regulate secondary metabolism. However, they are usually produced in very small amount, which has hindered their structural elucidation and application for antibiotic overproduction. 1,4-Butyrolactone, an easily accessible and cheap analogue of γ-butyrolactones, was applied to the fermentation of validamycin A by Tan et al. (2013) to enhance validamycin production. The production of validamycin was interestingly increased. The concentration and addition time of 1,4-butyrolactone were optimized in the fermentation of S. hygroscopicus 5008. The results showed that the optimal concentration is 10 mmol/L and addition time is not critical for the inducing activity of 1,4-butyrolactone. Upon the addition of 1,4-butyrolactone in flask experiments, the validamycin A production was increased approximately 30% at the end of a 5-day fermentation (P , 0.05) (Fig. 2.52A). The enhancement of validamycin A production by 1,4-butyrolactone was also demonstrated in a 3-L stirred bioreactor. Time course of validamycin A fermentation in the bioreactor with or without the addition of 1,4-butyrolactone is shown in Fig. 2.52B. At the end of fermentation, the validamycin A production reached 15 g/L, increased also by 30% compared with the control without added 1,4-BL, indicating that the addition of 1,4-butyrolactone could be an efficient strategy to improve validamycin A production. As shown in Fig. 2.52C, 1,4-butyrolactone addition significantly increased the transcription of the pleiotropic regulatory gene adpA-H both at 72 and 96 h of fermentation. Moreover, the transcription of validamycin A biosynthetic genes valABC was significantly enhanced at 72 and 96 h of fermentation, and the transcription of valG was also significantly increased at 72 h of fermentation. Therefore, it appears that 1,4-butyrolactone may stimulate the transcription of adpA-H and subsequent transcription of validamycin A gene cluster in S. hygroscopicus 5008. In order to demonstrate the applicability of this strategy, 1,4-butyrolactone was added to the fermentation of a high-yielding validamycin A producer S. hygroscopicus TL01. As expected, the addition of 1,4-butyrolactone enhanced the production of validamycin A and validoxylamine A

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FIGURE 2.52 Exogenous 1,4-butyrolactone addition on the validamycin A fermentation and gene transcription in S. hygroscopicus 5008. (A) Time course of validamycin A fermentation in the shake flasks with or without 1,4-butyrolactone addition. (B) Time course of validamycin A fermentation in the 3-L stirred tank bioreactor with or without 1,4-butyrolactone addition. (C) Quantitative real-time RT-PCR analysis of valABC, valKLMN, valG, and adpA-H transcription (mean fold of gene transcriptional level over untreated control) after 1,4-butyrolactone addition; P , 0.05.

(Fig. 2.53A), which is the immediate intermediate of validamycin A and could be enzymatically converted to validamycin A by the glucosyltransferase ValG (Yu et al., 2005). At the end of fermentation in flasks, the addition of 1,4-butyrolactone significantly enhanced validamycin A production by 15% and the production of validoxylamine A by 70% (Fig. 2.53B).

2.9.2.4 Precursor Supply There is a close relationship between amino acid metabolism and aminoglycoside antibiotic biosynthesis. Nine kinds of amino acids were added during the fermentation of S. hygroscopicus 5008 to investigate effects of amino acids on biosynthesis of validamycin A (Feng et al., 2013). Among these nine kinds of amino acids, seven kinds of them could promote validamycin A production. The addition of isoleucine was found to promote validamycin A production the most to 16.76 g/L, which increased by 49% compared to the control. Besides, the addition of methionine showed inhibition on

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(A)

(B) Validamycin A

mAu

Validoxylamine A

25

800 400 0 mAu

TL01 2

4

6

8

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1200 800

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1200

Validamyin A Validoxylamine A

20 15 10 5

400 0

0 2

4

6

8

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TL01

TL01+1,4-BL

FIGURE 2.53 Exogenous 1,4-butyrolactone addition on the fermentation of validamycin A in the industrial strain TL01. For the TL01 fermentation, 10 mmol/L of 1,4-butyrolactone was added to the medium at 12 h. (A) HPLC profiles of S. hygroscopicus TL01 with or without 1,4-butyrolactone addition. (B) Effects of 1,4-butyrolactone addition on the production of validamycin A and validoxylamine A in S. hygroscopicus TL01. P , 0.05.

validamycin A biosynthesis. Compared to the control, validamycin A productivity in methionine addition fermentation decreased by 25% to 8.47 g/L. To further understand the impact of amino acids on validamycin A biosynthesis, a series of analysis on protein accumulation, sugar utilization, and activities of key enzymes in carbon metabolism related to validamycin A precursor synthesis was done. The results revealed that addition of amino acids could affect activities of key enzymes in carbon metabolism and redirect their flux, which benefits to accumulation of validamycin A precursor and stimulates biosynthesis of validamycin A. To enhance validamycin production, aromatic amino acids were added to the medium to increase the supply of precursor for validamycin (Wei and Zhong, 2013). Phenylalanine, tryptophan, and tyrosine were added to the medium to study the effect of aromatic amino acids on validamycin production. Addition time and concentration of phenylalanine were optimized to find out the optimal condition. The G6PDH activity that related to validamycin precursor metabolism was analyzed to study its mechanism. The results showed that phenylalanine had the best effect on validamycin production. The optimal condition was 0.1 g/L phenylalanine added at 24 h, which enhanced validamycin production by 60%. The addition of phenylalanine enhanced validamycin production greatly at 37 C, but not so much at 42 C. G6PDH activity was higher at 42 C than 37 C, and it was found that the addition of phenylalanine had no significant effect on glucose-6-phosphate dehydrogenase activity. Validamycin production was enhanced 60% by addition of phenylalanine. The addition of phenylalanine might inhibit the consumption of intermediates in the PPP, which resaved precursor for validamycin production. Precursors of many antibiotics came from the PPP. In this respect, this result also had the reference value for enhancing other antibiotics’ production.

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FIGURE 2.54 Biosynthesis of UDP-glucose and the glycosylation of validoxylamine A. Ugp, UDP-glucose pyrophosphatase; ValG, glucosyltransferase.

Another precursor is UDP-glucose. From the validamycin biosynthesis route, sedoheptulose-7-phosphate from the pentose phosphate pathway and validoxylamine A are a precursor and an intermediate for validamycin biosynthesis, respectively. The final step in the biosynthesis of validamycin A is the attachment of glucose to validoxylamine A, catalyzed by the glucosyltransferase ValG (Fig. 2.54). In the valG knockout mutants, validamycin A production was completely abolished, and validoxylamine A was significantly accumulated. In vitro experiments had shown that ValG is an active glucosyltransferase and utilizes uridine diphosphate-glucose (UDP-glucose) as the preferred glycosyl donor. During the fermentation of wild-type 5008, 3 g/L validamycin A and 0.5 g/L validoxylamine A accumulated at the end of the fermentation. It seems quite straightforward that the validamycin titer could be increased by efficient conversion of the accumulated validoxylamine A to validamycin A. However, the reasons for the accumulation of validoxylamine A were still elusive. So the supply and flux of the precursor UDP-glucose were considered and investigated (Zhou et al., 2011). 5 mmol/L validoxylamine A and different concentrations of UDP-glucose (5, 10, or 50 mmol/L) were added into a cell-free extract of TL01 (Fig. 2.39A). When a high concentration of UDP-glucose (50 mmol/L) was added into the reaction, almost all of the validoxylamine A was converted into validamycin A, which proved that the glycosylation step could be

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enhanced and more validamycin A could be produced when UDP-glucose was not limiting. UDP-glucose was also added into the fermentation broth at a 5-mL scale. In 5-day small-scale fermentation, the validamycin A and validoxylamine A production was 1.77 and 0.76 g/L, respectively. The glycosylation was enhanced when 10, 50, or 100 mg UDP-glucose was added during fermentation. Validamycin A production reached 2.66 g/L and less than 0.1 g/L of validoxylamine A accumulated when 100 mg of UDP-glucose was added into the small-scale fermentation broth (Fig. 2.39B).

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Mahmud, T., Lee, S., Floss, H.G., 2001. The biosynthesis of acarbose and validamycin. Chem. Rec. 1 (4), 300310. Mahmud, T., Tornus, I., Egelkrout, E., Wolf, E., Uy, C., Floss, H.G., et al., 1999. Biosynthetic studies on the α-glucosidase inhibitor acarbose in Actinoplanes sp.: 2-epi-5-epi-valiolone is the direct precursor of the valienamine moiety. J. Am. Chem. Soc. 121 (30), 69736983. Minagawa, K., Zhang, Y., Ito, T., Bai, L., Deng, Z., Mahmud, T., 2007. ValC, a new type of C7-cyclitol kinase involved in the biosynthesis of the antifungal agent validamycin A. ChemBioChem 8 (6), 632641. Montefoiori, D.C., Robinson, W.E., Mitchell, W.M., 1988. Role of protein N-glycosylation in pathogenesis of human immunodeficiency virus type 1. Proc. Natl. Acad. Sci. USA 85, 92489252. Murakami, T., Burian, J., Yanai, K., Bibb, M.J., Thompson, C.J., 2011. A system for the targeted amplification of bacterial gene clusters multiplies antibiotic yield in Streptomyces coelicolor. Proc. Natl. Acad. Sci. USA 108, 1602016025. Nishimura, Y., Satoh, T., Adachi, H., Kondo, S., Takeuchi, T., Azetaka, M., et al., 1997. Synthesis and antimetastatic activity of l-iduronic acid-type 1-N-iminosugars. J. Med. Chem. 40 (16), 26262633. Qiu G., Chang H.; (Zhejiang Qianjiang Biochemical Co., Ltd., Peop. Rep. China). assignee. 2011 20110410. Production method for increasing fermentation level of jinggangmycin. Application: CN patent 2011-10088294, 102191302. Qu, S., Kang, Q., Wu, H., Wang, L., Bai, L., 2015. Positive and negative regulation of GlnR in validamycin A biosynthesis by binding to different loci in promoter region. Appl. Microbiol. Biotechnol. 99 (11), 47714783. Rao, Y.M., Sureshkumar, G.K., 2001. Improvement in bioreactor productivities using free radicals: HOCl-induced overproduction of xanthan gum from Xanthomonas campestris and its mechanism. Biotechnol. Bioeng. 72, 6268. Ren, H., Dai, L., Liu, D., 2003. Fermentation of Jinggangmycin A using liquefaction and saccharification solution of rice powder. Nongyao 42 (5), 1213. San Paolo, S., Huang, J., Cohen, S.N., Thompson, C.J., 2006. Rag genes: novel components of the RamR regulon that trigger morphological differentiation in Streptomyces coelicolor. Mol. Microbiol. 61 (5), 11671186. Shen, Y., 1981. Development of agricultural antibiotics, jinggangmycins. Kangshengsu 6 (1), 5861. Shen, Y.C., 1996. Research and development on jinggangmycins for 25 years. Zhiwu Baohu (Plant Protect.) 22 (4), 4445. Sheu, K.F., Frey, P.A., 1978. UDP-glucose pyrophosphorylase. Stereochemical course of the reaction of glucose 1-phosphate with uridine-5’[1-thiotriphosphate]. J. Biol. Chem. 253, 33783380. Shi, W., Xia, Z., Wang, K., 1988. A new species of Streptomyces producing validamycin. Weishengwu Xuebao 28 (2), 179182. Singh, D., Kwon, H.-J., Rajkarnikar, A., Suh, J.-W., 2007. Glucoamylase gene, vldI, is linked to validamycin biosynthesis in Streptomyces hygroscopicus var. limoneus, and vldADEFG confers validamycin production in Streptomyces lividans, revealing the role of VldE in glucose attachment. Gene 395 (1-2), 151159. Singh, D., Seo, M.-J., Kwon, H.-J., Rajkarnikar, A., Kim, K.-R., Kim, S.-O., et al., 2006. Genetic localization and heterologous expression of validamycin biosynthetic gene cluster isolated from Streptomyces hygroscopicus var. limoneus KCCM 11405 (IFO 12704). Gene 376 (1), 1323.

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Stratmann, A., Mahmud, T., Lee, S., Distler, J., Floss, H.G., Piepersberg, W., 1999. The AcbC protein from Actinoplanes species is a C7-cyclitol synthase related to 3-dehydroquinate synthases and is involved in the biosynthesis of the α-glucosidase inhibitor acarbose. J. Biol. Chem. 274, 1088910896. Suami, T., Ogawa, S., Chida, N., 1980. The revised structure of validamycin A. J. Antibiot. 33 (1), 9899. Takano, E., 2006. γ-Butyrolactones: Streptomyces signalling molecules regulating antibiotic production and differentiation. Curr. Opin. Microbiol. 9, 287294. Takano, E., Nihira, T., Hara, Y., Jones, J.J., Gershater, C.J.L., Yamada, Y., et al., 2000. Purification and structural determination of SCB1, a γ-butyrolactone that elicits antibiotic production in Streptomyces coelicolor A3(2). J. Biol. Chem. 275, 1101011016. Takeuchi, M., Kamata, K., Yoshida, M., Kameda, Y., Matsui, K., 1990a. Inhibitory effect of pseudo-aminosugars on oligosaccharide glucosidases I and II and on lysosomal alphaglucosidase from rat liver. J. Biochem. 108 (1), 4246. Takeuchi, M., Takai, N., Asano, N., Kameda, Y., Matsui, K., 1990b. Inhibitory effect of validamine, valienamine and valiolamine on activities of carbohydrases in rat small intestinal brush border membranes. Chem. Pharm. Bull. 38 (7), 19701972. Tan G., Bai L., Zhong J.; (Shanghai Jiao Tong University, Peop. Rep. China). assignee. 2014 20140109. Improved manufacture of validamycin with Streptomyces hygroscopicus 5,008 deficient in SHJG7,318 and SHJG4,003 genes. Application: CN patent 2014-10009500, 103740789. Tan, G.-Y., Bai, L., Zhong, J.-J., 2013. Exogenous 1,4-butyrolactone stimulates A-factor-like cascade and validamycin biosynthesis in Streptomyces hygroscopicus 5008. Biotechnol. Bioeng. 110 (11), 29842993. Tan, G.-Y., Peng, Y., Lu, C., Bai, L., Zhong, J.-J., 2015. Engineering validamycin production by tandem deletion of γ-butyrolactone receptor genes in Streptomyces hygroscopicus 5008. Metab. Eng. 28, 7481. Terabe, S., Otsuka, K., Ichikawa, K., Tsuchiya, A., Ando, T., 1984. Electrokinetic separations with micellar solutions and open-tubular capillaries. Analyt. Chem. 56 (1), 111113. Tomlin, C.D.S., 1997. The Pesticide Manual. Eleventh Edition. p. 1606. Uchiyama I., 2007. MBGD: a platform for microbial comparative genomics based on the automated construction of orthologous groups. 35: D343D346. Wang, C., Tian, Z., Zhang, Z., Xu, D., Shen, Y., Han, C., 2015. Determination of validamycin A in agricultural food samples by solid-phase extraction combined with liquid chromatography-atmospheric pressure chemical ionisation-tandem mass spectrometry. Food Chem. 169, 150155. Wang, S., Chen, S., Yu, Z., 2001. Selection of nitrogen source and proportion of carbon and nitrogen of jinggangmycin of Streptomyces hygroscopicus. Huazhong Nongye Daxue Xuebao 20 (6), 554556. Wehmeier, U.F., Piepersberg, W., 2004. Biotechnology and molecular biology of the alphaglucosidase inhibitor acarbose. Appl. Microbiol. Biotechnol. 63 (6), 613625. Wei, Z.-H., Bai, L., Deng, Z., Zhong, J.-J., 2011. Enhanced production of validamycin A by H2O2-induced reactive oxygen species in fermentation of Streptomyces hygroscopicus 5008. Bioresour. Technol. 102 (2), 17831787. Wei, Z.-H., Bai, L., Deng, Z., Zhong, J.-J., 2012a. Impact of nitrogen concentration on validamycin A production and related gene transcription in fermentation of Streptomyces hygroscopicus 5008. Bioprocess Biosyst. Eng. 35 (7), 12011208.

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Wei, Z.-H., Wu, H., Bai, L., Deng, Z., Zhong, J.-J., 2012b. Temperature shift-induced reactive oxygen species enhanced validamycin A production in fermentation of Streptomyces hygroscopicus 5008. Bioprocess Biosyst. Eng. 35 (8), 13091316. Wei, Z.-H, Zhong, J.-J, 2013. Enhanced validamycin production by addition of aromatic amino acids. Zhongguo Kangshengsu Zazhi 38 (3), 200203. Willey, J.M., Gaskell, A.A., 2011. Morphogenetic signaling molecules of the streptomycetes. Chem. Rev. 111, 174187. Wray, L.V.J., Atkinson, M.R., Fisher, S.H., 1991. Identification and cloning of the glnR locus, which is required for transcription of the glnA gene in Streptomyces coelicolor A3(2). J. Bacteriol. 173, 73517360. Wu H. 2012. Comparative functional genomics for validamycin overproduction. 176 pp. Wu, H., Qu, S., Lu, C., Zheng, H., Zhou, X., Bai, L., et al., 2012. Genomic and transcriptomic insights into the thermo-regulated biosynthesis of validamycin in Streptomyces hygroscopicus 5008. BMC Genomics 13, 337. Xu, H., Minagawa, K., Bai, L., Deng, Z., Mahmud, T., 2008a. Catalytic analysis of the validamycin glycosyltransferase (ValG) and enzymatic production of 4’’-epi-Validamycin A. J. Nat. Prod. 71 (7), 12331236. Xu, H., Yang, J., Bai, L., Deng, Z., Mahmud, T., 2009. Genetically engineered production of 1,1’-bis-valienamine and validienamycin in Streptomyces hygroscopicus and their conversion to valienamine. Appl. Microbiol. Biotechnol. 81 (5), 895902. Xu, P.-J., Zhan, R., Tao, B., Zhang, H.-Y., Jiang, S.-R., 2008b. Determination of validamycin A residues in soil by precolumn derivatization and capillary gas chromatography. Fenxi Huaxue 36 (2), 249252. Yan X., Huo C., Yin H., Xu W., Lin Y.; (Zhejiang Tonglu Huifeng Biosciences Co., Ltd., Peop. Rep. China). assignee. 2014 20140731. Method for controlling microbiological contamination in jinggangmycin fermentation process. Application: CN patent 2014-10372186, 104152513. Yan, X., Pu, Y., 1980. Jinggangmycin producing strain 15. Kangshengsu 5 (1), 2224. Yang, J., Xu, H., Zhang, Y., Bai, L., Deng, Z., Mahmud, T., 2011. Nucleotidylation of unsaturated carbasugar in validamycin biosynthesis. Org. Biomol. Chem. 9 (2), 438449. Yang, Y.H., Song, E., Kim, E.J., Lee, K., Kim, W.S., Park, S.S., et al., 2009. NdgR, an IclR-like regulator involved in amino-aciddependent growth, quorum sensing, and antibiotic production in Streptomyces coelicolor. Appl. Microbiol. Biotechnol. 82, 501511. Yu, L., An, X., 2007. Screening on the high yield validamycin producing strain by implantation with N1 and Ti1 ion sources. Fushe Yanjiu Yu Fushe Gongyi Xuebao 25 (6), 371374. Yu, X., Wu, P., Han, Z., Hou, H., Liu, H., 2011. Determination of validamycin A in rice and rice husks by liquid chromatography. Xiandai Nongyao 10 (2), 3739. Yu, Y., Bai, L., Minagawa, K., Jian, X., Li, L., Li, J., et al., 2005. Gene cluster responsible for validamycin biosynthesis in Streptomyces hygroscopicus subsp. jinggangensis 5008. Appl. Environ. Microbiol. 71 (9), 50665076. Zhang, C.-S., Stratmann, A., Block, O., Bruckner, R., Podeschwa, M., Altenbach, H.-J., et al., 2002. Biosynthesis of the C(7)-cyclitol moiety of acarbose in Actinoplanes species SE50/ 110. 7-O-phosphorylation of the initial cyclitol precursor leads to proposal of a new biosynthetic pathway. J. Biol. Chem. 277 (25), 2285322862. Zheng, L., Zhou, X., Zhang, H., Ji, X., Li, L., Huang, L., et al., 2012. Structural and functional analysis of validoxylamine A 7’-phosphate synthase ValL involved in validamycin A biosynthesis. PLoS One 7 (2), e32033.

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Zheng, Y., Chen, X., Wang, Z., Shen, Y., 2004. Production of validamycins from crude substrates by Streptomyces hygroscopicus in an external-loop airlift bioreactor with a low height-to-diameter ratio. Chin. J. Chem. Eng. 12 (1), 102107. Zheng, Y., Wang, Z., Chen, X., 2000. Studies on gas holdup in an external-loop airlift bioreactor and its application to jinggangmycin fermentation. Zhongguo Kangshengsu Zazhi 25 (2), 105108. Zhong J., Liao Y.; (Shanghai Jiao Tong University, Peop. Rep. China). assignee. 2008 20080626. High yield production method of validamycin by fermentation. Application: CN patent 2008-10039565, 101298623. Zhong J., Wei Z., Bai L., Deng Z.; (Shanghai Jiao Tong University, Peop. Rep. China). assignee. 2010 20100827. Method for improving jinggangmycin yield through addition of hydrogen peroxide during fermentation. Application: CN patent 2010-10264100, 101914600. Zhong, S.-h, Qi, F.-p, Fan, M.-l, Xu, M.-c, 2009. Studies on the natural screen of validamycin strain and its producing capability. Hunan Shifan Daxue Ziran Kexue Xuebao 32 (2), 8588. Zhong, S.-h, Xu, M.-c, Xu, G.-y, 2008. Study on optimum culture medium of validamycin by orthogonal test. Hunan Shifan Daxue Ziran Kexue Xuebao 31 (1), 6971. Zhou, T.-C., Kim, B.-G., Zhong, J.-J., 2014a. Enhanced production of validamycin A in Streptomyces hygroscopicus 5008 by engineering validamycin biosynthetic gene cluster. Appl. Microbiol. Biotechnol. 98 (18), 79117922. Zhou, T.-C., Zhong, J.-J., 2015. Production of validamycin A from hemicellulose hydrolysate by Streptomyces hygroscopicus 5008. Bioresour. Technol. 175, 160166. Zhou, W.-W., Ma, B., Tang, Y.-J., Zhong, J.-J., Zheng, X., 2012. Enhancement of validamycin A production by addition of ethanol in fermentation of Streptomyces hygroscopicus 5008. Bioresour. Technol. 114, 616621. Zhou W., Li W., Tu P., Gao H., Liu Y., Zheng X.; (Zhejiang University, Peop. Rep. China). assignee. 2014b 20140421. Optimization of fermentation of validamycin. Application: CN patent 2014-10159547, 103937856. Zhou W., Zhong J.; (Shanghai Jiao Tong University, Peop. Rep. China). assignee. 2010 20100517. Fermentation method for manufacturing jingangmycin. Application: CN patent 2010-10173832, 101851653. Zhou, X., Wu, H., Li, Z., Zhou, X., Bai, L., Deng, Z., 2011. Over-expression of UDP-glucose pyrophosphorylase increases validamycin A but decreases validoxylamine A production in Streptomyces hygroscopicus var. jinggangensis 5008. Metabol. Eng. 13 (6), 768776. Zhu S, Lin K, Mao G, Zhang Y, Li X, Luo X, et al.; (Wuhan Tianhui Bio-Engineering Co., Ltd., Peop. Rep. China; Wuhan Lushiji Bio-Engineering Co., Ltd.). assignee. 2008 20060829. Fermentation method for manufacturing validamycin A. Application: CN patent 200610112671, 101134976.

Chapter 3

Bioactivities of Validamycins and Related Natural Compounds Validamycins and related natural compounds have good bioactivities, such as antifungal activities, enzymatic inhibitory activities, and insecticidal activities.

3.1 ANTIFUNGAL ACTIVITIES 3.1.1 Antifungal Activity Against Rhizoctonia solani Validamycins and validoxylamines are highly active against R. solani by the dendroid-test method and the greenhouse test (Iwasa et al., 1971a) (Table 3.1). According to Table 3.1, the antifungal activities of validamycins A, E, and F were the highest, and the activity of validoxylamine B was the lowest. Validamycins E and F were highly active against Pellicularia sasakii by the dendroid-test method and the greenhouse test. Although validamycins C and D, as well as validoxylamines A and B, showed very low activity by the dendroid-test method as shown in the table, they showed considerable activity by the greenhouse test. However, validamycin H (Asano et al., 1990a) exhibited fivefold weaker activity against R. solani by the dendroid-test method than validamycins E and F, which were α-D-glucopyranosylvalidamycin A. The glucoside introduction into the hydroxymethyl group of validoxylamine A (validamycins C and D) caused the reduction of activity. The reduction of activity in validamycin H might be due to the suppression of permeability of the antibiotic into the pathogen by the glucoside introduction into the hydroxymethyl group (C-6ʺ) of the glucosyl residue (Asano et al., 1987). These results provided interesting evidence for structure activity relationship in the validamycin group.

3.1.2 Antifungal Mechanism Against R. solani Validamycin A did not significantly suppress the growth of R. solani on a nutritionally rich medium, but it caused an abnormal branching of hyphae and the cessation of colony development on a water agar (Iwasa et al., 1971b). Validamycin and Its Derivatives. DOI: http://dx.doi.org/10.1016/B978-0-08-100999-4.00003-4 © 2017 Elsevier Ltd. All rights reserved.

115

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TABLE 3.1 Minimum Concentration Causing Abnormal Branching at the Tips of Hyphae of R. solani in Dendroid-Test Method Compounds

Minimum Concentration (μg/mL)

Validamycin A

0.01

Validamycin B

0.50

Validamycin C

10

Validamycin D

25

Validamycin E

0.01

Validamycin F

0.01

Validamycin G

0.50

Validamycin H

0.05

Validoxylamine A

1.00

Validoxylamine B

50

Validoxylamine G

2.50

Validamycins did not inhibit the growth of fungal mass causing no change in the amount of protein, nucleic acid, and cell wall components, but altering the morphology of the fungus (Nioh and Mizushima, 1974). They significantly inhibited the extension of the main hyphae but not of the primary and secondary branches (Nioh and Mizushima, 1974; Shibata et al., 1981). Another proposal of action mode was that validamycins could induce the activities of endochitinase and β-1,3-glucanase and inhibit the activity of trehalase in rice plants (Zhang et al., 2003).

3.1.2.1 Effect of Validamycins on the Morphology and Components of R. solani 3.1.2.1.1 Effect on the Morphology of P. sasakii (Nioh and Mizushima, 1974; Shibata et al., 1980, 1982; Trinci, 1985) Despite the lack of inhibitory effect against R. solani on agar media in ordinary assay methods, validamycin inhibited remarkably the extension of the disease, and development of the hyphae on the rice plants seemed to be thoroughly inhibited. Modified Pfeffer’s agar was poured into the inner part and various media with or without validamycin were used in the outer one. An agar disk of P. sasakii was placed in the center of the inner part, incubated at 27 C, and after 24, 40, and 64 h incubation, the diameter of the mycelium that developed on the medium in the outer part was measured. As shown in

117

Bioactivities of Validamycins and Related Natural Compounds Chapter | 3

TABLE 3.2 Effect of Validamycin on the Growth of P. sasakii in Modified Dish Technique Outer Medium

Diameter of Mycelium (mm) 24 h

40 h

64 h

Validamycin added

57

76

90

No addition

78

90

90

Modified Pfeffer’s agar without carbon source

Validamycin added

51

61

66

No addition

73

90

90

Modified Pfeffer’s agar without nitrogen source

Validamycin added

40

44

52

No addition

80

90

90

Water agar

Validamycin added

32

35

36

No addition

70

90

90

Modified Pfeffer’s agar

Table 3.2, when the medium in the outer part was water agar, the mycelium of P. sasakii on validamycin-containing medium developed to some extent but an abnormal branching occurred at the tips of the hyphae, and further development was inhibited. Microscopic observation of this part indicated that branching at nearly right angle to main axis occurred at the tips of the hyphae and something like a fine drop of water was also observed among the branching hyphae (Fig. 3.1). This phenomenon was also observed, though weakly, when the medium in the outer part was modified Pfeffer’s agar without nitrogen source, but was not observed in the case of modified Pfeffer’s agar or that without carbon source. With nutrient-deficient medium in the inner part and nutrient-rich medium containing validamycins in the outer part, abnormal branching did not occur. When the medium containing plant constituents, such as rice straw infusion agar, was employed, the phenomenon was observed only with nutrient-deficient medium with validamycins in the outer part. Therefore, the hyphae on the latter underwent abnormal branching at the tips, and subsequent growth was inhibited when P. sasakii growing from nutrient-rich medium developed onto nutritionally very poor media containing validamycins. The occurrence of this phenomenon in other phytopathogenic fungi was examined by the modified dish technique. The minimum concentration causing abnormal branching is shown in Table 3.3. P. sasakii, R. solani, and Pellicularia praticola were sensitive to validamycins. This sensitivity to validamycin seemed to be limited to P. sasakii and related species.

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FIGURE 3.1 Hyphae of P. sasakii in modified dish technique. (A) Validamycin in the outer part (0.2 μg/mL) and (B) no validamycin. Reprinted with permission courtesy of Japan Antibiotics Research Association (JARA).

TABLE 3.3 Effects of Validamycins A and B on Some Phytopathogenic Fungi Tested Organisms

Minimum Concentration Causing Abnormal Branching (μg/mL) Validamycin A

Validamycin B

Pellicularia sasakii

0.01

0.5

Rhizoctonia solani

0.02

50

Pellicularia praticola

5

Corticium rolfsii

.100

Stereum fasciatum

.100

Glomerella cingulata

.100

.100

Alternaria kikuchiana

.100

.100

Fusarium oxysporum f. niveum

.100

.100

Sclerotinia screlotiorum

.100

.100

Botrytis cinerea

.100

Phytophthora infestans

.100

Bioactivities of Validamycins and Related Natural Compounds Chapter | 3

Radius of fungal colony (cm)

20

(A)

20

15

15

10

10

5

5

1

2

3

4

5

6

7

119

(B)

1

2

3

4

5

6

7

Days after inoculation FIGURE 3.2 Effect of validamycins on development of colony of P. sasakii. Effect of validamycins was determined by the dendroid-test method. The radius of inoculated agar disk has been subtracted. (A) Growth on water agar; (B) growth on modified Pfeffer’s agar medium. x Without validamycins;  with validamycins (0.1 μg/mL).

The development rate of a colony as determined by its radius was constant over 7 days, while the rate was considerably reduced after 1-day cultivation in the presence of validamycins (Fig. 3.2A) (Nioh and Mizushima, 1974), and smaller and denser colonies appeared. Validamycins also inhibited the development of the fungal colony on modified Pfeffer’s agar medium, although the inhibition was less than that on the water agar (Fig. 3.2B). Fig. 3.3 shows the growth of a primary hypha of the main axis in a colony in the presence and absence of validamycins during the first 20-h cultivation. Data were taken from Fig. 3.4. There was no difference between the cultures with and without validamycins during the first 11 h of cultivation. After that the growth rate of the primary hypha in the presence of validamycins began to decrease abruptly, while elongation of the primary hypha continued linearly in a colony in the absence of validamycins. Morphological changes produced by validamycin on the growing tip of the individual hypha were traced from the photomicrographs during the period of 8 20 h of cultivation (Fig. 3.4). Once a branching from a primary hypha was initiated near the tip of a hypha, elongation of the primary hypha between the agar disk and the branching point stopped almost completely. Consequently, the growth of hyphae could be traced by marking specific branching points. In the culture without validamycins, the primary hypha continued to elongate straight with occasional branching. On the other hand, the growth of the primary hypha retarded significantly once elongation of a branch in the primary hypha was initiated in the culture with validamycins,

120

Validamycin and Its Derivatives 16 14

Length of hydpha (mm)

12 10 8 6 4 2

8 10 12 14 16 18 20 Hours after inoculation FIGURE 3.3 Effect of validamycins on development of primary hypha of main axis of P. sasakii. Length of the hypha was measured in tracing of photomicrographs in Fig. 3.4. The radius of inoculated agar disk was subtracted. x Without validamycins;  with validamycins (0.1 μg/mL).

and the growth of the branched hyphae also retarded by the secondary branching from the hyphae, and so forth, thus the fungal hyphae formed dendroid mycelia. In order to examine whether validamycins inhibited the growth of the fungus as a whole, the total length of a mycelium developed from a single hypha was measured as a function of time (Table 3.4). A little delay in the elongation of hyphae was observed in the early stage of the cultivation in the presence of validamycins, but no inhibition was found in the increase in total length of the hyphae during 16 20 h of cultivation. On the other hand, the extension of the primary hypha during this period was inhibited to 72% by validamycins. The total length of the first secondary hyphae, i.e., hyphae directly branching from the primary hypha, and that of the rest of the secondary hyphae were measured separately (Fig. 3.5). Validamycins significantly suppressed the elongation of the first secondary hyphae as well as that of the primary hypha, while elongation of the rest of the secondary hyphae was even stimulated in the presence of validamycins after 16 h of cultivation. This result supported the idea that validamycins stimulate the growth of branched hyphae but do not suppress significantly the total growth

Bioactivities of Validamycins and Related Natural Compounds Chapter | 3

121

(A)

14 h

20 h

12 h

18 h

10 h

16 h

1 mm

8h 9h

(B)

9h 16 h

8h

18 h

20 h

13.5 h 1 mm 11 h

FIGURE 3.4 Development of a hyphal tip of P. sasakii. The tracing figures were traced from the photomicrographs of the fungus cultured on water agar at 25 C for the period designated in the figures. Figures on the hyphae are branch numbers counting from the inoculated point. The bottom of 8-h hyphae was at the periphery of agar disk. (A) Without validamycins; (B) with validamycins (0.1 μg/mL).

of hyphae. The density of the hyphae per unit area of the fungal colony was calculated as shown in Table 3.4. The hyphal density of the colony of a 20-h culture in the presence of validamycin was about three times as large as that without validamycins. As microscopic observation of the mycelium led to

TABLE 3.4 Effect of Validamycins on Elongation and Branching of a Mycelium of P. sasakiia Validamycins

Not added

Added

a

Hours of Cultivation

Length of a Primary Hypha (mm)

Total Length of a Mycelium (A) (mm)

Number of Total Branching Points on a Mycelium (B)

A/B

Hyphalb Density

8

2.9

4.1

5

0.82

0.08

12

6.0

20.2

49

0.41

0.12

16

10.3

71.2

176

0.40

0.17

18

12.2

104.5

240

0.44

0.18

20

13.9

128.0

329

0.39

0.17

8

1.8

5.5

11

0.50

0.20

12

2.7

12.1

33

0.37

0.25

16

5.6

37.6

103

0.37

0.25

18

6.3

70.2

236

0.30

0.38

20

6.6

93.5

336

0.28

0.47

The length and the number of branches were measured from microphotographs of mycelium grown from a single hypha on water agar with or without validamycins. Hyphal density was expressed as length of hyphae per unit area (mm/mm2). The value was corrected for the area of agar disk.

b

Bioactivities of Validamycins and Related Natural Compounds Chapter | 3

Length of hypha (mm)

(A) 60

60

40

40

20

20

8

12

16 18

20

123

(B)

8

12

16

18

20

Hours after inoculation FIGURE 3.5 Effect of validamycins on length of primary, first secondary, and rest of secondary hyphae of P. sasakii. The total length of each hypha was measured separately on the photomicrographs used in Table 3.4. (A) Without validamycins; (B) with validamycins. x Primary hypha; Δ sum of the first secondary hyphae; 3 sum of the rest of secondary hyphae.

the assumption that hyphal branching was much accelerated by validamycins at the periphery of a colony; number of branching points was measured over the entire period of cultivation with or without validamycins. Table 3.4 also shows data on branching. There seemed to be no difference in the total number of branching points between the two cultures. An average length of hyphae per branching remained fairly constant (ca. 0.4 mm) over the entire period of cultivation in the absence of validamycins. Although the mycelium grown in the presence of validamycin had hyphae of about the same average length per branching in the early stage of the cultivation, the length became shorter in the latter stage of the cultivation when the extensive growth of the late secondary hyphae occurred (ca. 0.3 mm). Difference between the cultures with and without validamycins was also observed in the angle between the primary hypha and the first secondary hypha. As Table 3.5 shows, the fungus in the culture with validamycins tended to form branches with wider angle than the culture without validamycins. In order to determine the effect of validamycins on the net increase in cellular constituents, the increase in protein and ribonucleic acid contents during the growth with and without validamycins was compared (Table 3.6). The increase in the amount of protein and ribonucleic acid was not affected significantly by the addition of validamycins.

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Validamycin and Its Derivatives

TABLE 3.5 Effect of Validamycins on Angle of Branches Occurred from a Primary Hypha of P. sasakiia No Validamycins Added

Validamycins Added

39

21

Mean

38.5

54.4

Standard deviation

15.9

15.4

Number of branches on a primary hypha

a

Measurement of the angles was made in a 20-h culture from Table 3.4.

TABLE 3.6 Amounts of RNA and Protein in P. sasakii Validamycins

RNA (μg/plate)

Protein (35S) (cpm/plate)

Not added

13.1

3860

Added

13.2

3220

TABLE 3.7 Contents of Glucose and Glucosamine in Cell Wall of P. sasakii Validamycins

Cell Wall (%)

Glucose (mg/10 mg Cell Wall)

Glucosamine (mg/10 mg Cell Wall)

Not added

16.5

3.15

1.03

Added

17.2

3.07

1.01

The cell wall components of P. sasakii grown in the presence and absence of validamycins were compared. Three neutral sugars (glucose, mannose, and xylose) and glucosamine were found in the wall hydrolysate of the fungus grown on either the water agar or modified Pfeffer’s agar medium with or without validamycins. There was no difference in the content of glucose and glucosamine in the cell wall of the fungus grown either in the presence or absence of validamycins (Table 3.7). 3.1.2.1.2 Effect of Validamycin A on Hyphal Extension and Hyphal Morphology of R. cerealis and R. solani (Trinci, 1985) Figs. 3.6 and 3.7 show the effects of validamycin A on the rate of expansion of colonies of R. cerealis and R. solani, and Tables 3.8 and 3.9 show the

50

Colony radius (mm)

40

30

20

10

0

50 100 150 Time since inoculation (h)

200

FIGURE 3.6 Effect of validamycin A concentration on the growth of R. cerealis colonies cultured on Vogel’s medium containing 5 mmol/L glucose. Validamycin A concentration: x, 0; Δ, 0.05; &, 0.10; K, 0.20; ▲, 0.40; ’, 0.80; ▼, 1.00 μmol/L.

100

Colony length (mm)

80

60

40

20

0

50

100 150 200 Time since inoculation (h)

250

FIGURE 3.7 Effect of validamycin A concentration on the growth of R. solani colonies cultured on Vogel’s medium containing 5 mmol/L glucose. Validamycin A concentration: x, 0; Δ, 0.05; &, 0.10; K, 0.20; ▲, 0.40; ’, 0.80; ▼, 1.00 μmol/L.

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Validamycin and Its Derivatives

TABLE 3.8 Effect of Validamycin A on the Expansion and Morphology of Colonies of R. cerealis Validamycin A Concentration (μmol/L)

Colony Radial Growth Rate (Kr, μm/h)

Morphology of Leading Hyphae at the Colony Margin Intercalary Compartment Length (μm)

Hyphal Radius (μm)

0

277

188 6 28

1.9 6 0.6

0.05

281

180 6 50

2.0 6 0.5

0.10

101

134 6 36

2.1 6 0.4

0.20

68

92 6 12

1.1 6 0.3

0.40

59

93 6 10

1.3 6 0.3

0.80

65

98 6 14

1.1 6 0.2

1.00

68

91 6 14

1.3 6 0.2

TABLE 3.9 Effect of Validamycin A on the Number of Branches Produced by Apical and Intercalary Compartments of Leading Hyphae at the Margin of Colonies of R. cerealis Validamycin A Concentration (μmol/L)

Mean Number of Branches per Compartment Apical Compartment

Intercalary Compartments First

Second

Third

Fourth

0

0

1.0

1.2

1.2

1.3

0.05

0

1.1

1.4

1.3

1.4

0.10

0

1.0

1.0

1.1

1.3

0.20

0

1.1

1.0

1.0

1.2

0.40

0

1.1

1.1

1.4

1.3

0.80

0

1.3

1.3

1.3

1.1

1.00

0

1.0

1.0

1.2

1.0

effects of the antibiotic on the morphology of leading hyphae at the margin of colonies of R. cerealis. A deceleration in the rate of hyphal extension of R. cerealis or R. solani caused by validamycin A was always correlated with an increase in hyphal density at the colony margin, which was visible to the

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127

naked eye. In the case of R. cerealis, a reduction in hyphal extension was always associated with a decrease in intercalary compartment length and a decrease in hyphal radius (Table 3.8); for technical reasons, measurements were not made of the effects of validamycin A on the morphology of R. solani hyphae. The correlation coefficient (γ 5 1 0.96%) between colony radial growth rate and intercalary compartment length was highly significant (P , 0.001), but the correlation coefficient (γ 5 0.69) between validamycin A concentration and colony radial growth was not significant (P . 0.05). For antibiotic concentrations between 0.2 and 1.0 μmol/L, there was on average a 77% decrease in colony growth rate, a 50% decrease in intercalary compartment length, and a 36% decrease in hyphal radius (treated values compared to control values of colonies cultured on medium lacking validamycin A); on average, the volume (423 μm3) of an intercalary compartment of a validamycin A-inhibited hypha was only about one-fifth that (2044 μm3) of an intercalary compartment of an untreated hypha. Therefore, Table 3.8 shows that, over the range 0.2 1.0 μmol/L, the effects of validamycin A on colony radial growth rate and hyphal morphology were independent of antibiotic concentration; the 0.1 μmol/L validamycin A result (Table 3.9) probably reflected the fact that the hyphae were fixed when they were in the deceleration phase of growth (Fig. 3.6). Since validamycin A is very soluble in water, the results in Table 3.8 could not be explained on the basis that the actual antibiotic concentrations in the medium were less than those expected. Figs. 3.6 and 3.7 show that for R. cerealis and R. solani the duration of the interval between inoculation and the onset of a deceleration in the rate of colony expansion was inversely related to antibiotic concentration. R. cerealis usually produced one branch per intercalary compartment and this branch was usually located just behind the distal septum. Tables 3.8 and 3.9 show that validamycin A treated hyphae produced intercalary compartments that were only about half as long as the compartments of untreated hyphae, but these short compartments continued to form branches in the normal way. So the increase in hyphal density caused by validamycin A was directly related to the observed reduction in intercalary compartment length. 3.1.2.1.3 Effect of Validamycins on the Growth and Morphology of Other Fungi (Nioh and Mizushima, 1974) The effect of validamycins on the growth of various fungi was examined. The diameter of fungal colonies after a 7-day growth was determined, and the results are shown in Table 3.10. Colony development in all the fungi that belong to Basidiomycetes was suppressed by validamycins. Some strains of Deuteromycetes and Ascomycetes were extremely affected, while other strains in the same class and Phycomycetes were not affected at all by validamycins. The extent of branching of hyphae along the periphery of the colony was also examined. All the fungi, in which the stimulation of branching

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Validamycin and Its Derivatives

TABLE 3.10 Effect of Validamycins on Growth and Branching of Various Fungi Strains

Inhibition (%)

Stimulation of Branching at Tip with Validamycina

Stereum roseum H4

71.0

1

Corticium rolfsii H3

78.3

11

Corticium rolfsii 21

73.3

11

Corticium gramineum 153

50.0

11

Corticium gramineum 154

90.4

11

Lentinus edodes H36

86.7

11

Cochliobolus miyabeanus 59

0

22

Pythium aphanidermatum 3

0

22

Phytophthora infestans 2

0

22

Helminthosporium sigmoideum 26

82.8

1

H. sigmoideum var. irregular 27

70.8

11

0

22

Sclerotium hydrophilum 145

81.0

11

Acrocylindrium sp.

16.4

1

Penicillium notatum P-1-1

0

22

Aspergillus oryzae O-2-1

0

22

Rhizopus nigricans R-1-1

3.3

22

Botrytis cinerea

a

11 Significantly stimulated; 1 Stimulated; 22 Not stimulated.

at the tip of hyphae by validamycin was observed, were those whose colony development was suppressed by validamycins (Table 3.10). 3.1.2.1.4 Effect of Treatment with Validamycin A on the Pathogenicity of R. solani (Endo et al., 1983) The hyphae treated with 0.63 5 μg/mL validamycin A were unable to penetrate into sheath tissues within 72 h after inoculation. Table 3.11 shows the results of naked eye and anatomical observation. A smaller spot than the lesions formed by untreated hyphae appeared at 72 h near the mycelial disk treated with 0.63 μg/mL validamycin A. In sections of these spots, deep yellow-brown or reddish brown stained epidermal and parenchyma cells and coagulated cytoplasm of the cells were observed, but no hyphae of R. solani

129

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TABLE 3.11 Effect of Validamycin A on Infection of R. solani by Inoculation After Dipping Mycelial Disks in Solution of Validamycin A Time After Inoculation (h)

Validamycin A Concentration (μg/mL) 0 a

0.63

1.25

2.5

5.0

0

0

0

0

0

0

24

I

I

I

0

0

48

III

I

I

0

0

72

IV

I or II

I or II

0, I, or II

0

a

Disease index: 0, little or no degeneration of sheath; I, browning of sheath; II, formation of small spot; III, formation of lesion; IV, appearance of new lesion formed in the upper part rather than at the inoculation site. The index of 0, I, and II indicates no infection of R. solani in rice sheath tissues; III and IV, infection of R. solani in rice sheath tissues.

were observed in the cells. In an elliptical small spot formed on the fifth sheath, opposite to the fourth sheath inoculated with a mycelial disk treated with 2.5 μg/mL validamycin A, hyphae and infection cushions were formed only on the surface of the sheath at 72 h. Degenerative changes, such as swelling or disappearance of chloroplasts, collapse of vessels, and shrinkage of parenchyma cells, were seen in the central part of this spot. The deep staining of parenchyma cells and chloroplasts by safranin, coagulation of cytoplasm in the cells, and shrinkage of the cells were observed in the marginal part of the spot. Little or no degeneration occurred in the sheaths treated with 5 μg/mL validamycin A at 72 h. Epidermal and parenchyma cells in healthy control sheaths were colored in pale yellow-brown at 72 h, but not in dark yellow-brown or reddish brown color. In the spray test, the results are shown in Table 3.12. When rice sheaths were sprayed with 10 μg/mL validamycin A at 4 h after inoculation with R. solani, epidermal and parenchyma cells became dark yellow-brown or reddish brown accompanying with coagulation of cytoplasm of the cells on the fourth day after inoculation. Small spots appeared on the sheaths sprayed with validamycin A at 8 h after inoculation. These spots seemed to have similar appearance to those formed by the hyphae dipped in solutions of validamycin A. In lesions formed on the sheaths sprayed 16 h after inoculation, the hyphal growth seemed to be stopped in the space of the sheaths. In other lesions, even when the hyphae penetrated into parenchyma cells, the cells were hardly damaged. Control sheaths, which were sprayed with 10 μg/mL validamycin A, had no pathological change as seen in healthy unsprayed sheaths. Effect of validamycin A on the enzymes of R. solani was investigated (Asano et al., 1987; Li et al., 2010; Robson et al., 1989; Shigemoto et al., 1989;

TABLE 3.12 Effect of 10 μg/mL Validamycin A Spray on Infection of R. solani into Rice Sheath Spray Time (h) After Inoculation of R. solani

No Penetrationa Browning of Sheath and Degeneration of Tissues

Penetrationa

Formation of Small Spot and Degeneration of Tissues

Formation of Lesion (I)

Formation of New Lesionb

Slight or No Damage of Tissues

Severe Damage of Tissues

Severe Damage of Tissues

0.5

1c

2

2

2

2

4

1

2

2

2

2

8

1

1

2

2

2

16

1

2

1

2

2

2

2

2

1

1

Unsprayed a

Sampling on the fourth day after inoculation of R. solani. Lesion formed in the upper part rather than (I). c Symbols: 1 , the phenomenon described in the above column was recognized; 2, not recognized. b

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131

Uyeda et al., 1985, 1986, 1988). In order to clarify the mechanism of the effect of validamycins and on the basis of a close relationship between endogenous β-D-glucan-degrading enzymes and the cell wall constitution or morphogenesis of a fungus, the effect of validamycin A on cell wall polysaccharides and some enzymes related to their metabolism was investigated (Uyeda et al., 1985). After various examinations on some enzymes, validamycin A was found to have no effect on the production of glucose-6-phosphate dehydrogenase, 6phosphogluconic acid dehydrogenase, glucosephosphate isomerase, glucosyltransferase, or glucoamylase. But prominent effects of the antibiotic were seen on the production of glucan synthetase and laminarinase, which are most likely related to the metabolism of cell wall glycan. Because of the presence of a β-1,3-linkage in the validamycin A molecule, the extracellular production of enzyme was increased in the culture filtrate, while significantly decreased in the mycelium in the presence of validamycin A. For the glucan synthetase, the culture was performed for 4 or 6 days in the presence of various concentrations of validamycin A, and the effects of the antibiotic on the intracellular production of the enzyme were examined. As shown in Table 3.13, the production of the enzyme significantly increased even with 1 μg/mL of the antibiotic. And the ratio of the increase due to validamycin was maximal at 10 μg/mL of the antibiotic. At a higher dose than 10 μg/mL, the effect rather decreased. Later, the effect of validamycin A on the β-D-glucan-degrading enzymes from R. solani was studied (Uyeda et al., 1986). β-D-Glucosidase and β-1,3glucanase are thought to be the main enzymes involved in the degradation of laminarin. R. solani was cultured in the presence of various concentrations

TABLE 3.13 Effect of Validamycin A on the Intracellular Production of Glucan Synthetase Cultivation Time (day) 4

6

Validamycin A in the Medium (μg/mL)

Glucan Synthetase Activity

Relative Activity (%)

0

0.096

100.0

1

0.141

147.1

10

0.162

168.5

50

0.140

146.3

0

0.233

100.0

1

0.316

135.7

10

0.385

165.2

50

0.271

116.3

132

Validamycin and Its Derivatives

TABLE 3.14 Effect of Validamycin A on the Extracellular Production of β-D-Glucosidase Cultivation Time (day) 6

9

Validamycin A in the Medium (μg/mL)

β-D-Glucosidase Activity

Relative Activity (%)

0

2.11

100.0

1

2.12

100.5

10

3.25

158.6

50

3.23

153.1

0

2.06

100.0

1

2.35

114.1

10

3.17

153.9

50

3.50

169.9

of validamycin A and then the enzyme activities in the filtrates were assayed. The results are shown in Table 3.14. It was found that the production of the β-glucosidase remarkably increased with more than 10 μg/mL of validamycin A. The ratio of increase was about 60% in this case, but in another instance the enzyme production in the culture with validamycin A (10 μg/mL) increased by more than 100%, compared with that in the control culture. To assay only the β-1,3-glucanase activity, it was necessary to perform the assay with pachyman as the substrate with inhibition of the β-D-glucosidase activity. Among various metal ions and inhibitors, Hg21, DFP, and chlorhexidine markedly inhibited the β-D-glucosidase activity. The inhibition by chlorhexidine was distinguishable from that of the others in the case of salicin as the substrate. Hereafter, the β-1,3-glucanase assay was performed with pachyman as the substrate in the presence of 250 μg of chlorhexidine. As a result, it was clarified that the extracellular production of β-1,3-glucanase slightly decreased in the presence of validamycin A. The ratio of decrease was less than 20%. Fig. 3.8 shows the time course changes of the intracellular production of β-D-glucan-degrading enzymes. In the control culture, the enzymatic activities rose until 8 days culture regardless of the kinds of substrates. While, in the cultures with validamycin, all the activities markedly decreased after 2 days culture. These results clarified that the intracellular production of β-D-glucosidase decreased in the presence of validamycin A. The ratio of decrease was as much as 70 80%. Furthermore, the intracellular production of β-D-glucan-degrading enzymes other than β-D-glucosidase may decrease in the presence of validamycin A. With regard to β-1,3-glucanase, the assay was

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133

β-D-Glucan-degrading enzyme activity (OD AT 600 nm/mg-protein)

20

10

0

2

4 6 Cultivation time (days)

8

10

FIGURE 3.8 Time course changes of the intracellular production of β-D-glucan-degrading enzymes. Enzymatic activities in a culture without (open symbols) or with (closed symbols) 10 μg/ mL of validamycin A were assayed with pachyman (x and K), laminarin (Δ and ▲), and salicin (& and ’) as substrates, respectively.

performed in the same way as with the culture filtrate. The effect of validamycin A on the intracellular production of β-D-glucan-degrading enzymes (with pachyman as the substrate) was not noticeably influenced by chlorhexidine. From the results, the intracellular production of β-1,3-glucanase was confirmed to decrease remarkably in the presence of validamycin A. Asano et al. (1987) studied the effect of validamycins on the glucosidases. No significant activity was exhibited against cellulase, pectinase, chitinase, α-amylase, or α- and β-glucosidases except trehalase from R. solani. Validoxylamine A exhibited the greatest inhibitory activity against trehalase. Validoxylamine A inhibited trehalase in a competitive manner with a Ki value of 1.9 3 1029 mol/L. The Ki value was about 1026 times smaller than the Km value (9.1 3 1024 mol/L) for trehalose. However, the attachment of the D-glucosyl residue to validoxylamine A diminished the inhibitory activity against trehalase. The pseudotetrasaccharides, such as validamycins C, E, and F, had no inhibitory effect on trehalase. The effect of validamycin A on several extracellular enzymes in the supernatant of stationary phase cultures of R. solani, including filter paper (FPase), carboxymethylcellulase (CMCase), xylanase, polygalacturonase (PGase), and cellobiase, was investigated by Robson et al. (1989). The results were shown in Table 3.15. 1 μmol/L-validamycin A caused

134

Validamycin and Its Derivatives

TABLE 3.15 Effect of Validamycin A on the Production of Some Extracellular Enzymes of R. solani A79 Enzyme

Enzymatic Activity (nkat/mg dry weight) Validamycin A Concentration in Medium (μmol/L) 0

1

FPase

0.13 6 0.01

CMCase

25

100

0.03 6 ,0.01

a

0.03 6 ,0.01

0.02 6 ,0.01a

1.13 6 0.12

0.03 6 ,0.01a

0.11 6 ,0.05a

0.02 6 ,0.01a

Cellobiase

0.83 6 0.04

0.22 6 0.02

0.17 6 ,0.08

0.12 6 0.02a

Xylanase

6.35 6 0.12

6.69 6 0.39

6.57 6 0.37

6.97 6 0.37

PGase

0.39 6 0.03

0.30 6 0.03

0.27 6 0.04

0.29 6 0.03

a

a

a

Significantly (using the t-test, P , 0.05) different from control lacking validamycin A.

a

significant (P , 0.05) reductions in the extracellular activities of FPase (a 77% reduction compared with the control), CMCase (97% reduction), and cellobiase (73% reduction), but had no significant (P . 0.05) effect on the xylanase or PGase activities of the culture supernatants. No further reduction in FPase or CMCase activity was observed when the validamycin A concentration was increased to 100 μmol/L, but a further significant (P , 0.05) decrease in cellobiase activity occurred at a validamycin A concentration of 100 μmol/L.

3.1.2.2 Induction of Validamycins for Endochitinase and β-1,3-Glucanase in Rice Plants The trehalase, endochitinase, and endo-β-1,3-glucanase in rice plants are very important enzymes for resisting pathogenicity. Zhang et al. (2003) investigated the effect of validamycin A on these three enzymes in rice plants. The results are shown in Table 3.16. Activity of trehalase in rice plants treated with 50 μg/mL validamycin A was significantly lower than that of the control with 14 days and recovered normal after 21 days. Activity of endochitinase treated by validamycin A was significantly higher compared with the control until the 21st day; moreover, the activity of endo-β-1,3-glucanase reached the highest level at the 3rd day after validamycin A spraying on the plants, then declined gradually. The result showed that there was high activity of endochitinase and β-1,3glucanase, and low activity of trehalase between treated and untreated rice at the same time, which was identical with control effect against rice sheath blight in the greenhouse. This finding provided a clue to reveal the

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135

TABLE 3.16 Comparative Change Rates of Three Enzymes with Efficacy Against R. solani After Validamycin A Spraying Days After Treatment

Trehalase

Endochitinase

Endo-β-1,3glucanase

Efficacy

3

299.770

123.64

155.88

179.22

7

229.420

151.02

11.92

181.72

14

269.490

117.19

117.54

170.31

21

20.005

116.39

13.92

113.27

mechanisms of validamycin A and realization of the new action mode for the antibiotic.

3.1.3 Antifungal Activity Against Fusarium oxysporum Soilborne diseases are important biotic constraints in sustainable crop production systems because the complexity of the soil environment makes their control with chemical fungicides difficult. Fusarium vascular wilt is a soilborne disease caused by F. oxysporum. The disease could be effectively controlled by a foliar spray of validamycin A or validoxylamine A. Validamycin A and validoxylamine A at 100 μg/mL were used to control Fusarium wilt of tomato by foliar spray in a pot test or large-scale pot experiment. The foliar spray of validamycin A or validoxylamine A controlled .80% of the internal symptoms of the disease when F. oxysporum was inoculated 7 or 21 days after treatment (Ishikawa et al., 2005). However, they lack anti-F. oxysporum activity in vitro and, thus, may work as plant activators on tomato because they have several qualities characteristic of systemic acquired resistance (SAR) inducers, i.e., they have no fungicidal activity against the pathogens (Table 3.17); they induce a broad range of disease resistance (Table 3.18); the resistance lasts for a long period; they induce several SAR molecular markers, such as SA accumulation and PR gene expression (Figs. 3.9 and 3.10); and they need a time lag between treatment and expression of efficacy (Table 3.19). A foliar spray of validamycin A or validoxylamine A on tomato seedlings in a nursery might be effective for the control of Fusarium wilt after the seedlings were transplanted into infested field soil. For disease control throughout the production cycle of tomato, a few additional sprays might be needed in the field, or validamycin A could be used in combination with other disease control measures in an integrated pest management program. The foliar spray of validamycin A and validoxylamine A represents a novel,

136

Validamycin and Its Derivatives

TABLE 3.17 Effect of Validamycin A or Validoxylamine A on Colony Diameter of F. oxysporuma Medium

Control

Validamycin

Validoxylamine A

Mycelial Growth (mm)b

Mycelial Growth (mm)

Inhibition (%)

Mycelial Growth (mm)

Inhibition (%)

PDA

39.0

36.7 ns

5.4

36.6 ns

5.8

Water agar

29.6

28.9 ns

2.3

28.3 ns

3.9

Czapektrehalosec

39.9

37.8 ns

5.7

36.5 ns

8.3

Czapeksucrosec

41.9

40.4 ns

3.3

39.2 ns

6.3

Czapekglucosec

39.5

39.8 ns

2 0.6

39.9 ns

20.97

Validamycin or validoxylamine A at 100 µg/mL; ns indicates that, for each medium, growth was not significantly different from the control according to Tukey’s honestly significant difference (P , 0.05). b Inoculation for 4 days at 28℃. The diameter of the mycelial mat is the average obtained from six replicates. The experiments were repeated three times with similar results. Data represent means of three separate experiments. c 0.1% (wt/vol) carbohydrate. a

convenient, and environmentally sound way to control soilborne diseases. By using validamycin A and validoxylamine A, it may become possible to reduce the use of more toxic pesticides.

3.1.4 Antibacterial Activity Against Pseudomonas solanacearum Validamycin A was effective to control tomato bacterial wilt caused by P. solanacearum (Ishikawa et al., 1996). After 7 days of incubation at 30 C, validamycin A inhibited bacterial multiplication only in media containing trehalose as the sole carbohydrate (Fig. 3.11). It did not inhibit the multiplication of P. solanacearum in media containing mannitol, glucose, mannose, inositol, sucrose, pyruvic acid, glycerol, or galactose as the sole carbohydrate. In liquid media containing trehalose as the sole carbohydrate, validamycin A strongly inhibited the multiplication of bacteria at concentration of 0.02 μg/mL and above. Direct injection of the validamycin A paste into stems 10 cm above the soil 7 days before inoculation inhibited wilting symptoms until 12 days after inoculation in greenhouse pot tests. Injecting the paste below the first

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137

TABLE 3.18 Control of Late Blight of Tomato by Foliar Spray of Validamycin A Treatment

Powdery Mildewa Disease Area (%)

Mock

9.44

Validamycin A (10 μg/mL)

0.0

Protection (%)

Late Blightb Disease Area (%)

Protection (%)

67.1 100

Validamycin A (100 μg/mL)

22.6

65.5

Validoxylamine A (100 μg/mL)

17.9

72.9

Trehalose (100 μg/mL)

65.9

0.9

a Tomato plants (cv. Odoriko) were sprayed to run off with validamycin A solution twice (14 and 28 days after seeding). Six plants with three replicates were used. Powdery mildew caused by Oidium spp. occurred by natural infection. The visible disease area with symptoms was rated from 0% to 100% in 5% increments 10 days after inoculation, the ratio of the diseased area to healthy area on the third to eighth leaves was recorded 64 days after seeding, and the average ratio of diseased area was calculated. b Seven days after a spray of validamycin A on 28-day-old tomato plants (cv. Odoriko) to run off, one drop of sporangium suspension (1 3 104 sporangia/mL) of Phytophthora infestans was placed on the first leaf. Inoculated tomato plants then were grown at 20℃ in a high-humidity chamber for 14 days. The ratio of diseased area to the total leaf area was estimated from 0% to 100% in 5% increments. In each plot, at least eight plants were used. The experiments were repeated three times with similar results.

inflorescence 7 days before inoculation inhibited disease development throughout the course of the experiment. After 20 days, 91.7% of the control plants had wilted, as opposed to 33.3% of those treated above the soil and only 25.0% of those treated below the first inflorescence (Fig. 3.12). Both validamycin A treatments by direct injection into the stem, either near (at 10 cm above the soil line) or far from the soil line (below the first inflorescence), were effective in delaying and reducing tomato bacterial wilt. In field tests, wilting occurred by 7 days after inoculation in the nontreated plot. The disease severity of nontreated control tomato plants at 15 and 23 days was 56% and 90%, respectively (Fig. 3.13). Almost all the plants died by 28 days. Foliar spraying with validamycin A at 250 μg/mL and 500 μg/mL resulted in wilted tomato plants by 20 days after inoculation. By 28 days, disease severity in the validamycin A treated plot at 250 μg/mL was 51% and at 500 μg/mL was 45%. Validamycin A delayed the appearance of disease symptoms for about 10 14 days.

138

Validamycin and Its Derivatives

Salicylic acid (µg/g fw)

0.6

VMA (100 µg/mL) VAA (100 µg/mL) Water

0.5 0.4 0.3 0.2 0.1 0 0

2

4

6 8 10 Days after treatment

12

14

16

FIGURE 3.9 Accumulation of total salicylic acid in tomato leaves sprayed with validamycin A (VMA) or validoxylamine A (VAA). Tomato plants (cv. Odoriko) were sprayed with VMA or VAA at 100 μg/mL 28 days after seeding. Leaves (1 2 g) were boiled for 10 min after addition of 103 volume of 2% acetic acid (pH 2.7). Three samples from three plants each were used for salicylic acid quantification. Free salicylic acid was detected with a UV detector at 245 nm. It was not detected in water-sprayed tomato leaves. The experiments were repeated three times with similar results.

H2O

VMA VAA

ASM

P4(PR-1) Tag(PR-2) Tos(PR-5) Total RNA FIGURE 3.10 RNA blot analysis of systemic acquired resistance (SAR) marker gene expression in tomato leaves treated with validamycin A (VMA) or validoxylamine A (VAA). Leaves of tomato plants (cv. Odoriko) were sprayed with H2O, VMA, or VAA at 100 μg/mL, or acibenzolar-S-methyl (ASM) at 125 μg/mL. Leaves were collected 7 days after application. Each lane was loaded with 2 μg of total RNA. The experiments were repeated three times with similar results.

In the hydroponics culture test, all the nontreated Oogatafukuju plants had wilted after 9 days (Table 3.20). On the other hand, only 25% of the plants in the validamycin A-treated plot had wilted after 12 days. By 12 days, 83% of the nontreated patio cultivar had wilted. Validamycin A completely inhibited wilting until 8 days when only 8% of the plants had wilted. No further increase in wilting was observed. Validamycin A was proven to be effective against tomato bacterial wilt when applied in foliar sprays, which are so far a more efficient and more feasible method of elimination soil complications in control.

Bioactivities of Validamycins and Related Natural Compounds Chapter | 3

139

TABLE 3.19 Control of Tomato Powdery Mildew Days After a Foliar Spray of Validamycin A Chemical, DBIa Mock

Disease Area (%)

Protection (%)

84.2

Validamycin A 7

3.4

96.0

5

6.3

92.5

4

3.8

95.4

3

51.5

38.8

2

54.3

35.5

1

70.0

16.8

7

56.5

32.9

5

77.3

8.1

2

70.2

16.6

1

77.9

7.5

Trehalose

a Tomato plants (cv. Odoriko), 28 35 days old, were sprayed with validamycin A or trehalose at 100 µg/mL or with water 1 7 days before inoculation (DBI) with conidia of Oidium spp. The disease area of the leaf was estimated from the symptom of disease on the first to fifth leaves in 5% increments 10 days after inoculation. In each treatment, at least eight plants were used.

FIGURE 3.11 Antibacterial activity of validamycin A against P. solanacearum embedded in Czapek agar medium containing trehalose as the sole carbohydrate. A paper disk contains about 30 μL of validamycin A solution. (A) Validamycin A 500 μg/mL; (B) 250 μg/mL; (C) 125 μg/mL; (D) 62.5 μg/mL.

140

Validamycin and Its Derivatives

Wilted plants (%)

100

Above the soil line

90

Below the first inflorescence

80

Nontreated

70 60 50 40 30 20 10 0

00

2

4

6 8 10 12 14 16 Days after inoculation

18

20

FIGURE 3.12 Effect of validamycin A against tomato bacterial wilt in greenhouse pot tests. A tomato stem was pierced with a minidrill to make a hole into which 0.2 g validamycin A paste (5% validamycin A, 70% CaCO3, 25% water) was syringed. Validamycin A paste was applied to nine tomato stems with two replicates either 10 cm above the soil or just below the first inflorescence (at 30 cm above the soil line) 7 days before the soil was drenched with a 500-mL bacterial suspension (1 3 108 cfu/mL).

100 VM-A (250 µg/mL)

90

VM-A (500 µg/mL)

Disease severity (%)

80

Nontreated

70 60 50 40 30 20 10 0 0

5

10 15 20 Days after inoculation

25

30

FIGURE 3.13 Control of tomato bacterial wilt by foliar sprays of validamycin A in field tests.

141

Bioactivities of Validamycins and Related Natural Compounds Chapter | 3

TABLE 3.20 Efficacy of Validamycin A Against Tomato Bacterial Wilt in Hydroponics Culture Cultivar

Validamycin A Treatment

Oogatafukuju

Patio

Wilted Tomato Plants (%) DAI

a

6

7

8

9

12

1

12.5

12.5

12.5

12.5

25.0

2

62.5

87.5

87.5

100.0

100.0

1

0.0

0.0

8.3

8.3

8.3

2

16.6

41.6

58.3

66.7

83.3

a

DAI means days after inoculation.

Diseased area (%)

30 VMA 15.6 µg/mL VMA 62.5 µg/mL VMA 250 µg/mL Control

20

10

0

0

4 8 Days after inoculation

12

FIGURE 3.14 Control of cabbage black rot by spraying validamycin A in a pot test.

3.1.5 Antibacterial Activity Against Xanthomonas campestris pv. campestris Black rot caused by X. campestris pv. campestris is one of the most serious diseases affecting crucifers. Foliar sprays of validamycin A controlled cabbage black rot effectively in pot and field trials (Ishikawa et al., 2004). In the pot test, a foliar spray application of validamycin A at 15.6 μg/mL controlled cabbage black rot caused by X. campestris pv. campestris (Fig. 3.14). The spraying of validamycin A at 62.5 or 250 μg/mL reduced the diseased area to 16% compared to the water spray 11 days after treatment. Validamycin A at each concentration exhibited no phytotoxicity to cabbages. In the field trial, disease severity and the proportion of diseased plants in the untreated control plot were 12.2% and 25.2%, respectively, due to a low disease pressure. Four spray applications of validamycin A at

142

Validamycin and Its Derivatives

62.5 μg/mL controlled 40% of cabbage black rot caused by natural infection in terms of disease severity and proportion of infected plants (Table 3.21). Validamycin A at 62.5 μg/mL exhibited no phytotoxicity to cabbage plants in the field trial. In cabbage leaves, validamycin A spray inhibited the proliferation of X. campestris pv. campestris multiplication, and might reduce extracellular polysaccharide production and virulence. These results indicate the potential efficacy of validamycin A against cabbage black rot.

3.1.6 Antimicrobial Activity Against Candida albicans More interestingly, validamycin A has been successfully applied in the fight against a phytopathogenic fungus, the prevalent human pathogen C. albicans (Guirao-Abad Jose et al., 2013). The MIC50 was 100 mg/L. Notably, this compound also caused a significant reduction in cytosolic neutral trehalase activity (Ntc1p) in C. albicans. This observation suggests that either validamycin A per se or its aglycone fraction (validoxylamine A) is able to cross the cell wall and plasma membrane in order to inhibit cytosolic hydrolases, although more experiments on specific transport are required to confirm this hypothesis. Although validamycin A cannot presently be considered as a suitable clinical antifungal, it is likely to be a promising substrate in the design of new compounds directed against the trehalose metabolism pathway as an antifungal target. This potential should promote future research into the development and testing of new drugs to more effectively combat life-threatening systemic infections caused by C. albicans.

3.1.7 Antimicrobial Activity Against Other Microorganisms Validamycins A and B, at 10,000 μg/mL, did not inhibit the growths of bacteria and fungi (Iwasa et al., 1971b), including Bacillus subtilis, Sarcina lutea, Staphylococcus aureus, Proteus vulgaris, Escherichia coli, Xanthomonas oryzae, Mycobacterium ATCC 607, Mycobacterium avium, Pellicularia sasakii, Pyricularia oryzae, Rhizoctonia solani, Colletotrichum lagenarium, Aspergillus niger, Alternaria kikuchiana, Penicillium chrysogenum, Saccharomyces cerevisiae, and Trichophyton mentagrophytes. In addition, 2177 strains of fungi and 762 strains of bacteria were not inhibited by the antibiotics under various assay conditions. Later, Robson et al. investigated the sensitivity of fungi to validamycin A. Various fungi were screened for sensitivity to validamycin A and those sensitive to the antibiotic are listed in Table 3.22. All the Basidiomycotina (and Deuteromycotina with a perfect basidiomycete stage) were sensitive to validamycin A, although the percentage reduction in colony diameter varied from only 12% for Phlebia rufa to 77% for Bjerkandera adusta. With the exception

TABLE 3.21 Control of Cabbage Black Rot by Validamycin A in a Field Trial Treatment

DSb

No. of Plants

% Control

DIa 0 Validamycin A

1

2

Diseased Plants (%)

% Control

3

51.0

5.3

2.0

1.0

6.9

45.7

10.3

3.0

2.0

12.2

43.4

14.0

44.4

62.5 μg/mL Untreated

25.2

Control a

DI: Disease index, which was recorded 80 days after transplantation according to the following: 0, no lesion; 1, 1 5% of area damaged in relation to the whole plant; 2, 6 20%; 3, .20%. DS: Disease severity.

b

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Validamycin and Its Derivatives

TABLE 3.22 Fungi Sensitive to Validamycin Aa Organismb

Colony Diameter of Treated Fungi (% of Control)

Organism

Colony Diameter of Treated Fungi (% of Control)

Bjerkandera adusta

23

Lentinus edodes

48

Pleurotus ostreatus

24

Stereum subtomentosum

62

Rhizoctonia cerealis c

28

Chaetomium globosumd

63

Coprinus cinereus

28

Agaricus silvicola

63

Flammulina velutipes

28

Hericium coralloides

66

Rhizoctonia solani A79 c

28

Phlebia gigantia

70

Lepiota procera

31

Phanerochaete chrysosporium

72

Chaetomium bostrychoidesd

38

Stereum hirsutum

77

Fusarium culmorumc

39

Entoloma arbortivus

78

Fistulina hepatica

40

Phlebia radiate

84

Schizophylum commune

42

Heterobasidion annosum

86

Daedalia quercina

47

Phlebia rufa

88

Pseudotrametes gibbosa

48

a Fungi were grown at 25℃ on Vogel’s (fungi other than Basidiomycotina) or modified Vogel’s (Basidiomycotina) media containing 5 mmol/L-glucose in the presence and absence of 25 µmol/L validamycin A. The values are expressed as a percentage of the diameter of colonies grown in the absence of validamycin A. b All fungi are Basidiomycotina except for a, Deuteromycotina and b, Ascomycotina. c Deuteromycotina d Ascomycotina.

of Fusarium culmorum, Chaetomium globosum, and Chaetomium bostrychoides, all Ascomycotina (and Deuteromycotina with a perfect ascomycete stage), Oocyectes and Mucorales tested were insensitive to validamycin A. Valienamine showed antibiotic activity against Bacillus sp., such as B. subtilis and B. cereus, on bouillon medium by the cylinder agar plate method. The results are shown in Table 3.23. However, the activity was not

Bioactivities of Validamycins and Related Natural Compounds Chapter | 3

145

TABLE 3.23 Antibiotic Activity of Valienamine Against B. subtilis by the Cylinder Agar Plate Method Concentration of Valienamine (mg/mL)

Diameter of Inhibition Zone (mm)

16

20

8

16

4

12

TABLE 3.24 Reversal of Valienamine Inhibition by Various Sugars Sugar (1 mg/mL)

Diameter of Inhibition Zone (mm)a

None

19

D-Glucose

0

D-Fructose

0

D-Galactose

Maltose

19 (13)b

Cellobiose

14

Lactose

20

Sucrose

(12)b

D-Mannitol D-Sorbitol

0 (16)b

a

Valienamine, 10 mg/mL. Inhibited incompletely.

b

observed with D-glucose, D-fructose, D-mannitol, and D-glucosamine. The addition of D-galactose and lactose had no effect on the activity, and that of maltose, sucrose, cellobiose, and D-sorbitol had some effects, as shown in Table 3.24. This phenomenon may be due to the antagonism of inhibition on the sugar metabolism.

3.2 ENZYME INHIBITORY ACTIVITIES 3.2.1 Inhibitory Activity Against Trehalase Since the validoxylamines and validamycins are structurally similar to trehalose, they have good inhibitory activity for α, α-trehalase (E.C. 3.2.1.28).

146

Validamycin and Its Derivatives

This enzyme is widely spread among animals, plants, insects, and microorganisms. And its important role becomes particularly apparent in the biological regulation of such mechanisms as the active transport of glucose into intestines, reserve supply of energy, germination of spores, etc. The inhibitory activity of validoxylamines on and inhibitory constants of validoxylamine A for various trehalases are shown in Table 3.25 and Table 3.26, respectively. The crude trehalases of porcine intestine, rat intestine, rabbit kidney, baker’s yeast, Mycobacterium smegmatis, and insect (Spodoptera litura) were employed in the test. Two dihydrovalidoxylamines A of D-gluco and L-ido configuration, which were obtained by the hydrogenation of validoxylamine A with Pt-H2, were used in the test, too. As shown in Table 3.25, the inhibitory effect of validoxylamines was found to be potent against the various trehalases, especially validoxylamine A and D-gluco-dihydrovalidoxylamine A. The inhibitory activity of competitive analogs can be expected to increase as the configurational structure approaches similarity to the substrate. This general proposition was confirmed in the test. However, D-gluco-validoxylamine A is less active than validoxylamine A and there is some difference in the activity between them. Generally, it may be seen that the double bond in validoxylamine A is not as essential to the activity as the configuration of the hydroxyl and hydroxymethyl groups. Lineweaver Burk plots showed competitive inhibition on each trehalase by validoxylamine A. The Ki values were found to be over 10,000 times smaller than the Kms, respectively, as shown in Table 3.26, and their affinities are the highest. On the other hand, the inhibitory activity of validamycins and validoxylamines against insect (S. litura) trehalase is shown in Table 3.27 and Fig. 3.15 (Asano et al., 1990b). All validamycins and validoxylamines, except validamycin C, showed a potent inhibitory activity against insect trehalase. Kinetic analysis with trehalose as the substrate indicated that all validamycins and validoxylamines were competitive inhibitors. Validoxylamine A was found to be the most potent inhibitor against the insect trehalase (Ki value: 4.3 3 10210 mol/L) in all tested compounds. The attachment of a hydroxyl group in C-5 or C-6 position of validoxylamine A (validoxylamine B or G) seemed to weaken the trehalase inhibitory activity. The introduction of D-glucose into C-4 or C-7 position of validoxylamine A (validamycin A or D), to the contrary, did not reduce the inhibitory activity so prominently, comparing with the results obtained with the trehalase in R. solani. Furthermore, the insect trehalase appeared to be very sensitive to validamycins E and F, while such pseudotetrasaccharides as validamycins C, E, and F had no inhibitory effect on the trehalase of R. solani. In contrast to the potent inhibition of trehalases, validoxylamine A showed no significant inhibition of other kinds of sugar hydrolases, such as cellulase,

TABLE 3.25 Inhibitory Activity of Validoxylamines on Various Trehalases Compound

IC50 (mol/L) Porcine Intestine 28

Rat Intestine

Rabbit Kidney

S.l. larva

4.9 3 10

1.1 3 10

3.0 3 10

1.1 3 10

4.8 3 1028

Validoxylamine B

3.4 3 1026

2.0 3 1026

4.0 3 1027

1.9 3 1026

2.6 3 1026

6.6 3 1026

Validoxylamine G

5.4 3 1026

3.0 3 1026

1.1 3 1026

3.2 3 1026

1.0 3 1026

5.9 3 1026

3.0 3 1028

3.1 3 1027

1.9 3 1028

7.4 3 1027

2.0 3 1027

5.3 3 1028

1.9 3 1024

1.0 3 1023

5.3 3 1024

8.4 3 1025

1.9 3 1025

8.4 3 1024

4.2 3 1027

2.4 3 1026

8.2 3 1029

7.4 3 1026

1.8 3 1026

3.7 3 1027

L-ido-Dihydrovalidoxylamine

Validamycin A

A

M.s.: Mycobacterium smegmatis, S.l.: Spodoptera litura.

29

M.s.

1.4 3 10

A

210

Baker’s Yeast

Validoxylamine A

D-gluco-Dihydrovalidoxylamine

29

28

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Validamycin and Its Derivatives

TABLE 3.26 Inhibition Constant of Validoxylamine A for Various Trehalases Trehalase Origin

Km (mol/L) 23

Ki (mol/L)

Porcine intestine

4.0 3 10

7.8 3 10210

Rat intestine

1.0 3 1022

3.1 3 1027

Rabbit kidney

1.3 3 1023

1.2 3 10210

Baker’s yeast

5.3 3 1023

2.7 3 10210

Mycobacterium smegmatis

24

9.0 3 10

4.9 3 1029

Spodoptera litura larva

2.0 3 1023

1.0 3 1029

TABLE 3.27 Inhibitory Activity of Validamycins and Validoxylamines Against Insect (S. litura) Trehalase Compound

IC50 (mol/L)

Ki (mol/L)

Validamycin A

27

3.7 3 10

4.7 3 1028

Validamycin B

1.8 3 1026

1.9 3 1027

Validamycin C

1.3 3 1024

2.8 3 1026

Validamycin D

27

2.6 3 10

3.2 3 1029

Validamycin E

1.2 3 1026

1.4 3 1027

Validamycin F

5.8 3 1026

3.3 3 1027

Validamycin G

7.9 3 1027

1.9 3 1027

Validoxylamine A

4.8 3 1028

4.3 3 10210

Validoxylamine B

6.6 3 1026

5.5 3 1028

Validoxylamine G

5.9 3 1026

1.2 3 1027

pectinase, chitinase, α-amylase, α-glucosidase, and β-glucosidase (Asano et al., 1987). Therefore, validoxylamine A appears to be specific for trehalases.

3.2.2 Inhibitory Activities Against D-Glucose Hydrolases The inhibitory activities (IC50s) against D-glucose hydrolases of valienamine were examined, and the results are listed in Table 3.28.

Bioactivities of Validamycins and Related Natural Compounds Chapter | 3

Validoxylamine A (8 × 10–9 M),

149

Validamycin A

none: Km =1.4 × 10–3 M.

(2 × 10–7 M), 12 10

1/V

8 6 4 2 0 0

0.2

0.4

0.6

0.8

1.0

1.2

–1

1/S (mM)

FIGURE 3.15 Lineweaver Burk plot of Spodoptera litura trehalase with validoxylamine A or validamycin A. Reprinted with permission courtesy of Japan Antibiotics Research Association (JARA).

TABLE 3.28 Inhibitory Activities of Valienamine Against Various Glucoside Hydrolases Enzyme (Origin)

Substrate

IC50 (mol/L)

α-Glucosidase (almond)

p-Nitrophenyl-α-glucoside

2.2 3 1023

α-Glucosidase (almond)

Maltose

8.8 3 1023

α-Glucosidase (yeast)

p-Nitrophenyl-α-glucoside

1.7 3 1024

α-Glucosidase (yeast)

Maltose

1.8 3 1025

Sucrase (yeast)

Sucrose

1.8 3 1023

Sucrase (porcine)

Sucrose

5.3 3 1025

α-Glucoamylase (Rhizopus sp.)

p-Nitrophenyl-α-glucoside

3.5 3 1024

α-Glucoamylase (Rhizopus sp.)

Starch

6.8 3 1023

α-Amylase (porcine)

Starch

Nonea

α-Amylase (sweet potato)

Starch

1.8 3 1022

α-Amylase (barley)

Starch

1.7 3 1022

Maltose

3.4 3 1024

Isomaltose

1.0 3 1023

Maltase (porcine) Isomaltase (porcine)

No inhibition was observed at a concentration of 2.0 3 1022 mol/L.

a

150

Validamycin and Its Derivatives

Different inhibitory activities against D-glucose hydrolases of valienamine, valiolamine, validamine, and hydroxyvalidamine were investigated by Kameda et al (Table 3.29). Among them, valiolamine with IC50 values in the range of 1026 to 1028 mol/L against porcine intestinal maltase, sucrose, and isomaltase was the most active. Valienamine was similar to validamine, while it was more active than hydroxyvalidamine and less active than valiolamine. To characterize the mechanism of inhibition by valienamine, validamine, and valiolamine, the inhibition kinetics were studied. Obtained from the calculation of the Lineweaver Burk plots, the kinetic constant (Km and Vmax) and the Ki values of valienamine, validamine, and valiolamine for inhibition of the activities of sucrose, maltase, glucoamylase, isomaltase, and trehalase are shown in Table 3.30. The apparent Ki values of valienamine, validamine, and valiolamine from sucrose, isomaltase, glucoamylase, maltase, and trehalase activities are 1027 to 1021 times smaller than the apparent Km values.

3.2.3 Inhibitory Activity Against Tyrosinase Tyrosinase (EC 1.14.18.1), which belongs to the type 3 copper protein family (Decker and Tuczek, 2000), is well known for its important role in the pathway of melanin biosynthesis because it catalyzes the first two reactions of the melanogenesis process, i.e., the hydroxylation of L-tyrosine to L-DOPA and the oxidation of L-DOPA to dopaquinone. As the reason of this function, tyrosinase is very important for insects to produce melanin, harden and stabilize the exoskeleton, and activate the immune response (Kramer et al., 2001; Suderman et al., 2006). Thus, finding a good inhibitor for tyrosinase would facilitate the control of insect growth. The inhibitory function of validamycin A, its kinetics, and its interaction with tyrosinase of mushroom were investigated by computational simulation (Wang et al., 2013). Validamycin A showed strong tyrosinase inhibition in a dose-dependent manner, with the half maximal inhibitory concentration (IC50) of 19.23 6 0.26 mmol/L. Tyrosinase was almost completely inactivated by 100 mmol/L validamycin A. When the assay was performed without validamycin A, the tyrosinase activity was nearly unaffected by the validamycin A in the preincubation step, indicating that validamycin A mediated tyrosinase inhibition was reversible. The inhibitory mechanism of validamycin A on tyrosinase activity was shown to be a competitive and noncompetitive mixed-type inhibitory mechanism, indicating that validamycin A can be combined with both free enzymes and enzyme substrate complexes. Comparing with other inhibitors that have the same inhibition mechanism, it is more effective than the fucoidan (IC50 5 11.5 mg/mL) (Wang et al., 2012) and isophthalic acid (IC50 5 40.9 mmol/L) (Si et al., 2011). While, it is less effective than o-toluic acid (IC50 5 2.56 mmol/L) (Huang et al., 2006), 5-methoxysalicylic acid (IC50 5 7.9 mmol/L) (Zhang et al., 2006), and arabinose (IC50 5 0.1 mmol/L) (Hu et al., 2012). With

TABLE 3.29 Inhibitory Activities of Valienamine and Its Related Aminocyclitols Against Various Glucose Hydrolases Enzyme (Origin)

Substrate

IC50 (mol/L) Valienamine 23

Valiolamine

Validamine

Cellobiose

8.8 3 10

8.1 3 10

1.5 3 10

7.4 3 1023

α-Glucosidase (yeast)

Maltose

1.8 3 1025

1.9 3 1024

5.8 3 1024

3.6 3 1024

Sucrase (yeast)

Sucrose

1.8 3 1023

.1.0 3 1022

.1.0 3 1022

.1.0 3 1022

Sucrase (porcine)

Sucrose

5.3 3 1025

4.9 3 1028

7.5 3 1026

4.2 3 1024

α-Glucoamylase (Rhizopus sp.)

Starch

6.8 3 1023

.1.0 3 1022

.1.0 3 1022

.1.0 3 1022

α-Amylase (porcine)

Starch

.1.0 3 1022

.1.0 3 1022

.1.0 3 1022

.1.0 3 1022

α-Amylase (sweet potato)

Starch

22

22

22

.1.0 3 1022

Maltase (porcine)

Maltose

3.4 3 1024

2.2 3 1026

1.1 3 1024

8.3 3 1023

Isomaltase (porcine)

Isomaltose

1.0 3 1023

2.7 3 1026

1.3 3 1024

.1.0 3 1022

.1.0 3 10

23

Hydroxyvalidamine

β-Glucosidase (almond)

.1.0 3 10

23

.1.0 3 10

TABLE 3.30 Kinetic Constants and Inhibitory Constants of Valienamine, Validamine, and Valiolamine on Activities of Carbohydrases in Rat Intestinal Brush Border Membranes Enzyme

Substrate

Km (mol/L)

Vmaxa

Ki Valienamine

3.4 3 10

85.6

3.0 3 10

3.2 3 10

3.2 3 1027

Maltase

Maltose

2.2 3 1023

237

9.6 3 1024

1.8 3 1024

2.9 3 1026

Soluble starch

7.7 3 1023b

119c

8.9 3 1024

1.6 3 1024

1.2 3 1026

Isomaltase

Isomaltose

5.7 3 1023

15.8

7.6 3 1024

8.8 3 1025

9.1 3 1027

Trehalase

Trehalose

1.1 3 1022

10.3

8.8 3 1024

2.7 3 1024

4.9 3 1025

None

None

Lactase a

Lactose

2.4 3 10

Micromoles of hydrolyzed substrate per milligram of protein per hour. Milligrams per milliliter. Micromoles of released glucose per milligram of protein per hour. d No inhibition was detected with 2.0 3 1023 mol/L pseudoaminosugars. b c

13.7

d

None

25

Valiolamine

Sucrose

22

24

Validamine

Sucrase

Glucoamylase

22

Bioactivities of Validamycins and Related Natural Compounds Chapter | 3

153

computational docking and simulation, it was predicted that validamycin A could bind directly with several residues in the active site of tyrosinase. The interacting residues HIS85, HIS244, GLU256, HIS259, and ASN260 were thought to be involved in the first stage of validamycin A binding.

3.3 INSECTICIDAL ACTIVITY OF VALIDOXYLAMINE A AND RELATED COMPOUNDS Injection of validoxylamine A into pupae induced a change in egg diapause determination (Takeda et al., 1988), and prevented glutinous material production (Takeda et al., 1990; Yao et al., 1991) in the silkworm, Bombyx mori. From in vivo experiments, 1-μg/pupa validoxylamine A injection inhibited 78% of trehalase activity in the colleterial gland arborescent region of the silkworm (Yao et al., 1991) and 10-μg/pupa validoxylamine A injection induced a total inhibition against ovary trehalase of the silkworm (Takeda et al., 1988). Therefore, 10-μg/larva validoxylamine A injection seems to give the strong inhibition against the trehalases in tissues and organs of the silkworm larva. Validoxylamine A also showed lethal activity caused by incomplete metamorphosis in certain lepidopteran species, B. mori (Kono et al., 1993), S. litura (Asano et al., 1990b; Kono et al., 1994b), Mamestra brassicae (Kono et al., 1994b, 1995), and Myzus persicae (Moriwaki et al., 2001). For B. mori, the lethal effect of validoxylamine A appeared at the later stages of the instar, especially wandering and spinning stages or later (Table 3.31). With 1-μg/larva injection of validoxylamine A, 20% of the treated larvae TABLE 3.31 Effect of Validoxylamine A on the Development of the Fifth Instar Silkworma Dose Injected (μg)

No. Treated

No. of Larvae Dead 1

2

3

4

5

6

No. of Cocoons Obtained

No. of Healthy Pupae

Days After Injection None

20

0

0

0

0

0

0

20

19

1

20

0

0

0

0

4

0

9

4

5

20

0

0

0

1

7

2

6

1

10

20

0

0

1

0

8

4

2

0

50

20

0

0

0

1

11

4

0

0

100

20

0

0

2

4

12

1

0

0

a

Validoxylamine A dissolved in 0.75% NaCl solution was injected into the body cavity of 1-day 5th instar larvae (10 males and 10 females).

154

Validamycin and Its Derivatives

were dead in larval stage, 35% of the larvae failed to spin the cocoon, and only 20% of the larvae succeeded in normal pupation. The lethal effect increased as the dosage of injection became higher, and consequently, no normal pupa were obtained at doses higher than 10 μg/larva and no cocoons at doses higher than 50 μg/larva. Characteristic symptoms observed in the later stages of the treated larvae were the extrusion of the midgut and hindgut from the anus and the failure of larval pupal molt. Effect of validoxylamine A injection (50 μg) upon the last instar larvae of S. litura was investigated (Asano et al., 1990b). The characteristic of abnormality in prepupae was the extrusion of hind- and midgut outside of the body after the cessation of feeding and death followed. Effect of injection of validoxylamine A relatives (10 μg/larva) on the morphogenesis of S. litura is shown in Table 3.32. Validoxylamine A is the most potent insecticidal compound among the validamycin complex. Validamycins A and D provided over 50% mortality, but validamycin A showed a lower adult ecdysis rate from normal pupae than validamycin D, although they had the same extent of inhibitory activity against trehalase in vitro. Validamycin C, which showed less activity (IC50 5 1.3 3 1024 mol/L) in vitro, exerted a fairly good activity in vivo. This seems to be due to the conversion of validamycin C into the potent insecticidal compounds in the insect body. The further attachment of the hydroxyl group to C-5 or C-6 position in validoxylamine A as

TABLE 3.32 Effect of Trehalase Inhibitor Injection (10 μg) to the Last Instar Larvae on the Later Development in S. litura Compound

Test Larvae

Death in Prepupae

Abnormal Pupaea

Normal Pupae

Adult Ecdysis/ Normal Pupae

Validamycin A

20

5

6

9

1/9

Validamycin B

20

0

0

20

17/20

Validamycin C

20

3

5

12

7/12

Validamycin D

20

9

4

7

5/7

Validamycin E

20

5

7

8

3/8

Validoxylamine A

20

14

6

0

Validoxylamine B

20

0

0

20

17/20

Validoxylamine G

20

2

2

16

14/16

None

20

2

0

18

16/18

a

No adult emerged from abnormal pupae.

155

Bioactivities of Validamycins and Related Natural Compounds Chapter | 3

seen in validoxylamines B and G and validamycins B and G had a marked effect on the efficacy of insecticidal activity. For M. brassicae (Kono et al., 1995), in 3 h after the injection of validoxylamine A (10 μg/insect) to the second day last instar larvae, their segmental muscle began to relax and the symptom sustained for 1 2 days. When the pulsation frequency of the dorsal vessel was counted 1 day after the injection, the frequency was 49.1 1 9.6/min (n 5 18), which was significantly less than that of the control larvae (60.4 1 5.7/min). The injected larvae deposited wet and sticky feces and some of them were attached together. The lethal effect of validoxylamine A injection appeared at the late stage of the larva. Eighty percent of 15 larvae became larval pupal intermediate (pupal cuticle was formed under the larval cuticle, but was not shed), 13% of the larvae partially shed the larval cuticle, and the remaining one larva pupated with the marginal part of the wing pad separated from the body cuticle. All of the treated individuals with abnormality died afterward. The insecticidal activity of validoxylamine A against insecticide susceptible (S) and resistant (R) strains of the green peach aphid, M. persicae, was investigated (Moriwaki et al., 2001). The leaf dipping method, two methods to determine the systemic activity using plants with roots (root soaking) and without roots (stalk-tip soaking), and the parafilm method, which allows the aphids to suck artificial diet solution containing validoxylamine A through the parafilm membrane, were employed for the tests. Mortality of first instar nymphs 2 days after treatment by leaf dipping method with 100 μg/mL validoxylamine A was about 10% in both S and R strains (Table 3.33). The stalk-tip soaking method, however, showed strong effects of 93.3% and 88.7% against S and R strains, respectively, at 100 μg/mL validoxylamine A (Table 3.34). At 20 μg/mL validoxylamine A in the root soaking method, 57.4% (S) and 49.9% (R) of female adults were dead within 7 days (Fig. 3.16). The production of nymphs by the treated female was strongly suppressed. By the parafilm method, 20 μg/mL validoxylamine A caused complete mortality in female adults within 5 days (Fig. 3.17).

TABLE 3.33 Insecticidal Activity of Validoxylamine A Against M. persicae by Leaf Dipping Method Chemicals

No. of Nymphs

No. Dead

Mortality (%)

S

R

S

R

S

R

Control

42

53

1

8

2.4

15.1

Validoxylamine A 100 μg/mL

40

54

6

6

15

11.1

a

a

Control: spreader diluted 5000-fold with water.

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Validamycin and Its Derivatives

TABLE 3.34 Insecticidal Activity of Validoxylamine A Against M. persicae by Systemic Method Chemicals

No. of Nymphs

No. Dead

Mortality (%)

S

R

S

R

S

R

Control

42

48

0

1

0

2.1

Validoxylamine A 100 μg/mL

45

53

42

47

93.3

88.7

a

a

Number of nymphs deposited

Control: spreader diluted 5000-fold with water.

8 7 6 5 4 3 2 1 0

(A)

a b

c

b

b

c 2

1

5

3

7

(B)

8 7 6 5 4 a 3 2 1 0

a b

a

b

1

a

Control 4 ppm 20 ppm 100 ppm

2 3 5 7 Days after VAA treatment

Days after VAA treatment

FIGURE 3.16 Number of nymphs deposited by a female adult by root soaking method. (A) S; (B) R. Values represent the mean 6 SE; Student’s t-test. a: P , 0.05, b: P , 0.01, c: P , 0.001.

Number of nymphs deposited

7 6

Control 4 ppm 20 ppm 100 ppm

5 4

a

3 2

b b

1 0

a

b

1

b b

3 4 2 5 6 Days after VAA treatment

7

FIGURE 3.17 Number of nymphs deposited by a female adult S strain by parafilm method. Values represent the mean 6 SE; Student’s t-test. a: P , 0.05, b: P , 0.01, c: P , 0.001.

Bioactivities of Validamycins and Related Natural Compounds Chapter | 3

157

Therefore, concentrations of validoxylamine A at the sucking site of the aphid were lower than that around the root where treatment occurred. For practical application of this chemical as insecticide, chemical modification is needed to improve the systemic characteristics. In P. americana, flight was drastically suppressed by an injection of validoxylamine A (Kono et al., 1994a). Trehalase is usually distributed in most tissues that require an energy source to generate a glycolytic flux. As shown in Table 3.35, when the cockroach was injected with 50 μg validoxylamine A/insect just after flight and kept under normal conditions for 1 day, it lost its ability to fly. All cockroaches treated with validoxylamine A 24 h previously, stopped flying within 1 2.5 min (,1.5 min on average). Their hemolymph trehalose was maintained at the elevated level even after flight. The result appeared to be due to the inhibition of muscle trehalase. Validoxylamine A injection of 5 and 50 μg/insect inhibited trehalase activity 74.0 6 8.2% (mean 6 SD, n 5 3) and 68.2 6 6.4% (n 5 3), respectively. A dose-dependent inhibition was not observed at these dose levels. The drastic effect of validoxylamine A on flight activity in the cockroach seems to depend on the specific mechanism that provides energy for flight. Furthermore, reproduction was strongly inhibited by validoxylamine A in P. americana (Kono et al., 1997) and L. migratoria (Tanaka et al., 1998). The effect of validoxylamine A (5 and 50 μg/insect) on oocyte development, deposition of oothecae, and hatchability in P. americana was examined for 14 days following the injection of validoxylamine A (Table 3.36). In the control groups of 10 females, 6 8 oothecae and 11 oothecae, respectively, were deposited during the first and second week.

TABLE 3.35 Effect of Validoxylamine A Injection on Flight Activity and Hemolymph Trehalose Concentration Category of Flight Activity Inactive

No. of Insect Used

Time of Flight (min) 1 day After Injection Mean 6 SD

Trehalose Concentrations (mmol/L) Immediately After Flight Mean 6 SD

1

,1

105.7

Active

4

1.3 6 0.5

94.1 6 15.5

Very active

3

1.5 6 0.9

77.2 6 37.3

Without Validoxylamine A Active

2

4.0 6 1.4

20.2 6 2.1

Very active

3

15.3 6 2.5

8.3 6 1.4

TABLE 3.36 Effect of Validoxylamine A on Oocyte Development and Ootheca Deposition in P. americana Dose (μg/insect)

No. of Females Treated

No. of Females Without Oothecaa

Control

10

8

First Week After Injection

Second Week After Injection

No. of Oothecae Deposited

No. of Oothecae Dented

Oocyte Developmentb

8

0

11 9

No. of Oothecae Deposited

No. of Oothecae Dented

Oocyte Developmentb

11

0

11 10

2

2

11 2

11 5

10

7

6

0

10

8

6

4

11 3 17

10

8

4

2

14 63 Died 1 50

10

8

6

4

11 2 15 63

10

7

6

3

11 2 63 23 Died 2

a

Numbers were checked at the beginning of the experiment. Ovaries were dissected to check the stage of oocyte development according to the criterion mentioned in the text at the end of the first or the second week.

b

Bioactivities of Validamycins and Related Natural Compounds Chapter | 3

159

Inhibitory effect of validoxylamine A on the oocyte development was also analyzed through the changes in hemolymph components, including vitellogenin, in the developmental cycle of oocyte of P. americana. Twenty five-day-old short-day mated females of L. migratoria were individually kept in small cages for 14 days at 30℃, and those that laid three eggpods during this period were injected with validoxylamine A or water on the day of the last oviposition. Locusts injected with water produced 6.4 eggpods on average during the 50-day observation period (Table 3.37). On the other hand, those injected with 50 and 100 μg of validoxylamine A deposited only 2.0 and 1.9 egg-pods on average, respectively. The differences in mean egg-pod production between validoxylamine A injected and water-injected locusts were statistically significant (Table 3.37). In either of the validoxylamine A injected groups, except for one individual that laid as many as 6 or 8 egg-pods, all locusts produced only 0 2 egg-pods. It appeared that most locusts could produce at least one egg-pod several days after injection of validoxylamine A, but the subsequent oviposition was suppressed strongly in most individuals. Eggs produced after validoxylamine A injection gave rise to hatchlings. Injection of 50 μg of validoxylamine A did not influence longevity significantly as compared with the results from water-injected controls. However, locusts injected with 100 μg of validoxylamine A lived significantly shorter than controls (Table 3.37). Validoxylamine A also affects all the major physiological processes responsible for oocyte development, including the juvenile hormone biosynthesis by the corpora allata, vitellogenin synthesis by the fat body, and incorporation of yolk material by the ovary in L. migratoria.

TABLE 3.37 Effects of Validoxylamine A on Ovipositing Activity and Longevity in Reproductively Active Females of L. migratoria at 30 C. Dose of Validoxylamine A

N

No. of Egg-Pods Produced

Longevity (days)b

(Mean 6 SD)

Range

(Mean 6 SD)

Range

7

6.4 6 1.0

5 8

37.7 6 6.4

33 50

50 μg

7

a

2.0 6 1.9

0 6

35.4 6 16.1

8 50

100 μg

7

1.9 6 2.7a

1 8

25.7 6 12.1a

19 50

0 (Water)

Females actively laying eggs were injected with 50 or 100 μg of validoxylamine A and observed for 50 days. A group of females was injected with water alone as controls. a Indicates that the value was significantly different from that of controls at 5% level by Mann Whitney U-test. b Included 1 surviving individual in each treatment that was assumed to have died on the last day of the observation period.

160

Validamycin and Its Derivatives

Recently, the inhibition of validamycin on trehalase activity of termite in vitro (Jin and Zheng, 2009) and in vivo (Odontotermes feae) (Tatun et al., 2014), and the mortality (Tatun et al., 2014), were investigated. Termites are the most efficient decomposers of cellulose by expressing the high cellulolytic activity from both endogenous and symbionts’ cellulases. Fungusgrowing termites are members of the Termitidae family, which has lost flagellates in their hindgut. Tokuda et al. (2005) reported that the fungusgrowing termite (Odontotermes formosanus Wasmann) showed only a trace of cellulase activity throughout the gut. It is implied that the fungus-growing termite does not have enough cellulase activity to survive on native cellulose because it shows only 15.3% and 5.2% of cellulase activity in the midgut and hindgut, respectively, compared with Coptotermes formosanus Shiraki (Tokuda et al., 2005), and had lower cellulase activity than other flagellatefree termites, which have different feeding habits (Li et al., 2013). It is of interest whether cellulolytic activities of fungus-growing termites fulfill their energy requirements. It has been reported that the carbon isotope ratios between termites and their symbiotic fungi have shown that almost all of the carbon assimilated by Odontotermes spp. derives from the fungi (Hyodo et al., 2003). Hence, nutrients derived from digestion of symbiotic fungal mycelia or nodules compensate for the shortage of energy produced from the breakdown of cellulose. Trehalose is widely distributed in fungi and it accumulates in both vegetative and reproductive stages (Thevelein, 1984); therefore, trehalose from fungi may be the primary source of energy for fungus-growing termites. The only enzyme known to be responsible for trehalose hydrolysis is trehalase. Trehalose metabolism is involved in many aspects of insect physiology; hence, any impairment of trehalase activity may affect metabolism, feeding, growth, and development. After the worker termites were fed with validamycin (0.3 g/mL), trehalase activity extracted from whole body dropped 50% compared with the termites fed with distilled water at 24 h posttreatment (Fig. 3.18A). Then trehalase activity decreased to a very low level from 2 to 3 days posttreatment. Moreover at 3 days after treatment, termites fed filter paper soaked with validamycin had died (89.7% mortality), while the control termites exhibited only 10% mortality (Fig. 3.18B). In addition, the samples were collected 3 days posttreatment and were then dissected to collect midgut, hindgut, and carcass, which were subjected to trehalase activity measurement (Fig. 3.18C). As described earlier, in the control groups, trehalase activity in midgut and hindgut were at high level (0.18 and 0.19 nmol/g protein/min, respectively), but trehalase activity extracted from midgut and hindgut of the termites fed with validamycin decreased by about fourfold compared with that of the control. In addition, trehalase activity in the carcass of termites fed with validamycin was about twofold lower than the control. The results revealed that validamycin compounds also had inhibitory activity on trehalase termite by mixing the compounds with the filter paper.

Bioactivities of Validamycins and Related Natural Compounds Chapter | 3

0.25

(B) Filter paper+DW 100 Filter paper+validamycin

0.20

80

Mortality (%)

Trehalase activity (nmol/µg protein/min)

(A)

0.15 0.10 0.05 0.00

161

Filter paper+DW Filter paper+validamycin

60 40 20

1 2 3 Days after validamycin application

0

Trehalase activity (nmol/µg protein/min)

(C) 0.25

Filter paper+DW Filter paper+validamycin

0.20 0.15 0.10 0.05 0.00 Midgut

Hindgut

Carcass

FIGURE 3.18 Trehalase activity in whole body homogenate of the worker termites, O. feae, after being fed on the filter paper soaked with validamycin (0.3 g/mL) and sterilized distilled water for 3 days (A), percentage mortality (B), and trehalase activity in midgut, hindgut, and carcass (C).

Trehalase activity decreased markedly to a very low level within 3 days, and interestingly, enzyme activity in the carcass of validamycin-ingested termites decreased about half of the control, indicating that validamycin was absorbed through the gut membrane to hemocoel and may be absorbed into other tissues including fat body, muscle, and epidermis. High mortality rate indicated that validamycin had insecticidal effect on fungus-growing termite by preventing the hydrolysis of trehalose, which caused lack of glucose, as described in many insects (Wegener et al., 2003). Taken together, validamycins are valuable tools for studying the role of trehalase enzyme in other termites and termite control. However, further experiments to examine the effects of validamycin on termites in the field are needed.

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Asano, N., Takeuchi, M., Kameda, Y., Matsui, K., Kono, Y., 1990b. Trehalase inhibitors, validoxylamine A and related compounds as insecticides. J. Antibiot. 43 (6), 722 726. Asano, N., Yamaguchi, T., Kameda, Y., Matsui, K., 1987. Effect of validamycins on glycohydrolases of Rhizoctonia solani. J. Antibiot. 40 (4), 526 532. Decker, H., Tuczek, F., 2000. Tyrosinase/catecholoxidase activity of hemocyanins: structural basis and molecular mechanism. Trend. Biochem. Sci. 25, 392 397. Endo, T., Matsuura, K., Wakae, O., 1983. Effect of validamycin A on infection of Rhizoctonia solani in rice sheaths. Nippon Shokubutsu Byori Gakkaiho 49 (5), 689 697. Guirao-Abad Jose, P., Sanchez-Fresneda, R., Valentin, E., Martinez-Esparza, M., Arguelles, J.-C., 2013. Analysis of validamycin as a potential antifungal compound against Candida albicans. Int. Microbiol. 16 (4), 217 225. Hu, W.J., Yan, L., Park, D., Jeong, H.O., Chung, H.Y., Yang, J.M., et al., 2012. Kinetic, structural and molecular docking studies on the inhibition of tyrosinase induced by arabinose. Int. J. Biol. Macromol. 50, 694 700. Huang, X.H., Chen, Q.X., Wang, Q., Song, K.K., Wang, J., Sha, L., et al., 2006. Inhibition of the activity of mushroom tyrosinase by alkylbenzoic acids. Food Chem. 94, 1 6. Hyodo, F., Tayasu, I., Inoue, T., Azuma, J.I., Kudo, T., Abe, T., 2003. Differential role of symbiotic fungi in lignin degradation and food provision for fungus-growing termites (Macrotermitinae: Isoptera). Funct. Ecol. 17, 186 193. Ishikawa, R., Fujimori, K., Matsuura, K., 1996. Antibacterial activity of Validamycin A against Pseudomonas solanacearum and its efficacy against tomato bacterial wilt. Jap. J. Phytopathol. 62 (5), 478 482. Ishikawa, R., Shirouzu, K., Nakashita, H., Lee, H.-Y., Motoyama, T., Yamaguchi, I., et al., 2005. Foliar spray of validamycin A or validoxylamine A controls tomato Fusarium wilt. Phytopathology 95 (10), 1209 1216. Ishikawa, R., Suzuki-Nishimito, M., Fukuchi, A., Matsuura, K., 2004. Effective control of cabbage black rot by validamycin A and its effect on extracellular polysaccharide-production of Xanthomonas campestris pv. campestris. J. Pestic. Sci. (Tokyo, Jpn.) 29 (3), 209 213. Iwasa, T., Higashide, E., Shibata, M., 1971a. Validamycins, new antibiotics. III. Bioassay methods for the determination of validamycin. J. Antibiot. 24 (2), 114 118. Iwasa, T., Higashide, E., Yamamoto, H., Shibata, M., 1971b. Validamycins, new antibiotics. II. Production and biological properties of validamycins A and B. J. Antibiot. 24 (2), 107 113. Jin, L.-Q., Zheng, Y.-G., 2009. Inhibitory effects of validamycin compounds on the termites trehalase. Pestic. Biochem. Physiol. 95 (1), 28 32. Kono, Y., Takahashi, M., Matsushita, K., Nishina, M., Kameda, Y., Hori, E., 1994a. Inhibition of flight in Periplaneta americana (Linn.) by a trehalase inhibitor, validoxylamine A. J. Insect. Physiol. 40 (6), 455 461. Kono, Y., Takahashi, M., Mihara, M., Matsushita, K., Nishina, M., Kameda, Y., 1997. Effect of a trehalase inhibitor, validoxylamine A, on oocyte development and ootheca formation in Periplaneta americana (Blattodea, Blattidae). Appl. Entomol. Zool. 32 (2), 293 301. Kono, Y., Takeda, S., Kameda, Y., 1994b. Lethal activity of a trehalase inhibitor, validoxylamine A, against Mamestra brassicae and Spodoptera litura. Nippon Noyaku Gakkaishi 19 (1), 39 42. Kono, Y., Takeda, S., Kameda, Y., Takahashi, M., Matsushita, K., Nishina, M., et al., 1993. Lethal activity of a trehalase inhibitor, validoxylamine A, and its influence on the blood sugar level in Bombyx mori (Lepidoptera: Bombycidae). Appl. Entomol. Zool. 28 (3), 379 386. Kono, Y., Takeda, S., Kameda, Y., Takahashi, M., Matsushita, K., Nishina, M., et al., 1995. NMR analysis of the effect of validoxylamine A, a trehalase inhibitor, on the larvae of the cabbage armyworm. Nippon Noyaku Gakkaishi 20 (1), 83 91.

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Kramer, K.J., Kanost, M.R., Hopkins, T.L., Jiang, H., Zhu, Y.C., Xu, R., et al., 2001. Oxidative conjugation of catechols with proteins in insect skeletal systems. Tetrahedron 57, 385 392. Li, M., Yang, Y., Yang, M., Zhou, E., 2010. Effects of jinggangmycin on cell wall degrading enzyme activity and soluble proteins of Rhizoctonia solani Kuhn. Huazhong Nongye Daxue Xuebao 29 (3), 272 276. Li, Z.Q., Liu, B.R., Zeng, W.H., Xiao, W.L., Li, Q.J., Zhong, J.H., 2013. Character of cellulase activity in the guts of flagellate-free termites with different feeding habits. J. Insect Sci. 13, 37 47. Moriwaki, N., Nishimori, T., Mori, Y., Kono, Y., 2001. Insecticidal activity of a trehalase inhibitor, validoxylamine A, against the green peach aphid, Myzus persicae. Nippon Oyo Dobutsu Konchu Gakkaishi 45 (3), 129 135. Nioh, T., Mizushima, S., 1974. Effect of validamycin on the growth and morphology of Pellicularia sasakii. J. Gen. Appl. Microbiol. 20 (6), 373 383. Robson, G.D., Kuhn, P.J., Trinci, A.P.J., 1989. Effect of validamycin A on the production of cellulase, xylanase and polygalacturonase by Rhizoctonia solani. J. Gen. Microbiol. 135 (10), 2709 2715. Shibata, M., Mori, K., Hamashima, M., 1982. Inhibition of hyphal extension factor formation by validamycin in Rhizoctonia solani. J. Antibiot. 35 (10), 1422 1423. Shibata, M., Uyeda, M., Mori, K., 1980. Reversal of validamycin inhibition by the hyphal extract of Rhizoctonia solani. J. Antibiot. (Tokyo) 33 (6), 679 681. Shibata, M., Uyeda, M., Mori, K., 1981. Stimulation of the extension of validamycin-inhibited hyphae by the hyphae extension factor present in Rhizoctonia solani. J. Antibiot. 34 (4), 447 451. Shigemoto, R., Okuno, T., Matsuura, K., 1989. Effect of validamycin A on the activity of trehalase of Rhizoctonia solani and several sclerotial fungi. Jan. J. Phytopath. 55 (2), 238 241. Si, Y.X., Yin, S.J., Park, D., Chung, H.Y., Yan, L., Lu¨, Z.R., et al., 2011. Tyrosinase inhibition by isophthalic acid: kinetics and computational simulation. Int. J. Biol. Macromol. 48, 700 704. Suderman, R.J., Dittmer, N.T., Kanost, M.R., Kramer, K.J., 2006. Model reactions for insect cuticle sclerotization: cross-linking of recombinant cuticular proteins upon their laccasecatalyzed oxidative conjugation with catechols. Insect Biochem. Mol. Biol. 36, 353 365. Takeda, S., Kono, Y., Kameda, Y., 1988. Induction of non-diapause eggs in Bombyx mori by a trehalase inhibitor. Entomol. Exp. Appl. 46 (46), 291 294. Takeda, S., Kono, Y., Kameda, Y., 1990. Non-glutinous egg production by using a trehalase inhibitor, validoxylamine A, in Bombyx mori. Nippon Sanshigaku Zasshi 59 (5), 360 365. Tanaka, S., Okuda, T., Hasekawa, E., Kono, Y., 1998. Suppression of oocyte development by a trehalase inhibitor, validoxylamine A, through inhibition of juvenile hormone biosynthesis and vitellogenesis in the migratory locust, Locusta migratoria L. Entomol. Sci. 1, 313 320. Tatun, N., Wangsantitham, O., Tungjitwitayakul, J., Sakurai, S., 2014. Trehalase activity in fungus-growing termite, Odontotermes feae (Isoptera: Termitideae) and inhibitory effect of validamycin. J. Econ. Entomol. 107 (3), 1224 1232. Thevelein, J.M., 1984. Regulation of trehalose mobilization in fungi. Microbiol. Rev. 48, 42 59. Tokuda, G., Lo, N., Watanabe, H., 2005. Marked variations in patterns of cellulase activity against crystalline-vs carboxymethyl-cellulose in the digestive systems of diverse, woodfeeding termites. Physiol. Entomol. 30, 372 380. Trinci, A.P.J., 1985. Effect of validamycin A and L-sorbose on the growth and morphology of Rhizoctonia cerealis and Rhizoctonia solani. Exp. Mycol. 9 (1), 20 27.

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Uyeda, M., Ikeda, A., Machimoto, T., Shibata, M., 1985. Effect of validamycin on production of some enzymes in Rhizoctonia solani. Agric. Biol. Chem. 49 (12), 3485 3491. Uyeda, M., Ikeda, A., Ogata, T., Shibata, M., 1986. Effect of validamycin on β-D-glucan-degrading enzymes from Rhizoctonia solani. Agric. Biol. Chem. 50 (7), 1885 1886. Uyeda, M., Suzuki, K., Tsuruta, H., Shibata, M., 1988. Effects of validamycin on glucan synthesis by cell-free extracts form Rhizoctonia solani. Agric. Biol. Chem. 52 (10), 2607 2608. Wang, Z.-J., Ji, S., Si, Y.-X., Yang, J.-M., Qian, G.-Y., Lee, J., et al., 2013. The effect of validamycin A on tyrosinase: inhibition kinetics and computational simulation. Int. J. Biol. Macromol. 55, 15 23. Wang, Z.J., Si, Y.X., Oh, S., Yang, J.M., Yin, S.J., Park, Y.D., et al., 2012. The effect of fucoidan on tyrosinase: computational molecular dynamics integrating inhibition kinetics. J. Biomol. Struct. Dynam. 30, 460 473. Wegener, G., Tschiedel, V., Scholder, P., Ando, O., 2003. The toxic and lethal effects of the trehalase inhibitor trehazolin in locust are caused by hypoglycemia. J. Exp. Biol 206, 1233 1240. Yao, X., Fugo, H., Takeda, S., 1991. Effect of validoxylamine A on the trehalase activity in the colleterial glands of the silkmoth, Bombyx mori. Nippon Sanshigaku Zasshi 60 (4), 296 301. Zhang, J.P., Chen, Q.X., Song, K.K., Xie, J.J., 2006. Inhibitory effects of salicylic acid family compounds on the diphenolase activity of mushroom tyrosinase. Food Chem. 95, 579 584. Zhang, S., Zhao, Q., Tang, W., Wolf, G.A., 2003. Influence of jinggangmycin A on the activities of resistance-related enzymes in rice. Zhiwu Baohu Xuebao 30 (2), 177 180.

Chapter 4

Chemical Synthesis of Validamycin and Related Natural Compounds Since the discovery of validamycins A and B, there have been a series of related compounds found in the fermentation broth, such as valienamine, valiolamine, validamine, hydroxyvalidamine, etc. Most of these new compounds have certain bioactivity, and can be applied in the agricultural and medical fields. However, there are some limitations of the quantity of these compounds in the fermentation broth. Therefore, chemical synthesis was taken into consideration by the chemist. By constant effort, the synthesis pathways of validamycin and these related active compounds were developed. Different kinds of chemicals were selected as the starting material to get the target compound, and the detailed methods were discussed as follows.

4.1 SYNTHESIS OF VALIENAMINE Valienamine was first found as a related compound of validamycin in 1972 by Horii Satoshi and Kameda Yukihiko (Kameda and Horii, 1972), and since then many synthetic efforts directed toward valienamine have been made by chemists all over the world. Some of these researches afforded the racemic form and the diastereoisomeric mixture, while others afforded the optically active compound. In this book, we mainly describe the optically active valienamine, (1)-valienamine (1) and β-valienamine (2) as shown in Fig. 4.1. On the basis of the different starting materials and reaction mechanisms, the synthesis of valienamine is divided into several methods as discussed in this chapter.

4.1.1 Synthesis From L-Quebrachitol Paulsen Hans et al. 1981 reported the synthesis of valienamine from L-Quebrachitol (3, 2-O-methyl-L-chiroinositol), which is a byproduct in the production of natural rubber. The synthesis route is shown in Scheme 4.1. To incorporate the side chain, 3 was converted with dimethoxypropane into the diisopropylidene compound 4, which could be catalytically oxidized with Validamycin and Its Derivatives. DOI: http://dx.doi.org/10.1016/B978-0-08-100999-4.00004-6 © 2017 Elsevier Ltd. All rights reserved.

165

166

Validamycin and Its Derivatives OH

OH OH

H 2N

OH

H 2N

OH

OH

OH

OH

1

2

FIG. 4.1 Chemical structures of (1)-valienamine (1) and β-valienamine (2). OH OMe

OH OMe OH OH O

3

O

OH

O

OR1 OR2 OBzl

BzlO BzlO

OR1

12: R1=R2=R3=H 13: R1=Bz,R2=R3=H 14: R1=Bz,R2=H,R3=Ms 15: R1=Bz,R2=R3=Ms

R1O 23

N3

OH OH

O O

O

O

BzlO BzlO

OBzl OR

O

RO

16: R=H 17: R=Ac

HO HO

OBzl OR O

18: R=H 19: R=Ac

OH

O

10: R=H 11: R=Bzl

9

RO

OBzl

O OR O

OH

OBzl OBzl

BzlO BzlO

O O OH

8

OR3

6

OH OH

O

O

O

5

O

7

O

O

O

OH OH OH

O

O

O

O

4

OH OH OMe

O

O OMe

OMe

O

OH

OH

O O

BzlO BzlO

OBzl OR2

R1O 20: R1=R2=Ac 21: R1=R2=H 22: R1=Bz, R2=H

NH2 Bz=-COC6H5 Bzl=-CH2C6H5

HO 1

SCHEME 4.1 Synthesis of valienamine from L-Quebranchitol.

ruthenium tetroxide/sodium periodate to the ketone 5 (with yield 63%). On the reaction with dimethyloxosulfonium methylide, 5 the epoxide 6 was stereoselectively formed (yield 57%). Then the reaction of 7 with the anion of the dithiane analogue N-methylthioformaldine (5-methylper-hydro-1,3,5dithiazine) afforded the product with the other (undesired) stereochemistry at the branching site. The ring-opening product 7 (89%) could be obtained by hydrolysis of 6 with aqueous alkali. Since the stereochemistry of the

Chemical Synthesis Chapter | 4

167

side chain in 7 was fixed, the methyl ether could now be cleaved with boron tribromide, and the completely deblocked product 8 was formed. The triisopropylidene compound 9 was yielded by the reaction of 8 with dimethoxypropane. Further selective hydrolysis of the trans-isopropylidene group led to the formation of 10, from which the tribenzyl ether 11 (75%) could be obtained. The benzoylation of the tetrol 12, obtained from 11 by acid hydrolysis, could be steered so that it selectively furnished the dibenzoate 13 (82%). The OH group at the branching site and the 6-OH group were considerably less reactive. On mesylation of 13, an inseparable mixture of monomesylate 14 and dimesylate 15 (Ms 5 CH3SO3) was obtained. After the treatment of this mixture with sodium ethoxide, it afforded a mixture of the epoxides 16 and 18, which could be isolated as the acetates 17 and 19. The double bond was then incorporated by reaction of 17 and 19 with sodium iodide, followed by an elimination reaction with phosphorus oxide trichloride, which led uniformly to the olefin 20 (69%). For the introduction of the amino group, after hydrolysis of 20 to 21, the side chain was protected by selective benzoylation to 22. It was possible to convert the free hydroxy group at the allyl position into an azido group with hydrazoic acid/triphenylphosphane/azodicarboxylate to give 23 (72%), which had the same stereochemistry of valienamine. The azido group in 23 was converted with triphenylphosphane into the phosphinimide, which could be hydrolyzed to obtain the amino group. In the end, cleavage of the benzyl ether protecting groups was achieved with sodium in liquid ammonia, and then valienamine 1 was isolated as the hydrochloride. Though there were more than 10 steps to get the final valienamine, this method succeeded in carrying out the first synthesis of valienamine with the correct stereoconfiguration (Chen et al., 2003).

4.1.2 Synthesis From D-Glucose D-Glucose is one of the most common carbohydrates and also an important raw material for the chemical industry. Considering the similar hexatomic ring to valienamine, D-Glucose is applied in synthesis work, and several pathways have been established to get optical active valienamine. This method involves different key steps, including a Ferrier rearrangement, an aldol-type cyclization of a nitrofuranose, an intramolecular HornerEmmons reaction, a ring-closing alkene metathesis, and an aldol condensation of a sulfone. The detailed synthetic routes were introduced in the following part. With the Ferrier rearrangement as the key step, several researchers have studied different synthetic routes: (1) Schmidt and Koehn (1987) reported a 15-step method (Scheme 4.2) with an overall yield of 5.06%. In this method, for the decisive diastereospecific allylic amination of an alkoxysubstituted cyctohexene, the (2,3)-sigmatropic

168

Validamycin and Its Derivatives OH

HO

NC

O O

40%

BzlO

BzlO X 73.1% BzlO

BzlO

HO OH

OBzl Y

OMe

EtS

X OBzl

79%

BzlO BzlO

26a: X = H, Y = OH 26b: X = OH, Y = H

58%

BzlO BzlO OBzl

OBzl 27

Y

OBz

78% EtS

Y

48.4%

26a: X = H, Y = OAc 26b: X = OAc, Y = H OBz

BzO

BzlO

X OBzl

25a: X = H, Y = OH 25b: X = OH, Y = H

24

BzlO

EtS

NH Tos

28

OR RO RO OR

NHR

1: R = H 30: R = Ac

SCHEME 4.2 Synthesis of valienamine from D-Glucose by Richard R. Schmidt and Arnim Kohn.

shift of a sulfimide group was chosen as the reaction step. The starting material is the cyclitol derivative 25 a,b, which could be obtained via Ferrier rearrangement as a 4:1 diastereomeric mixture in six simple steps from the commercially available methyl α-D-glucopyranoside (24, yield 40%). For introducing the C1 side-chain, and preparatory to the sigmatropic shift, the diastereomeric mixture was directly converted in an HCl-catalyzed reaction with ethanethiol into the thioketal derivative, which afforded 26 a,b (4:1 diastereomeric mixture) on reaction with trimethylsilyl cyanide and tin chloride as catalyst. Reduction of the nitrile group with diisobutylaluminum hydride, then further reduction with LiAlH4 and selective benzoylation with benzoyl cyanide (BzCN) afforded the cyclitol derivative 27 a,b. By regiospecific dehydration with triphenylphosphane/diethyl azodicarboxylate (DEAD), compound 28 was obtained. Imination of the thioether group with chloramine T in a two-phase process with benzyltriethylammonium chloride in dichloromethane afforded valienamine derivative 29 diastereospecifically. The protecting groups on 29 were removed with sodium in liquid ammonia to give valienamine 1, which was characterized by peracetylation to the known compound 30. (2) In 1988, Yoshikawa Masayuki et al. applied a substitution reaction for an acetoxyl group at the β-position of a nitro group to a synthesis of valienamine, with an overall yield of 26.82% from 31 (Scheme 4.3) (Yoshikawa et al., 1988). The aldol-type cyclization of a nitrofuranose was the key step in the synthesis. The reaction was started with the

169

Chemical Synthesis Chapter | 4 OH

BzO

NO2 OAc

AcO

BnO

OH

BzO 60%

NO2

OAc

60%

NHAc

AcO

BnO

BnO

32

BzO NHAc 91%

BnO

OAc 33

AcO

AcO

89%

NHAc

AcO

OAc

31

OH

BzO

HO

AcO

OAc

NHAc

AcO

92%

OAc

34

NH2

HO

HO

OH 1

30

SCHEME 4.3 Synthesis of valienamine from D-Glucose by Yoshikawa Masayuki.

treatment of a pseudonitrosugar (31) with liq. NH3 in tetrahydrofuran (THF) at 278 C for 2 h, and subsequent acetylation of the product yielded 32, a white powder. Compound 33 was obtained by the elimination of the nitro group in 32 with n-Bu3SnH (60%), and then the dehydration of 33 with SOCl2 in pyridine at 2 C for 15 min selectively gave 34 (89%). After removal of all the protecting groups in 34, the product was acetylated to furnish pentaacetylvalienamine (30, 91%), which was confirmed to be identical with the authentic sample by qualitative comparisons. Finally, optically active valienamine (1) was afforded by the deacetylation of 30 with 80% aq. NH2NH2 (in a sealed tube, yield 92%), and the structure was identical with the standard sample. (3) Nicotra et al. (1989) reported a brief synthesis route of valienamine with a total yield of 22%, using enone 35 as the starting material. The detailed reagents and conditions in Scheme 4.4 were concluded CH2OBn

OBn

O

Cl OBn OBn

OBn

81%

OBn OBn

38

CH2OBn

56%

d N3

CH2OAc

CH2OH

OBn

OBn

OAc

OH

f

e OBn

NH2

OH

NH2

OBn

OBn 39

OBn

OBn 36: R=H 37: R=Ac

35

OBn

c OBn

OBn

CH2OBn

79%

OBn

b

a

40

OAc

NHAc OAc

OH 1

SCHEME 4.4 Synthesis of valienamine from D-Glucose by Nicotra Francesco.

30

170

Validamycin and Its Derivatives

as follows: (a) benzyloxymethylmagnesium chloride; (b) thionyl chloride, reflux; (c) sodium azide; (d) hydrogen sulfide in pyridine-water; (e) sodium in liquid ammonia; (f) Ac2O, pyridine. With this method, the desired product was obtained with the five-step reaction. (4) Park and Danishefsky (1994) reported an interesting synthesis of valienamine and the exploration of the stereoelectronic factor by an SN2 reaction (Scheme 4.5). The readily available TIPS glucal 41 was OTIPS 74%

HO

PMBO

PMBO

PMBO

O

41

PMBO

PMBO

PMBO

PMBO

O

PMBO

OMe

PMBO

93%

OMe

O

PMBO

46

PMBO

47

48 OC(O)NHBn PMBO

PMBO OAc

OAc 52

OAc

OAc

49

50

51

O

CH2

PMBO

100%

98%

PMBO

PMBO

OC(O)NHBn

OMe

OH

92%

OMe

PMBO

97%

OAc

PMBO

PMBO

72%

O

OAc

O PMBO

94%

OMe

OAc

45

I

PMBO

76%

OAc

O

44 OTs

PMBO O

OH

43 OH

PMBO

OMe

O

42 OTIPS

76%

O

O

O

HO

OTIPS

OTIPS

OTIPS

O PMBO

PMBO

PMBO 45%

85% PMBO PMBO

NHBn

O

+

PMBO

PMBO

NHBn

O

NHBn

O

OH

53

O

O

54

55

O 56

OH O

OAc

PMBO PMBO AcO PMBO NHBn

O

PMBO NBn O

AcO NHAc OAc

O O 57

58

30

SCHEME 4.5 Synthesis of valienamine from D-Glucose by Tae Kyo Park and Samuel J.

Chemical Synthesis Chapter | 4

171

converted to the bis PMB derivative 42. The PMB derivative 42 was transformed to compound 45 through the formation of oxirane 43 and methyl glycoside 44. The oft-used feature of glycal methodology provided easy access to uniquely unprotected oxygens at C2 of a β-glucoside. Conversion of 45 to 49 was achieved by common chemical synthesis methods. From compound 49 to 50, a Ferrier transformation occurred, and the β-aldol thus was converted to 50 by mesylation and elimination. Compound 51 was given by the reduction of the ketone under Luche conditions, and readily converted to 52 through the action of benzylisocyanate. Cleavage of the acetate afforded 53, which was used to provide 54 by an oxidation reaction with PDC. Methylenation of 54 by a modified Lombardo reaction gave rise to 55. Epoxidation of 55 with mCPBA afforded a separable mixture of 56 and 57 with poor selectivity (56:57 5 1:2, 85% yield). Meanwhile, the availability was useful for probing the topography in the intramolecular SN2 reaction. In the presence of 18-C-6, the reaction of compound 57 with KHMDS directly gave rise to the desired valienamine derivative 58 (75% yield). In the last, pentaacetylvalienamine (30) was obtained by the acetylation of the deprotection product 58 (51% yield). Finally an overall yield of ,5.50% was obtained. However, further study found this reaction with spiroepoxide 56 was unsuccessful for the synthesis of 57, which rather reflected a strong stereoelectronic preference. Besides the Ferrier rearrangement, the intramolecular HornerEmmons reaction was also an important pathway to obtain valienamine from D-glucose. This reaction started with tetra-O-benzyl-D-glucono-1,5-lactone, which was readily available from D-glucose, and was carried out by Fukase Hiroshi and Horii Satoshi in 1992 (Fukase and Horii, 1992). The detailed route is shown in Scheme 4.6. In the first step, compound 59 was treated with 2 equiv of lithium dimethyl methylphosphonate to prepare the (dimethoxyphosphoryl)heptulopyranose derivative 60. Before oxidation, the pyranose ring of the heptulose derivative 60 was reductively opened with sodium borohydride (NaBH4), to give the heptitol derivative 61. The newly formed C-2 and the released C-6 hydroxyl groups of 61 were oxidized to afford 62 with a reagent combination of DMSO, trifluoroacetic anhydride, and Et3N. The intramolecular cyclization of 62 was accomplished with potassium carbonate in the presence of 18-crown-6, and gave the branched unsaturated inosose derivative 63. Next, to obtain the final product, the synthesis mainly focused on the introduction of the axial amino group to the oxo group of 63. In this case, the α,β-unsaturated keto group of 63 was reduced stereoselectively to an allylic equatorial secondary hydroxyl group with NaBH4/cerous chloride in ethanol, and then followed by cooling in a dry iceacetone bath to generate 64. The resulting branched unsaturated inositol derivative 64 was applied in a Mitsunobu reaction proceeding with

172

Validamycin and Its Derivatives BnOH2C

BnOH2C

O

BnO

D-glucose

BnO

BnO OBn

CH2P(OCH3)2 OBn

O

59

BnOH2C

OH

OH

BnO BnO

OH

60

BnOH2C

O

BnO

O

O

BnO

CH2P(OCH3)2 OBn

O

O

BnO

CH2P(OCH3)2 OBn

OH

61

O

62

BnOH2C BnO BnOH2C

BnOH2C

BnO

BnO

BnO

BnO

OBn

BnO OBn

OH

O

O

N

O

OBn

63

64

65

HOH2C

BnOH2C HO

BnO

HO

BnO OBn 66

OH NH2

NH2

1

SCHEME 4.6 Synthesis of valienamine from D-Glucose by Fukase Hiroshi and Horii Satoshi.

complete inversion of the configuration, and converted to the phthalimido derivative 65. Namely, the free hydroxyl group at the allylic position of 64 was replaced with a phthalimido group in the DEAD/triphenylphosphane (Ph3P) system. Removal of the phthaloyl group of 65 with hydrazine, and subsequent removal of the O-benzyl protecting groups with sodium in liquid ammonia, gave an unsaturated pseudoaminosugar that was identical to compound 1. However, there was no further information about the yields in this reference. Vasella et al. reported a synthesis route to prepare valienamine in seven steps and with an overall yield of 17% from commercially available 2,3,4,6-tetra-Obenzyl-D-glucopyranose (67) (Kapferer et al., 1999). The ketone 68 was readily obtained from 67, and then stereoselective addition of vinylmagnesium bromide to the 1,3,4,5-tetra-O-benzyl-6,7-dideoxy-L-xylo-hept-6-en-2-ulose gave the diene (69, 86%). Ring-closing alkene metathesis of the diene in the presence of 0.15 equiv of Grubb’s catalyst gave the cyclohexene (70, 58%). To transform 70 into (1)-valienamine, a method for the conversion of allylic alcohols to allylic amines by a [3,3]-sigmatropic rearrangement of allylic cyanates was applied. Thus, treatment of the tertiary allylic alcohol 70 with trichloroacetyl isocyanate in CH2Cl2 at 0 C, followed by hydrolysis with K2CO3 in aqueous MeOH, gave 86% of the carbamate 71. Then dehydration of 71 with Ph3P, Et3N, and CBr4 in

Chemical Synthesis Chapter | 4 OBn

OBn

O

O BnO OBn 67

OBn OBn

OBn

OBn OBn

OH OBn

OBn

OBn

68

OBn

OBn

173

OBn 69

OBn

OBn OBn

BnO BnO

BnO BnO

OH

70

BnO BnO

OCONH2

OCN

OBn 71

OBn

BnO BnO

OBn 72

N 73

C O

OH

OBn OBn BnO BnO

NHR 74: R = Z 75: R = Ac

OH HO HO

NH2 1

SCHEME 4.7 Synthesis of valienamine from D-Glucose by Vasella.

CH2Cl2 at 220 C afforded the isocyanate 73 by spontaneous rearrangement of the bona fide cyanate 72. The protected (1)-valienamine 74 was obtained from 73, which was treated in situ with PhCH2OH in 70% yield. Alternatively, in situ treatment of 73 with Me3Al yielded 77% of Nacetyl-tetra-O-benzylvalienamine 75. The benzyl carbamate 74 was deprotected and gave (1)-valienamine (1) in 78% yield as a slightly yellow solid (Scheme 4.7).

4.1.3 Synthesis From 2,3,4,6-Tetra-O-Benzyl-D-Glucose 2,3,4,6-tetra-O-benzyl-D-glucose 76 was synthesized from D-glucose, and was commercially available as the starting material of valienamine. There were mainly three approaches reported by researchers: (1) An efficient synthesis of valienamine was described by Young-Kil Chang in 2005 (Chang et al., 2005). Valienamine was synthesized starting from commercially available 2,3,4,6-tetra-O-benzyl-D-glucose in nine steps as show in Scheme 4.8, using ring-closing metathesis of (4S,5S,6S)-4,5,6-tribenzyloxy-7-(benzyloxymethyl)octa-1,7-dien-3-ol as a key step. The reaction of 76 with ethanethiol containing trifluoroacetic acid (TFA) gave diethyl dithioacetal 77 in 77% yield. The hydroxyl on the dithioacetal 77 was oxidized with aceticanhydride and DMSO to give the corresponding ketone 78 in 94% yield. Exposure of 78 to ylide CH2 5PPh3, formed in situ, smoothly afforded olefin 79 in 88% yield. Hydrolysis of the dithioacetal function in 79 was accomplished by the treatment with HgO/HgCl2 in CH3CN/H2O (10/1) solvent to prepare

174

Validamycin and Its Derivatives OBn

O BnO

SEt

BnO O

BnO

BnO

BnO

OH

BnO

OH SEt

BnO

OBn

SEt

SEt

OBn

OBn 77

76 OBn

OBn

78 OBn

SEt

BnO

BnO SEt OBn

O

OBn

OBn

79

OBn

80 OBn

OBn

OBn

OH

BnO OH

BnO OBn

BnO

N3

OBn BnO

81

OBn

BnO

82

OBn

OH

NH2

BnO

BnO

OBn 84

OBn 83

NH2

HO

HO

OH 1

SCHEME 4.8 Synthesis of valienamine from 2,3,4,6-tetra-O-Benzyl-D-glucose by Young-Kil Chang.

the aldehyde 80, which was not stable enough for column chromatography, and used in the following synthesis without further purification. An inseparable epimeric mixture (7:3 by 1H NMR) of allylic alcohol 81 was obtained by addition of vinylmagnesium bromide to the aldehyde 80 in 79% yield from 79 in the two steps. The desired (1R)-cyclohexenol derivative 82 was provided by the ring-closing metathesis of the diastereomeric diene 81 with Grubbs’ second-generation catalyst in refluxing CH2Cl2 in 61% yield. The absolute configuration of 82 was confirmed by comparing its 1H NMR spectrum and specific rotation with the reported values. The azide 83 was provided in 83% yield by the treatment of the alcohol 82 with diphenylphosphoryl azide (DPPA) in the presence of DBU followed by addition of 1 equiv of sodium azide. Treatment of 83 with triphenylphosphineammonium hydroxide led to the reduction of the azido group to furnish allylic amine 84 in 80% yield. Since the presence of the alkene moiety in 84 precluded the use of catalytic hydrogenolysis, debenzylation of 84 was carried out with sodium in liquid ammonia at 278 C to provide valienamine 1.

Chemical Synthesis Chapter | 4

175

(2) Ian Cumpstey reported a synthesis route for the precursor of valienamine named (1R,2S,3S,4R)-2,3,4-Tri-O-benzyl-5-(benzyloxymethyl)-cyclohex5-ene-1,2,3,4-tetrol, which was synthesized in eight steps from tetrabenzyl glucose 76 as shown in Scheme 4.9 (Cumpstey, 2005). The key steps of this method were considered to be the selective protection of one of two secondary alcohols in the acyclic derivative 85 and the formation of the cyclohexene ring using ring-closing metathesis mediated by Grubbs’ second generation catalyst, with the double bond being in the correct position for valienamine. Thus, treatment of commercially available Tetrabenzyl glucose 76 with vinylmagnesium bromide gave 85 and its enantiomer as a 2.2:1 mixture in favor of the desired R diastereomer. To differentiate the two alcohol functionalities of the resulting diol 85, it was found that excellent regioselectivity could be achieved using 3,4-dimethoxybenzyl chloride (DMBCl) with sodium hydride at 0 C in DMF, and 86a and 86b were obtained as an inseparable 10:1 mixture of regioisomers in favor of the 2-O-dimethoxybenzyl derivative 86a, along with the diprotected compound 87. Pivaloylation of the alcohols 86 and removal of the DMB protecting groups gave the inseparable alcohols 89a and 89b. Swern oxidation of the alcohols 89a and 89b gave the OR1 R2O

OH HO O

BnO

BnO

OH

OBn

BnO

BnO

BnO

BnO

OBn

OBn

OBn

OBn 76

86a: R1 = DMB, R2 = H 86b: R1 = H, R2 = DMB 87: R1 = R 2 = DMB

85

OR1 R2O

OR1 R2O

OBn

PivO

O

BnO

BnO

BnO

OBn

BnO

BnO

OBn

BnO

OBn

OBn

89a: R1 = H, R2 = Piv 89b: R1 = Piv, R2 = H

88a: R1 = DMB, R2 = Piv 88b: R1 = Piv, R2 = DMB

OBn OBn 90

PivO OPiv

BnO

BnO

BnO

OBn OBn 91

OBn

BnO OBn 92

OH

BnO

BnO

OBn OBn 93

SCHEME 4.9 Synthesis of valienamine from 2,3,4,6-tetra-O-Benzyl-D-glucose by Ian Cumpstey.

176

Validamycin and Its Derivatives

ketone 90 and its isomer, which were easily separated by a flash column chromatography, in 81% and 7% yields, respectively. Wittig methylenation of the ketone 90 gave the diene 91, which smoothly underwent ring-closing metathesis mediated by Grubbs’ second-generation catalyst to give the carbasugar 92. The alcohol 93 was afforded by the straightforward deacylation of the pseudoanomeric protecting group, and might be transformed into valienamine 1 in three steps using Fukase’s procedure (Fukase and Horii, 1992). In summary, this synthesis of the precursor 93 to valienamine 1 was in eight steps from commercially available tetrabenzyl glucose 76 and in 7.7% overall yield. What’s more, the key intermediate 93 could also be prepared from L-sorbose with several steps, but the synthesis route was found to be with a low yield (Cumpstey et al., 2008). (3) In 2013, Qing Ri Li et al. succeeded in accomplishing the total synthesis of (1)-valienamine concisely from tetrabenzyl glucose via a highly diastereoselective amination of chiral benzylic ether using chlorosulfonyl isocyanate, intramolecular olefin metathesis, and diastereoselective reduction of cyclic enone using L-selectride as the key steps (Scheme 4.10) (Li et al., 2013). The total synthesis of (1)-valienamine (1) began with the reduction of 76 and subsequent protection of primary alcohol using TBDPSCl to afford compound 95 in high yields. Swern oxidation of 95 and subsequent Wittig reaction furnished olefin 96, which was subjected under standard desilylation conditions to give the corresponding product 97 in 92% yield. After oxidation of primary alcohol, an inseparable diastereomeric mixture of allylic alcohol 98 (diastereoselectivity of 2.5:1 ratio by 1H NMR analysis) was provided by the treatment of aldehyde with vinylmagnesium bromide in THF at 0 C. Intramolecular olefin metathesis of diastereomeric diene 98 with second generation Grubbs’ catalyst in refluxing CH2Cl2 gave a separable diastereomeric mixture of 99a and 99b in 60% and 25% yields, respectively. To obtain an essential synthetic precursor 101 for the formation of 102, the allylic alcohols 99a and 99b were oxidized to give the enone 100, which was subjected under reduction conditions using a bulky hydride reagent, such as L-selectride. As expected, L-selectride attacked the ketone exclusively from the sterically accessible β-face of enone 100 to give the desired product 99b in 80% yield with an excellent level of diastereoselectivity. After benzylation of the primary alcohol, the diastereoselectivity of the reaction of cyclic 1,2-syn-polybenzyl ether 101 with chlorosulfonyl isocyanate was examined under various reaction conditions. After further optimization, the best results were obtained by the use of 101 (1 equiv), chlorosulfonyl isocyanate (3 equiv), Na2CO3 (4.5 equiv) in toluene (0.25 M) at 0 C for 15 h. The cyclic 1,2-syn-amino alcohol 102 was obtained in 61% yield with a high diastereoselectivity (10:1). Retention of stereochemistry could be explained by SNi mechanism

Chemical Synthesis Chapter | 4 OBn

BnO

BnO OBn

BnO

177

OBn

OBn HO

OH

HO

OTBDPS

BnO O

OBn

OH

76

OBn

OBn

94

BnO

95

BnO

BnO

OBn

OBn OH

OBn

OBn

96

OBn

OBn

97

OBn 98

OBn

OBn

OBn

BnO

BnO

OH

OBn

OTBDPS OBn

OBn

BnO OBn

OBn

OBn

+ BnO

BnO

BnO OH

OH 99a

O 100

99b

OBn

OBn

BnO

OBn

BnO

BnO

OBn

OBn

OBn

BnO

BnO

BnO

OH

OBn

NHCbz

99b

101

102

OH HO OH HO NH2 1

SCHEME 4.10 Synthesis of valienamine from 2,3,4,6-tetra-O-Benzyl-D-glucose by Qing Ri Li.

through a four-centered transition state. Finally, debenzylation of 102 with BCl3 in the presence of MeOH afforded the aim product (1)-valienamine (1) in 72% yield.

4.1.4 Synthesis From D-Xylose Tatsuta et al. synthesized valienamine by using compound 103 as the starting material, with an aldol condensation of a sulfone as the key step (Scheme 4.11) (Tatsuta et al., 2000). The compound 104 was prepared from D-xylose by bromine oxidation and tritylation, and silylated and de-O-tritylated to provide the lactone 104. The PfitznerMoffatt oxidation of

178

Validamycin and Its Derivatives

TrO

HO

O O

OH D-xylose

O

OTBS

78.3%

OH

73%

O

MeO

O

OTBS

OTBS

103

94%

OTBS

104

105 O

MeO MeO

MeO

O

OTBS

MeO

O

OTBS TBSO

OH SO2Ph

92%

OTBS

SO2Ph

70%

MeO

SO2Ph

TBSO OTBS

OTBS

OTBS OTBS

106

107

108

O

OH

TBSO

TBSO

84%

OH

80%

82%

OH

TBSO

TBSO OTBS

OTBS

109

110

OMOM

OMOM

HO

OMOM

HO OMOM

HO

81%

HO

OMOM HO

OH 111

100%

OMOM HO

N3

NH2

112

113

OH HO 100%

OH HO NH2 1

SCHEME 4.11 Synthesis of valienamine from 2,3,4,6-tetra-O-Benzyl-D-glucose by Tatsuta.

104 followed by treatment with orthoformate afforded the acetal 105, which reacted with lithiated methyl phenyl sulfonate to give the furanose 106. This was efficiently converted to the cyclohexenone 108 by ring-opening with TBSOTf and then ring-closing of the resulting enol silyl ether 107 with SnCl4. The cyclohexenone 108 was subjected to the Michael reaction with tributylstannyl lithium followed by trapping of the produced anion with formaldehyde to incorporate the hydroxymethyl group into the molecule, to obtain the α-hydroxymethyl-cyclohexenone 109 in 84% yield. The α-alcohol 110

Chemical Synthesis Chapter | 4

179

was obtained by the stereoselective reduction of the carbonyl group in 109, which was carried out by Zn(BH4)2 in ether with 80% yield. The alcohol 110 was then converted to the properly protected alcohol 111, in which the allyl hydroxy group at C-1 was expected to be more reactive than the others. In this case, Mitsunobu inversion of the allyl alcohol 111 using HN3 mainly gave the α-azide 112. Mild hydrogenation of 112 with 1 atm of hydrogen over Raney Ni gave the corresponding amino compound 113 without any significant reduction of the olefin. Finally, the hydrochloride of (1)-valienamine (1) was obtained by the deprotection of 113 with methanolic hydrogen chloride. The overall yield in the synthesis from 103 to 1 was calculated to be 15.44%.

4.1.5 Synthesis From Cyclohexane Skeleton In 1985, Ogawa et al. used (1R)-(2)-1,2,3-Tri-O-acetyl-(1,3/2,4,6)-4-bromo6-bromomethyl-1,2,3-cyclohexanetriol as the starting material to synthesize valienamine (Scheme 4.12) (Ogawa et al., 1985c). Dehydrobromination of 114 with 1,8-diazabicyclo undec-7-ene (DBU) in toluene exclusively gave the diene 115, which was directly treated with an equimolar amount of m-chloroperbenzoic acid (m-CPBA) in dichloromethane to oxidize the exo-methylene group. A silica gel column was applied for the separation of the mixture to give the spiro oxiranes 116 (27%). The chloride 117 was given by the chlorination of 116 with the concentrated hydrochloric acid in tetrahydrofuran (THF) and subsequent acetylation. Treatment of 117 with sodium azide in DMF at 60 C selectively afforded the azide 118 in quantitative yield. In the last, the reduction of 118 with hydrogen sulfide, followed by the conventional acetylation, gave the desired product 30 in 94% yield. This synthesis route contained five steps with a total yield of 24.11%. CH2Br

H2C

O

Br OAc

OAc

OAc

OAc

OAc

OAc OAc

OAc 114

115

Cl

AcOH2C

CH2OAc OAc OAc

AcOH2C

OAc

OAc N3

OAc OAc 117

OAc 116

NHAc

OAc

OAc 118

OAc 30

SCHEME 4.12 Synthesis of valienamine from cyclohexane skeleton by Ogawa.

180

Validamycin and Its Derivatives

Knapp et al. (1992) reported a method utilized the diol 119 as the starting material to prepare valienamine (Scheme 4.13). The two hydroxyls of 119 were O-benzylated, and then the double bond was epoxidized with 3.5:1 stereoselectivity. The major epoxide 120 was isomerized to allylic alcohol 121 by the SharplessReich protocol, with attack by PhSeNa occurring virtually exclusively at C-1 because of steric hindrance to attack at C-2. Hydroxyl-directed epoxidation of 121, followed by O-benzylation, afforded the epoxide 122. Another application of the SharplessReich procedure was applied for the rearrangement to give the allylic alcohol 123. Inversion of the allylic hydroxyl 123 was accomplished by a Mitsunobu sequence, and the desired allylic alcohol substrate 124 was obtained in about 23% from 119. Condensation of the potassium salt of 124 with p-methoxybenzyl isothiocyanate, followed by iodomethane quench, gave the carbonimidothioate 125, iodocyclization of which gave, after aqueous sodium sulfite quench, the iodooxazolidinone 126. Oxidation of 126 to the corresponding iodoso compound 127 resulted in spontaneous syn elimination of HOI, with formation HO

BnO

HO

BnO

BnO

BnO

BnO

82%

86%

83%

HO

O 119

BnO

BnO O

120

121

122

BnO

BnO

BnO

BnO

BnO

BnO N

O

OH

OH

123

124

SMe

BnO

I

BnO

N O

N

126

O 128

NHR

RO Ar

O

O 127

Ar

RO

BnO Ar

O

RO

BnO

Ar

O

125

BnO

O

N

BnO

BnO

BnO

71%

65%

84%

BnO

I

BnO

BnO

BnO

74%

RO 1: R = H 30: R = Ac

SCHEME 4.13 Synthesis of valienamine from cyclohexane skeleton by Knap.

181

Chemical Synthesis Chapter | 4

of the unsaturated oxazolidinone 128. Finally, oxidative removal of the N-p-methoxybenzyl group, basic hydrolysis of the oxazolidinone, and debenzylation gave 1, which was isolated and characterized as its known peracetate 30. Therefore, compound 30 was synthesized in 10 steps with an overall yield of 7.89%. In 1998, Trost et al. synthesized valienamine with compound 129 as the starting material (Scheme 4.14) (Trost et al., 1998). In this procedure, a new protocol evolved, invoking the importance of cocatalysis for the palladium-based cis-hydroxyamination sequence and a new application of the asymmetric palladium-catalyzed hydroxycarboxylation sequence. The inability to perform the DielsAlder reaction asymmetrically led to a different asymmetric synthesis of the pivotal epoxide intermediate in enantiomerically pure form, which was derived from asymmetric palladium-catalyzed reactions. With the desymmetrization of meso enedicarboxylates, the net equivalence of an asymmetric cis-hydroxycarboxylation led to the enantiomerically pure epoxide, and (1)-valienamine was then available in 12% overall yield.

OSMDBT

OSMDBT

CO2C2H5

OSMDBT

CO2C2H5 O

129

131

132

OSMDBT

OSMDBT CO2C2H5

TBDMSO

OSMDBT

CO2C2H5

TBDMSO

134

TBDMSO

CO2C2H5

CO2C2H5

(

136

NHR

NTos

L NTos

RO

O

Pd

O

OR RO OR

L+

)

L 135

OSMDBT

OSMDBT TBDMSO

O

L+ Pd

O

L

133

-

-

Pd

O

CO2C2H5

)

L O

CO2C2H5

O

130

TBDMSO

OSMDBT TBDMSO

CO2C2H5

O 137

1: R=H 30: R=Ac

SCHEME 4.14 Synthesis of valienamine from cyclohexane skeleton by Trost.

4.1.6 Synthesis From (2)-Quinic Acid The research group of Professor Shing reported the synthesis of valienamine with (2)-quinic acid (138) as the starting material. Based on different

182

Validamycin and Its Derivatives

reaction mechanism, there were mainly two paths to obtain valienamine from 138 as follows: (1) In the previous study, the work has shown that the diol 139 could be obtained from (2)-quinic acid 138 in six steps with 34% overall yield (Scheme 4.15) (Shing et al., 1999; Shing and Wan, 1996). Then, the tribenzyl ether 140 was obtained by the selective benzylation of the secondary alcohol in diol 139 with benzyl bromide in the presence of sodium hydride. To furnish the double bond in valienamine, a solution of the tribenzyl ether 140 in pyridine was added dropwise to the thionyl chloride solution in CH2Cl2 at 0 C to give the desired alkene 141 in OBn HO

OBn OH

CO2H

OH 34%

HO

OBn OH

70%

85% O

OH

OBn

O

13 8

85% O

OBn

O

OH

OBn

OBn

OBn O

O

139

14 0

14 1

OBn OBn

OBn

OBn

OBn OBn 97% O HO

OBn

OBn 95% OBn

OBn S

OH

98%

N3

O

OBn

N3

OBn

OH

OMs

O 14 2

144

14 3 OBn

OBn

OBn 61% OBn

OBn

OBn 100%

N3

145

OBn 97%

N3

OBn

68% H 2N

OH

OH

146

147

148

OH OH

H 2N

OBn

OAc

OH OH 1

SCHEME 4.15 Synthesis of valienamine from (2)-quinic acid by Shing.

Chemical Synthesis Chapter | 4

183

70% yield. Hydrolysis of the cyclohexylidene blocking group in 141 was carried out using aqueous TFA in CH2Cl2 to give the diol 142 in 85% yield. The cyclic sulfite 143 was afforded by a second reaction with thionyl chloride in the conventional way in excellent yield. To introduce the azido group at the allylic position, a regiospecific SN2 reaction of lithium azide on 143 was conducted to afford a single adduct 144 in 97% yield. To get a high yield of 146, 144 was reacted with mesylchloride to give 145. The elimination reaction of the resultant mesylate 145 in the presence of tetrabutylammonium acetate yielding the desired azidoacetate 146 was greatly improved to 61%. Since the current chirality of all the functional groups was now established, the next step was to deacetylate 146 to afford 147 with a catalytic amount of potassium carbonate in methanol. The azido group in 147 was then reduced readily to the amine 148 with PPh3 and NH4OH in pyridine. Finally, valienamine 1 was afforded by the debenzylation of 148 with sodium in liquid ammonia at 278 C. (2) Valienamine was also produced directly from a cyclohexane precursor bearing an allylic acetate moiety. A regio- and stereospecific palladiumcatalyzed reaction was proven to effectively install the amino function (Scheme 4.16). At last, compound 30 was synthesized from (2)-quinic acid in 20 steps with an overall yield of 11%, and then deacetylated to furnish valienamine 1 (Kok et al., 2001).

4.1.7 Synthesis From (1SR,2RS,3SR)-6-Methylenecyclohex-4Ene-1,2,3-Triol In this method, valienamine was synthesized by racemic modification from (1SR,2RS,3SR)-6-methylenecyclohex-4-ene-1,2,3-triol, which was acetylated to give compound 166 (Scheme 4.17) (Ogawa et al., 2004b). Treatment of 166 with a slight excess of bromine in carbon tetrachloride afforded the 1,4-addition product 167 with 90% yield. Then compound 167 was treated with sodium acetate to give 168 without purification, following by the treatment with sodium azide in DMF at room temperature to give the rac-azide 169 in 85% yield. Compound 169 was treated with 2,2-dimethoxypropaneTsOH in DMF to afford a mixture of the 2,3-O-isopropylidene derivatives stereoselectively, which were found to be separable on a silica gel column with 1:6 EtOAc/hexane as an eluent to give 170 with the desired configuration in 40% yield. Removal of the acetyl and isopropylidene groups 170 was effected by treatment with 4M hydrochloric acid at reflux temperature to give azide 171 in 80% yield. Finally, reduction of the azido group 171 with triphenylphosphine in 70% aqueous THF afforded the free amine 1 in 86% yield.

184

Validamycin and Its Derivatives O

HO

O

94% HO

98%

86% O

O

OH

1 38

99% O

OH

O O

O

O

OH

149

150

1 51

OH

CO2Me

CO2Me

HO

CO2Me

HO

HO

CO2H

OBn

OBn OH

93% O

O

98%

O

O

92%

O

OH

152

O

1 54

OBn

15 5 OBn

OBn

OH

OAc OBn

OBn 83% O

OBn

O

153

OBn

O

OBn

O

OAc OBn

OAc OBn 100%

O

OBn

87% from 158

HO

OBn

O

98%

OH

O

OBn

O

OBn O

OH S

1 56

157

OBn

1 58

OBn

OBn OAc OBn

90%

88%

HO

AcO

OBn

OBn

16 1

OH OBn

60% OBn

16 4

1 63

OBn OBn

OBn OAc

162

OBn

OAc

AcO

OAc

OH 160

OBn

OBn

81%

BnHN

OBn OH

OAc OBn

OBn

159

OH 69 %

BnHN

OBn

H 2N

OH 165

OH OH 1

SCHEME 4.16 Synthesis of valienamine from (2)-quinic acid by Kok.

4.1.8 Synthesis From Tartaric Acid With the L-tartaric acid as the starting material, Yuan-Kang Chang et al. provided a concise approach to (1)-valienamine by an evolved strategy invoking a new application of the [3,3] sigmatropic rearrangement of allylic azides and the presence of a C2 symmetry element within a pool of chiral

185

Chemical Synthesis Chapter | 4

AcO

H2 C

AcO

CH2OAc

BrH2C

AcO

AcO

AcO

AcO

166

OAc

167

CH2OAc AcO

Me

AcO

N3 OAc

Br

Br OAc

OAc

Me

168

CH2OH

CH2OAc

HO

HO

O

HO OH

OAc 170

OH N3

N3 169

CH2OH

HO

O

171

NH2 1

SCHEME 4.17 Synthesis of valienamine from (1SR,2RS,3SR)-6-methylenecyclohex- 4-ene1,2,3-triol.

substrates (Chang et al., 2009). Cyclic diol 175, readily prepared from C2symmetric L-tartaric acid 172, was applied as a versatile bridging intermediate en route to aminocyclitols and envisioned using azide to introduce an amino group in the equivalent of a protected form. This synthesis based on a C2-symmetric pool of chiral substrates requires 8 steps from very cheap L-tartaric acid 172 to give (1)-valienamine in 8.4% overall yield. As shown in Scheme 4.18, the synthesis commenced with commercially available and cheap L-tartaric acid 172. Reduction of its derivative dimethyl 2,3-O-isopropylidenetartrate 173 with DIBALH, followed by a highly diastereoselective divinylzinc addition to the in situgenerated dialdehyde, afforded the desired vinyl carbinol 174. Subsequent RCM with the second generation Grubbs’ catalyst afforded the corresponding cyclic diol 175. For the construction of allylic azide 177, the in situgenerated allylic epoxide 176 was chosen to apply as the valuable key intermediate. Gratifyingly, sequential addition of triflimide/sodium hydride and a solution of sodium azide in 1.5:1:1 DMF/EtOH/H2O at 90 C to cyclic vinyl carbinol 175 led to the 1,4type azido alcohol 177 in 70% yield, with no 1,2-type azido alcohol. Elaboration of 177 to (1)-valienamine was initiated by oxidation of 177 with DessMartin periodinane to produce enone 178 (96%). The availability of 178 allowed for direct introduction of a hydroxymethyl group via the BaylisHillman reaction. The desired hydroxymethylcyclohexenone 179 was obtained by the treatment of 178 with formaldehyde solution (37% in H2O) in THF at room temperature followed by addition of imidazole (1.5 equiv) and aqueous NaHCO3 (1 M) resulting in efficient formation of in 65% yield. Controlled reduction of enone 179 with NaBH4 in methanol solution containing cerium chloride at 278 C gave alcohol 180 with excellent stereoselectivity (.16:1) in 80% yield. Finally, (1)-valienamine 1 was provided by reduction of azido alcohol 180 with LiAlH4.

186

Validamycin and Its Derivatives

OH

O

O

O

OH OH HO

O

O OMe MeO

O

172

O O

HO

O

HO

OH

O

173

OH

174

175

O

O

O O

N3

O N3

O

N3

O

O O O

OH

O

HO

176

177

178

O N3

OH O

OH

H 2N

OH HO 180

179

OH HO 1

SCHEME 4.18 Synthesis of valienamine from L-Tartaric acid by Yuan-Kang Chang.

Lo et al. (2012) reported a new enantioconvergent strategy directed toward the synthesis of valienamine, which was developed by using a C2-symmetric element within the chiral pool and by applying an iodinepromoted cyclization of an unsaturated carbonimidothioate for the regio- and diastereocontrolled installation of amino and hydroxy units. Following the reported procedures, the synthesis commenced with commercially available D-tartaric acid 181 (Scheme 4.19). Reduction of dimethyl 2,3-O-isopropylideneD-tartrate 182 with DIBALH, followed by a highly diastereoselective divinylzinc addition to the in situgenerated dialdehyde, gave the desired vinyl carbinol 183. The second-generation Grubbs’ catalyst was then used for the RCM, and subsequent oxidation with DessMartin periodinane proceeded to give cyclic enedione 184. Controlled reduction of enedione 184 with Zn (BH4)2 in DME/Et2O at 278 C gave a mixture of hydroxyenones 185a and 185b in 92% yield with 13:1 diastereoselectivity, which could be separated on a chromatograph. Next, this hydroxyenone 185a was subjected to methylenation with the ylide derived from phosphonium iodide, and only the desired dienol 186a was obtained. With enantiomerically pure dienol 186a in hand, the stage was set for installation of the amino and hydroxy units. Treatment of 186a with sodium hydride in THF at 0 C followed by addition of benzyl isothiocyanate (1.5 equiv) and methyl iodide resulted in efficient

187

Chemical Synthesis Chapter | 4

O

OH

O

O

OH

OH

O

O

OH O

O

O

HO

OMe OMe

O

181

HO O

182

183

O

O O

O

+

185a

184

O

O

O

O

O

HO

HO

O

+

HO

HO 186a

185b

O

186b

O

O

O

O

O

O N

N NBn

O

O

O O

O

O

Bn

I

MeS 187

Bn

188

OH 189

OH HO OH

HO NH2 1

SCHEME 4.19 Synthesis of valienamine from L-Tartaric acid by Hong-Jay Lo.

formation of desired carbonimidothioate 187 in 88% yield. The exposure of 9 to I2 in THF at room temperature smoothly and cleanly afforded the desired cyclization to afford 7-iodooxazolidinone 188 in 87% yield. Sequential exposure of iodide 188 to sodium acetate in DMF and hydrolysis under basic conditions resulted in efficient formation of the allylic alcohol 189. In the end, simple removal of the N-benzyloxazolidinone and acetal groups completed the synthesis of valienamine 1.

4.1.9 Synthesis From L-Serine Starting with L-serine, stereoselective total synthesis of (1)-valienamine is conducted utilizing Sharpless asymmetric dihydroxylation, diastereoselective Carreira alkynylation, and ring-closing enyne metathesis (RCEYM) as key steps (Scheme 4.20) (Krishna and Reddy, 2009). The known compound 190,

188

Validamycin and Its Derivatives HO

CO2Me

MOMO

CO2Me

OH O

NBoc

O

O

O

O

193

HO

MOMO

MOMO

MOMO

OMOM O

OMOM

NBoc

194a

MOMO

MOMO

+

NBoc

NBoc

192

HO

OMOM

OMOM

NBoc

191

MOMO

OH

OMOM

NBoc

190

MOMO

CO2Me

O

+

NBoc

194b

OMOM O

NBoc

195a

MOMO

195b

MOMO

MOMO

MOMO BocHN OMOM

HO

NHBoc

MOMO

NHBoc

196

197

OMOM

MOMO

OMOM

199

OMOM 198

OH

BocHN

OMOM

OMOM

OAc

AcHN

OAc

AcO

OAc

30

OH

H 2N

OH

HO

OH

1

SCHEME 4.20 Synthesis of valienamine from L-serine.

obtained from L-serine on Sharpless asymmetric dihydroxylation (AD-mixα, OsO4, MeSO2NH2, t-BuOHH2O, 0 C) provided a separable mixture 191 of diastereomers in 99:1 ratio, which was protected as its MOM ether 192 (MOMCl, DIPEA, CH2Cl2, r.t., 93%). Next, reduction of 192 with LiAlH4 in THF afforded the primary alcohol 193 (75%). Alcohol 193 on Swern oxidation and quenching the ensuing aldehyde with TMSacetylenic anion (TMSacetylene, n-BuLi, THF, 278 C) gave the propargylic alcohol, which upon TMS deprotection (K2CO3, MeOH) furnished 194a and 194b (70% overall yield for three steps) as an inseparable diastereomeric mixture (8.0:2.0, anti/syn). Later, the diastereomeric mixture of 194a and 194b was subjected to a Mitsunobu reaction (p-NO2C6H4COOH, DIAD, TPP, THF, r.t.) followed by methanolysis (K2CO3, MeOH, r.t.) to provide the aimed diastereomer as the major product (4:1 ratio). However, practical separation of 194a and 194b diastereomeric mixture into individual entities was accomplished after derivatization (MOMCl, DIPEA, CH2Cl2, 0 C to r.t.) as MOM ethers 195a and 195b. To continue the synthesis, the free primary

189

Chemical Synthesis Chapter | 4

alcohol 196 was provided with the selective cleavage of acetonide group on 195b using CuCl2  2H2O in acetonitrile at 0 C (92% yield). The enyne 197 was furnished with Swern oxidation of 196 followed by Wittig olefination (Me1PPh3I2, t-BuOK, THF, 0 C, 64% yield). The critical RCEYM reaction of 197 was conducted with the Grubbs’ II catalyst in toluene at 110 C under an ethylene atmosphere for 12 h, and gave the vinylcyclohexene derivative 198 in high yield (92%). The selective dihydroxylation of the vinylic olefin 198 (OsO4, NMO, acetone-H2O) and the oxidative cleavage of the ensuing diol (NaIO4, MeOH-H2O, r.t.) gave the corresponding aldehyde, which was reduced (NaBH4, MeOH, 0 C) to afford the carbocyclic methanol 199 (62% overall yield for three steps) without any purification of the intermediates. Finally, global deprotection of MOM and Boc protecting groups with TFA in CH2Cl2 at room temperature gave valienamine 1.

4.1.10 Synthesis From Garner’s Aldehyde Starting from Garner’s aldehyde, a synthesis of (1)-valienamine was achieved in 10 steps and 23% overall yield. A unique feature of the synthetic route is that an acyclic precursor was constructed, using diastereoselective antireductive coupling reaction of alkyne and Garner’s aldehyde as the key step, which was then cyclized in an intramolecular aldol reaction to form the valienamine skeleton (Zhou et al., 2012). As depicted in Scheme 4.21, Boc

OTBS

O

OHC

N

84%

+

N

TBSO

73% O

Boc

200

OH 202

201 Boc OH

Boc N

TBSO

O

93%

O

TBSO

OH

OMOE

OMOE N

90%

HO NHBoc

OH

OH

OEOM

EOMO

203

EOMO

204

OEOM 205

O CHO EOMO

O

66%

NHBoc

EOMO OEOM 206

CH2OH

EOMO

68%

NHBoc

EOMO

HO

NH2

HO

OEOM 207

SCHEME 4.21 Synthesis of valienamine from L-serine Garner’s aldehyde.

OH 1

190

Validamycin and Its Derivatives

the desired allylic alcohol 202 was obtained in good yield (84%) by the treatment of 200 with zirconocene hydrochloride in THF, followed by addition of Garner’s aldehyde 201 and ZnBr2. The ratio of anti/syn was determined by HPLC system, and found to be virtually complete diastereocontrol (anti/ syn .15:1). Dihydroxylation of allylic alcohol using osmium tetroxide with TMEDA produced the syn,syn-triol 203 in an acceptable 73% yield with a small amount of syn, anti diastereomer (15%). The diastereoselectivity was good (syn/anti 5 4.5:1B5:1), and the unwanted syn, anti diastereomer was separable by column chromatography at this point. When AD-mix-β was used, the product obtained was a 2:1 mixture of the diastereomeric triols, with the desired syn, syn diastereomer as the major product. With all the stereocenters in place, it was ready to prepare for the cyclization. Exposure of triol 203 to EOMCl afforded compound 204 in 93% yield. The 13C NMR spectra of compound 204 were complex due to the presence of the Boc protecting group, which resulted in a number of rotational isomers at 23 C. Selective deprotection of TBS ethers by exposure of 204 to TsOH in MeOH, followed by removal of the N,O-acetonide with CuCl2 in MeCN, provided diol 205 in 90% yield in the two steps. In order to execute the wanted aldoltype cyclization, the terminal dihydroxy group needed to be oxidized to the level of aldehyde. A protocol involving DessMartin periodinane oxidation was found to be superior to other conditions, and the desired dialdehyde 206, was directly converted to the six-membered ring structure 207 without further purification through an intramolecular aldol condensation reaction under mild conditions (cat. TsOH, piperidine, r.t., 2 h), followed by β-elimination of the formed hydroxyl group with MsCl and Et3N. Attempts at other intramolecular aldol condensation cyclization conditions failed to give the desired compound 207. Thus, the valienamine skeleton was constructed by the intramolecular aldol-type cyclization. Controlled reduction of enal 207 with NaBH4 in methanol solution containing cerium chloride, followed by removal of the EOM ether and Boc in TFACH2Cl2, provided (1)-valienamine 1.

4.1.11 Synthesis From (2)-Shikimic Acid Starting from the naturally abundant (2)-shikimic acid 208, a stereoselective synthesis of (1)-valienamine 1 was described by Wei Ding et al. in 2015. (1)-Valienamine 1 was synthesized via 13 steps in 38.3% total yield, and the synthetic route for the stereoselective synthesis of (1)-valienamine 1 is shown in Scheme 4.22. Firstly, (2)-shikimic acid was converted into epoxy compound 209 via four steps in 79.7% overall yield according to a previously reported procedure (Quan et al., 2013). Epoxide 209 was then treated with hot water at 80 C by Qu’s method to furnish ethyl 3-epi-5-Omethanesulfonyl shikimate 210 in 96% yield (Wang et al., 2008). In the water-mediated ring-opening of epoxide 209, water preferentially attacked the much more reactive allylic (C-3) position on the opposite side of the

Chemical Synthesis Chapter | 4

HO

CO2Et

CO2H

HO

HO

CO2Et

OH

O HO

HO OMs

OH 208

HO OMs

209

211 OH

BzO N3

213 OAc

215 OH

HO

HO

OBz BzO

N3

214 OH

BzO

OAc OH OBz

BzO

N3

212

BzO OBz

BzO OMs

OH

BzO

OBz

OBz BzO

OMs

210

BzO

BzO

191

OH HO

OH HO

N3

N3

216

217

NH2 1

SCHEME 4.22 Synthesis of valienamine from (2)-shikimic acid.

epoxide, and thus highly regio- and stereoselective epoxide opening of compound 209 occurred. Subsequently, the compound 210 was treated with 2.5 equiv of diisobutylaluminum hydride (DIBAL-H) in CH2Cl2 at 10 C, and the ester group of compound 210 was selectively reduced, leaving the methanesulfonyl group (Ms) intact. The intermediate compound 211 was exposed to 4.0 equiv of benzoyl chloride, 5.0 equiv of triethylamine, and a catalytic amount of DMAP at 0 C to room temperature in ethyl acetate, and compound 212 was thus obtained in 86% yield over two steps. Compound 212 was then treated with 4.0 equiv of sodium azide at 90 C in the presence of 2.0 equiv of triethylammonium hydrochloride in a mixed solvent of dimethyl sulfoxide and water (DMSO/H2O 5 5:1) to furnish the azido compound 213 in 90% yield via an SN2-type nucleophilic substitution. Meanwhile, the (R)-configuration of C-5 was inverted to the (S)-configuration via a Waldentype inversion. Next, when compound 213 was treated with 1.5 equiv of sodium periodate (NaIO4), 1.0 equiv of sulfuric acid, and 0.002 equiv of ruthenium trichloride at 05 C in a mixed solvent of ethyl acetate, acetonitrile, and water (EtOAc/CH3CN/H2O 5 3:3:1), dihydroxyl compound 214 was afforded in 91% yield via Rh-catalyzed stereoselective dihydroxylation. To afford compound 215, compound 214 was subsequently treated with 1.2 equiv of acetic anhydride, 2.0 equiv of triethylamine, and a catalytic amount of N,N-dimethylaminopyridine (DMAP) at 0 C in ethyl acetate, and the desired compound 215 was synthesized in 92% yield via selective acetylation of the secondary hydroxyl group at the C-4 position. Compound 215 was then treated with 5.0 equiv of thionyl chloride (SOCl2) and 3.0 equiv of pyridine at reflux (41 C) in CH2Cl2, and the elimination occurred smoothly

192

Validamycin and Its Derivatives

to give compound 216 in 85% yield. When a solution of compound 216 in a mixed solvent of methanol and concentrated aqueous ammonia (CH3OH/ NH3.H2O, 4:1) was stirred at room temperature for approximately 24 h, the three benzoyl groups and one acetyl group of compound 216 were cleanly removed to give an intermediate 217. The compound 217 was immediately exposed to H2 atmosphere in methanol at room temperature for 3 h in the presence of Lindlar catalyst to give the target compound ( 1 )-valienamine 1.

4.1.12 Synthesis From Acarbose, Validamycin, and Their Derivatives One of the production methods of valienamine was a chemical production method that uses validamycin as a raw material and NBS (N-bromosuccinimide). However, this method had a low production yield and difficulties in removing byproducts, as well as difficulties in the purification process, because DMSO was used as the solvent. In 2003, Sutaek-Dong et al. reported an invention to provide a new method of producing valienamine with significantly high conversion rate by selectively hydrolyzing acarbose, validamycin, and their derivatives (Hur et al., 2004). In their invention, a heterogeneous solid catalyst, such as a strong acidic cation exchange resin, a zeolite-based catalyst or an anion exchange resin, was applied and then removed in a convenient manner. For example, a high-pressure reactor with a volume of 100 mL was charged with 1 g of pure acarbose derivative (disaccharide or trisaccharide); thereto successively added were 20 mL of water for dissolving the acarbose derivative and 2 g of a strong acidic cation exchange resin Amberlyst 131, and then the mixture was reacted at 80 C for 12 h or more and filtrated to remove the reaction solution. To the filtered cation exchange resin, 100 mL of 0.5 N ammonia water was added and stirred for 300 min, then the mixture was filtered, and the filtrate was purified through a weak acidic cation exchange resin to give 0.23 g of valienamine. Similarly, validamycin and its derivatives could be subjected as the starting material. Meanwhile, zeolite and anion exchange resin could also be applied as the catalyst.

4.2 SYNTHESIS OF EPI-VALIENAMINE In the above part, (1)-valienamine was discussed with the different synthesis methods starting from various materials. However, as the distinct geometry configuration of OH and NH2 on valienamine, valienamine has several diastereomers, besides (1)-valienamine, and some of the diastereomers are applied as an important intermediate in the medical synthesis process for their particular bioactivity. Considering this, in this part, we mainly describe the preparation of epi-valienamines, including 1-epi-valienamine, 2-epi-valienamine, 4-epi-valienamine and 1,2-bis-epi-valienamine (Fig. 4.2).

Chemical Synthesis Chapter | 4 OH

193

OH

HO

HO OH

HO

OH

HO NH2

NH2

218: 1-epi-valienamine

240: 2-epi-valienamine

OH

OH

HO

HO OH

HO

OH

HO NH2

260: 4-epi-valienamine

NH2

265: 1,2-bis-epi-valienamine

FIG. 4.2 Structure of 1-epi-valienamine, 2-epi-valienamine, 4-epi-valienamine, and 1,2-bisepi-valienamine.

4.2.1 Synthesis of 1-epi-Valienamine In 2004, Seiichiro Ogawa synthesized 1-epi-valienamine by racemic modification from (1SR,2RS,3SR)-6-methylenecyclohex-4-ene-1,2,3-triol. Similar to the synthesis of valienamine, compound 169 was obtained by the method described in the above (Scheme 4.23) (Ogawa et al., 2004b). O-Deacetylation of 169 under Zemple´n conditions gave the tetrols 220, which were treated with 2,2-dimethoxypropane-TsOH in DMF to afford, after acetylation, a mixture of the 2,3-O-isopropylidene derivatives 219, selectively. These compounds were found to be separable on a silica gel column with 1:6 EtOAc/ hexane as an eluent, giving 219 (42%). Removal of the acetyl and isopropylidene groups of 219 was effected by treatment with 4M hydrochloric acid at reflux temperature to furnish the respective azides 220 (74%). Reduction of the azido group of 220 with triphenylphosphine in 70% aqueous THF afforded, after purification over a column of Dowex 50 W 3 2 (H1) resin with 5% aqueous NH3 as eluent, the free amines 218 in 86% yields.

194

Validamycin and Its Derivatives

AcO

H2C

AcO

BrH2C

AcO

AcO

AcO

Br

Br OAc

OAc

OAc 177

167

CH2OAc

AcO

CH2OAc

AcO

Me

AcO

N3 OAc

Me

168

CH2OAc

O

CH2OH

HO

N3 HO

O

N3 HO

NH2

OH

OAc

169

CH2OH

HO

219

OH

220

218

SCHEME 4.23 Synthesis of 1-epi-valienamine from (1SR,2RS,3SR)-6- methylenecyclohex-4ene-1,2,3-triol.

Cumpstey et al. (2008) reported a low-yielding synthesis of 1-epi-valienamine as shown in Scheme 4.24. The treatment of 76 with vinylmagnesium bromide afforded the alkene diol 221 in the yield 29%. Treatment of the diol 221 with O

BnO

OR1 R2O

OH HO

OH BnO

BnO

OBn

BnO

BnO

OBn

OBn

BnO

OBn

222a: R1=DMB, R2=H 222b: R1=H, R2=DMB

221

76 O DMBO

DMBO

BnO

HO

BnO

BnO

OBn OBn

OBn

BnO

BnO

OBn

OBn

OBn

BnO

OBn

OBn

224

223

225 O

OH

BnO

N

BnO

NH2

BnO O

BnO

BnO

OBn

OBn OBn

OBn 226

227 NH2

HO

HO

OH OH 218

SCHEME 4.24 Synthesis of 1-epi-valienamine by Ian Cumpstey.

BnO

OBn OBn 228

Chemical Synthesis Chapter | 4

195

dimethoxybenzyl chloride and sodium hydride in DMF resulted in the selective protection of the allylic alcohol OH-6, albeit with a lower selectivity, and afforded the regioisomers 222a and 222b, which could be partially separated by column chromatography. Oxidation of 7b with PCC was more successful and gave ketone 223 in good yield. Wittig methylenation of this ketone gave the diene 224. The attention then turned to the metathesis reaction. The diene 224 was deprotected to give C-6-epimeric allylic alcohol 225. Metathesis of the diene 225 now proceeded quickly and with a lower catalyst loading to give the respective carbocycle 226 in good yield. The route to 1-epi-valienamine from 226 followed an analogous route: nitrogen was introduced at C-1 of 226 with inversion of configuration by Mitsunobu reaction of the allylic alcohol with phthalimide. Deprotection of phthalimide 227 with ethylene diamine gave the free amine 228, which was then deprotected under Birch conditions to give 1-epi-valienamine 218. Meanwhile, the key intermediate 226 could also be provided by the reaction starting with L-sorbose (Scheme 4.25) (Cumpstey et al., 2008). Hemiketal 229 is readily produced in three steps from L-sorbose in excellent yield. Acylation of the hemiacetal 229 with either pivalyl chloride or benzoyl chloride resulted in the exclusive high-yielding formation of the OH-6 protected ketones 230 or 231, respectively. These ketones 230 and 231 underwent Wittig reaction afforded the respective alkenes 232 and 233. In the case of the benzoate-protected compound 233, however, some OH O BnO

L-sorbose

BnO

BnO

OBn

BnO

OBn

OR

232: R=Piv 233: R=Bz 234: R=H

OH BnO

BnO

OBn

BnO

OBn

OBn

OBn

OBn

235

236

237

OH BnO + BnO

OBn OBn 226

SCHEME 4.25 Synthesis of 1-epi-valienamine from L-sorbose.

OR

OBn OBn

OH BnO

OBn

O

BnO

OBn OBn

O

BnO

BnO

230: R=Piv 231: R=Bz

229

BnO

O

196

Validamycin and Its Derivatives

cleavage of the ester was seen, resulting in the formation of the free C-6 alcohol 234. Oxidation of the primary alcohol in 234 under Swern conditions furnished the aldehyde 235, which was treated directly with vinylmagnesium bromide to give the diene 236 as an epimeric mixture. The diastereomers underwent ring-closing metathesis mediated by Grubbs’ second generation catalyst to give the cyclohexenes 237 and 226, which could be separated by column chromatography. The compound 237 could be applied in the synthesis of valienamine mentioned in Section 4.1.3. In 2013, Qing Ri Li et al. reported a total synthesis of (2)-1-epi-valienamine, which was concisely accomplished from readily available D-glucose, similar to the synthesis of valienamine discussed previously. The key steps were considered to be a highly diastereoselective amination of chiral benzylic ether using chlorosulfonyl isocyanate, intramolecular olefin metathesis, and diastereoselective reduction of cyclic enone using L-selectride (Li et al., 2013). Based on the results in Section 4.1.3, the cyclic allylic alcohol 99a was applied in this synthesis via a three-step synthesis, as illustrated in Scheme 4.26 (Li et al., 2013). After benzylation of 99a under standard reaction conditions, cyclic 1,2-anti-polybenzyl ether 238 was treated with chlorosulfonyl isocyanate under optimal reaction conditions (toluene, 0 C, 24 h) to give the corresponding 1,2-anti-amino alcohol 239 in 75% yield with an excellent level of diastereoselectivity. Benzyl and Cbz protection groups were removed using BCl3 to provide (1)-1-epi-valienamine 218. OBn

OBn

BnO

OBn

BnO

OBn

OBn

BnO

BnO OH

OBn BnO

OBn

99a

BnO

238

NHCbz 239

OH HO OH HO NH2 218

SCHEME 4.26 Synthesis of 1-epi-valienamine from D-glucose.

4.2.2 Synthesis of 2-epi-Valienamine 2-epi-Valienamine 240, as the unnatural diastereomer of valienamine, was considered to be an α-mannosidase inhibitor. The construction of 2-epi-valienamine was first reported by Shing et al. (1999) (Scheme 4.27). The synthesis started from (2)-quinic acid, and compound 144 was obtained according to the method discussed above in Section 4.1.6. The regio- and stereochemical

197

Chemical Synthesis Chapter | 4 OBn

OBn OH

CO2H

HO

OH

O

OH

OBn

138

85%

70% O

O

OBn

O

OH

OBn

OBn 85%

34% HO

OBn OH

OBn O

O

139

141

140 OBn

OBn

OBn

OBn

OBn OBn

95% O S

OH

OBn

97%

OBn

OBn

HO

OBn

97%

O

N3

OBn

H2N

OBn

OR

OH

144: R=H 241: R=Ac

242

O 142

143 OR OR

70%

RHN

OR OR 240: R=H 243:R=Ac

SCHEME 4.27 Synthesis of 2-epi-valienamine from (2)-quinic acid.

assignments were based on 1H NMR spectral analysis of the azido acetate 241 formed from 144. The reduction of the azido group in 144 was effected with triphenylphosphine (PPh3), aqueous ammonia, and pyridine, affording amine 242 in excellent yield. For the presence of a double bond in 242, debenzylation of 242 was carried out with sodium in liquid ammonia at 278 C to give 2-epi-valienamine. The overall yield of 243 from (2)-quinic acid was 11% in 14 steps. Ramstadius et al. (2009) described another synthesis route of 2-epivalienamine from D-mannose as shown in Scheme 4.28. Starting from a partially protected mannose hemiacetal 244 with OH-2 free, which was synthesized from mannose in six steps on a multigram scale without the need for chromatography, vinyl Grignard addition gave the triol 245 as an inseparable diastereomeric mixture (3:1). Protection with an isopropylidene acetal (2,2-dimethoxypropane, DMF, CSA, rt; heating to 70 C) afforded the required five-ring compound 246 as the major component of an inseparable mixture. The diene 248 was obtained by the oxidation to the ketone 247, followed by Wittig methylenation. Before attempting metathesis, the isopropylidene protection was removed to furnish diastereomers 249a and 249b, which could now easily be separated by flash chromatography. The carbocycle 250 was

198

Validamycin and Its Derivatives

O

OH

BnO

BnO BnO

OH

BnO

OH

OH

BnO

O

O

O

OH BnO

OBn

OH

BnO

OBn 244

245

BnO

O

BnO

OBn 246

BnO

O

247

BnO

OH

O OBn

OH

+ BnO

O

BnO

OH

BnO

OBn

OBn 248

249a

249b

OH

OAc

BnO

OAc

BnO

BnO

OAc

OH

BnO

OAc

BnO BnO

OH OBn

OBn

OBn

251

252

OBn 250

BnO O BnO

N3

BnO BnO

OBn 253

OH

NHAc AcO AcO

OBn 254

NH2

HO OAc

HO

OH

OAc

OH

243

240

SCHEME 4.28 Synthesis of 2-epi-valienamine from D-mannose.

provided by treatment of diene 249a with the Grubbs’ second generation ruthenium complex in variable yields (22%76%), along with unidentified byproducts. The diol 249a was protected as a diacetate 250, which was ringclosed by Grubbs’ second generation complex in toluene at 60 C to give carbocycle 251 in 82% yield. The HoveydaGrubbs second generation ruthenium complex gave good cyclization results with both the diacetate and the diol under the same conditions. The acetates were removed from 251 by methanolysis to afford the respective carbocyclic diol 252. A better yield (71%) of the epoxide 253 was obtained by DPPA. Epoxide opening with azide gave the known α-lyxo configured derivative 254, which has been converted into 2-epi-valienamine 240 and its peracetate 243 by Shing.

4.2.3 Synthesis of 4-epi-Valienamine In the study of Palakodety Radha Krishna and P. Srinivas Reddy, they use L-serine as the starting material, and finally get 4-epi-valienamine 260 as shown in Scheme 4.29 (Krishna and Reddy, 2009). By a similar method, the intermediate 195a was applied as the reactant and subjected to the same set

Chemical Synthesis Chapter | 4 HO

CO2Me

MOMO

CO2Me OH

NBoc

O

190

O

193

HO

MOMO

MOMO

MOMO

OMOM O

OMOM

NBoc

194a

MOMO

MOMO

+

NBoc

NBoc

O

192

HO

OMOM

OMOM

NBoc

O

191

MOMO

OH

OMOM

NBoc

O

MOMO

CO2Me

+

NBoc

O

194b

OMOM NBoc

O

195a

MOMO

195b

MOMO

MOMO

MOMO BocHN OMOM

NHBoc

HO

199

MOMO

NHBoc

255

256

OMOM

MOMO

OMOM 258

OMOM 257

OH BocHN

OMOM

OMOM

OH

OAc AcHN

OAc

AcO

OAc 259

H2N

OH HO

OH 260

SCHEME 4.29 Synthesis of 4-epi-valienamine from L-serine.

of transformations as valienamine (mentioned in Section 4.1.9) to afford a nonnatural 4-epi-valienamine with 68% overall yield, which might be considered as a potential α-galactosidase inhibitor.

4.2.4 Synthesis of 1,2-bis-epi-Valienamine Also reported by Ramstadius et al. (2009), the synthesis route of 1,2-bis-epivalienamine 265 from D-mannose was shown in Scheme 4.30. The compound 252 was the key intermediate in this preparation, and synthesized as mentioned previously. Then the diol 252 was converted into its diimidate 261. Treatment of the diimidate with a Lewis acid at low temperature resulted in the formation of a major product. Adding water to the reaction mixture after the complete consumption of diimidate 261 resulted in hydrolysis of the first-formed oxazoline to give the amide 263. Subjection of the trichloroacetamide 263 to Birch conditions cleaved the benzyl ethers and reduced the trichloroacetamide group to an acetamide 264. The acetamide in 264 was cleaved with LiOH to give the 1,2-bis-epi-valienamine 265.

200

Validamycin and Its Derivatives HN OH

BnO BnO

O NH

BnO

OH

CCl3

BnO

O

OBn

OBn

261 O

BnO

262

CCl3 NH

BnO

CCl3 O

BnO

CCl3

OBn

252

N

BnO

NHAc

BnO BnO

OH

HO

OH

OBn 263

NH2

HO

OH

OBn

OH

264

265

SCHEME 4.30 Synthesis of 1,2-bis-epi-valienamine from D-mannose.

4.3 SYNTHESIS OF VALIOLAMINE Since the discovery of valiolamine, several total syntheses of valiolamine have been reported with different starting material.

4.3.1 Synthesis From D-Glucose (1L)-(1,3,4/2)-4-Azido-1,2,3-tri-O-benzyl-6-(trityloxymethyl)-5-cyclo-hexene1,2,3-triol (266), which was derived from D-glucose, was utilized as the synthetic precursor to synthesize chiral valiolamine 274 (Scheme 4.31) OTr

OTr

OBn

OTr

OBn

OBn

N3

OBn

NHAc

OBn

266 OTr

OAc 271

OAc

OH OBn NHCbz

OH OAc NHCbz

OAc

OBn

270

269

OAc

OH OBn OBn

OAc

OBn 272

OAc OH OAc OAc

OBn

268

OTr

NHCbz

NHCbz

OBn

267

OH

OBn

NH2

OBn

OH OBn

OTr

NHAc OAc

274

SCHEME 4.31 Synthesis of valiolamine from D-Glucose.

NHAc OAc

273

Chemical Synthesis Chapter | 4

201

(Ogawa et al., 1983c). The compound 266 was reduced with lithium aluminum hydride in boiling diethyl ether, unexpectedly, to give (1D)-(1/2,3)3-amino-1,2-di-O-benzyl-5-(trityloxymethyl)-6-cyclohexene-1,2-diol (267) as the major product. Its N-acetyl and N-benzyloxycarbonyl derivatives, 268 and 269, were obtained. Compound 269 was oxidized at 6070 C with a catalytic amount of osmium tetroxide in the presence of trimethylamine N-oxide, giving a valiolamine derivative 270 in 58% yield. Compound 270 underwent acetylation of only the secondary hydroxyl group at C-2 by treatment with acetic anhydride and base, giving 271. The trityl group of 271 was removed with acetic acid, and the resulting primary hydroxyl group was acetylated to give the diacetate 272. The diacetate 272 was catalytically hydrogenated with palladium-on-carbon for simultaneous removal of the benzyl and the benzyloxycarbonyl groups, and the product was then acetylated in the usual way, giving 273. 2,3,4,6-Tetrabenzyl-D-glucono-1,5-lactone was another intermediate derived from D-glucose and readily available. With it as the starting material, valiolamine could be synthesized as shown in Scheme 4.32. HOH2C

BnOH2C O

HO

BnOH2C O

BnO

HO

BnO

SCH3

BnO

OH

OH

O

BnO

OBn

O

OBn

275

BnOH2C OH

O

BnO

SCH3

BnO

SCH3

BnO OBn

OH

SCH3 OBn

277

H3CS O

BnOH2C

SCH3

276

BnOH2C BnO

OH

BnO

278

BnOH2C

SCH3

SCH3

BnO

BnO

BnOH2C

SCH3

BnO OBn

SCH3

O

O

BnO OH OBn 280

279

BnO

O

OH OBn 281

HOH2C HO HO OH OH

NH2

274

SCHEME 4.32 Synthesis of valiolamine from 2,3,4,6-Tetrabenzyl-D- glucono-1,5-lactone.

O

202

Validamycin and Its Derivatives

Shing Tony and Cheng Hau (2008) described a new and stereoselective intramolecular direct aldol reaction of diketones derived from carbohydrates has been developed to construct carbocycles with D-gluco-, D-galacto-, Dmanno-, and L-ido-configurations (Scheme 4.33). The stereochemical outcome of the aldol reaction of the diketone is dependent on the base used. First, the formation of carbocycles (aldols) from D-glucose was conducted, and it is noteworthy that L-proline-catalyzed the direct aldol reaction of diketone 282 prepared from D-glucose in 6 steps with 30.8% overall yield, to give cyclohexanones 283 regioselectively. The constitution of 283 was confirmed by conversion into known 274. The remaining stereocenter was installed by a reductive amination of 283 to give protected valiolamine 284. After acid hydrolysis, the valiolamine 274 was obtained in 88% yield with specific rotation.

O

O

OH

O O

six steps

O

O

O

D-glucose O

O

MeO

O OMe

282

O

O

MeO

OMe

283

OH HO

O

OH

NH2 HO O

NH2

O

MeO

OMe

HO

284

OH

274

SCHEME 4.33 Synthesis of valiolamine from D-glucose by Shing.

4.3.2 Synthesis From (2)-Quinic Acid (2)-Quinic acid was utilized as the starting material to synthesize valiolamine, as reported by Shing et al. As shown in Scheme 4.34, the total yield was 8.1% after 14 steps (Shing and Wan, 1996).

Chemical Synthesis Chapter | 4

203

OBn BnO

OAc

CO2H

HO

BnO

OAc

OAc OAc

O O

OBn

OBn

OH

HO

HO

O

OBn

O OH

OH

138

285

BnO

286

BnO

OAc

N3

OBn OH 288

HO

BnO

OAc

OAc

287

OAc

OAc

N3

OBn

OAc

N3

OBn

OTf

OAc

289

290

OH OH

H2N

OH OH 274

SCHEME 4.34 Synthesis of valiolamine from (2)-Quinic acid.

4.3.3 Synthesis From Valienamine and Validamine Valiolamine 274 was stereoselectively synthesized from valienamine 1 in a good yield of 67.06%, as shown in Scheme 4.35. N-(Benzyloxycarbonyl) valienamine (291), an acyclic carbamate derivative of valienamine, was treated with bromine to afford the bromocyclitol cyclic carbamate 293. This cyclization reaction is thought to proceed by a mechanism similar to halolactonization. This regioselective intramolecular cyclization reaction is explained by Markovnikov’s rule. The bromocyclitol cyclic carbamate 293 was dehalogenated reductively with sodium borohydride, and the resulting dehalogenated cyclic carbamate 294 was hydrolyzed with barium hydroxide to give 274 (Horii et al., 1985). Validamine 295 could also be converted into valiolamine, as shown in Scheme 4.36. The 7-deoxy-7-iodo derivative 296d of tri-O-acetyl-N-(benzyloxycarbonyl)validamine, prepared from 295 by a four-step sequence via 296a296c, was treated with silver fluoride in pyridine to give the exomethylene derivative 297a. Hydrolysis of 297a with ammonium hydroxide

204

Validamycin and Its Derivatives

HOH2C HOH2C

Br

HOH2C 91%

HO

90%

HO

HO

HO

OH

HO

HO

OH

H 2N

NH

C

OH

OBn

NH

O C

O

1

O

291

HOH2C

HOH2C

HOH2C

Br

89%

92%

HO

HO

HO

HO

HO

HO OH

O

Bn

292

OH

O

NH

OH

OH

NH

NH2

O

O

O

O 293

294

274

SCHEME 4.35 Synthesis of valiolamine from valienamine.

HOH2C

CH2

CH2R1

HO

R2O

R2O

HO

R 2O

R 2O

OH

OR2

NH2

HN

C

OR2

HN

C

OBn

OBn O

296a: R1=OH, R2=H 296b: R1=OTs, R2=H 296c: R1=OTs, R2=Ac 296d: R1=I, R2=Ac

295

O 297a: R2=Ac 297b: R2=H

XH2C

Br

HOH2C R 2O

R 2O OR2 O

O

O

OR2

NH

NH

HO OH

C

C

298

HO

R 2O

R 2O

Bn

OH NH2

O

299a: X=Br, R2=H 299b: X=Br, R2=Ac 299c: X=OAc, R2=Ac

274

SCHEME 4.36 Synthesis of valiolamine from validamine.

gave the de-O-acetate 297b. The exomethylene derivatives 297a and 297b were treated with bromine to give the (bromomethyl)cyclitol cyclic carbamates 299b and 299a, respectively, presumably via the transient intermediate 298. The (bromomethyl)cyclitol cyclic carbamate 299b was treated with silver acetate to give the acetoxymethyl derivative 299c, which was identical to

Chemical Synthesis Chapter | 4

205

the tetra-O-acetate of 274. Hydrolysis of 299a and 299c with barium hydroxide gave 274 (Horii et al., 1985). In 2013, Li Ji reported an efficient and practical synthesis of (1)-valiolamine starting from readily available aminocyclitol (1)-valienamine in five steps and up to 80% total yield in gram-scale quantities (Ji et al., 2013). The general route was shown in Scheme 4.37. AcO OH

OAc O

HO

Ac2O

HO

NH2

AcO

[O]

AcO

AcO

AcO

NHAc

OH

OAc

1

30

300

AcO

HO OH

[H]

NHAc OAc

OH

AcO

HO

AcO

NHAc

HO

OAc 301

NH2 OH 274

SCHEME 4.37 Synthesis of valiolamine from valienamine by Li Ji.

4.3.4 Synthesis From (2)-vibo-Quercitol Convenient and practical synthesis of (1)-valiolamine from (2)-vibo-quercitol, 1-deoxy-epi-myo-inositol, readily obtained by bioconversion of myo-inositol, is described by Seiichiro Ogawa (Scheme 4.38) (Ogawa et al., 2004a). (2)-vibo-Quercitol 302, which could readily be obtained by stereospecific microbial dehydration of myo-inositol, is biochemically oxidized under the influence of Gluconobacter sp. AB10277 to produce about 80% yield of crude 2-deoxy-scyllo-inosose, 2L-(2,4/3,5)-2,3,4,5-tetrahydroxycyclohexan1-one 303. Treatment of 303 with 2 molar equiv of diazomethanediethyl ether was carried out in methanol for 7 h at room temperature. On addition of excess diethyl ether, a sole spiro epoxide 304 crystallized out from the reaction mixture in 44% yield. Hydrolysis of 304 with 3 M aqueous potassium hydroxide for 6 h at 100 C gave, after chromatography, a crystalline (2)-β-valiol 305 in 32% yield. On the other hand, nucleophilic substitution of 304 with excess of sodium acetate in 80% aqueous DMF for 3 h at 120 C afforded, after acetylation with acetic anhydride in pyridine, the penta-Oacetyl derivative (B100%), which was treated with methanolic sodium

206

Validamycin and Its Derivatives

HO

OH

O

OH

O

HO

OH

OH

HO HO

OH

HO

OH

HO

OH

HO

OH

OH

OH

OH

OH

302

303

304

305

OH

OH

OH

OH

OH

O Ph

O O

OH

O

Ph

O

OH

Ph

O

O

O

306

O

307

308

OH

OH

OH N3

O Ph

OTs

O

O

O

NHAc

O Ph

O

O

309

O O

310

NH2

HO HO

OH OH

274

SCHEME 4.38 Synthesis of valiolamine from (2)-vibo-quercitol.

methoxide to give 305 quantitatively. Then, benzylidenation of 305 was carried out by treatment with α,α-dimethoxytoluene and TsOH-H2O in dry DMF for 4 h at room temperature, giving the desired compound 306 (59%). Treatment of 306 with 2.5 molar equiv of 2-methoxypropene in the presence of TsOH-H2O in DMF for 4 h at room temperature gave a mixture of the products, which were easily fractionated on a silica gel column to give307 in 41% yield. Treatment of 307 with excess p-toluenesulfonyl chloride in pyridine gave the tosylate 308 (B100%). Direct nucleophilic substitution of 308 with an azide anion in the presence of 15-crown-5 ether in DMF proceeded smoothly at 120 C to afford a single azide 309 (88%) selectively. Formation of elimination products was not observed. Hydrogenation of 309 with Raney nickel catalyst in ethanol in the presence of excess acetic anhydride gave the amide 310 (76%). This compound was deacylated with 2 M hydrochloric acid at 80 C to give, after purification over a column of Dowex 50 W 3 2 (H) resin with 5% aqueous ammonia, valiolamine 274 (90%) as a syrup.

4.3.5 Synthesis From myo-Inositol In 2007, Seiichiro Ogawa and Miki Kanto described a convenient and practical synthesis of valiolamine and its related carbaglycosylamine glycosidase inhibitors from myo-inositol (Ogawa and Kanto, 2007). The general synthesis route was shown in Scheme 4.39.

Chemical Synthesis Chapter | 4 O

OH

OH

O OH

OH

OH

HO

OH

HO

207

biocatalysis

HO

311

302

303

O

OH

HO

O

O

OH

OH

HO

OH

O

TsO O

314

O

Ph

315

Ph

OH

HO

HO

HO O

O

N3

O O

316

O

OH

313

Ph

HO

OH

O

312

Ph

HO

HO

OH

OH

OH

OH

HO

OH

HO

HO

OH OH

AcHN

O

OH

H2N

OH

O

OH

317

274

SCHEME 4.39 Synthesis of valiolamine from myo-inositol.

In 2011, an efficient formal synthesis of rac-valiolamine starting from readily available myo-inositol was reported by Rajendra C. Jagdhane and Mysore S. Shashidhar (Jagdhane and Shashidhar, 2011). In all the synthetic steps only one regioisomer is formed, which circumvents laborious purification of products. Regioselective benzylation of myo-inositol orthoformate, super-hydride-mediated deoxygenation of a cyclitol derivative, and stereoselective addition of dichloromethyllithium to an inosose are the key reactions in the synthesis. Unfortunately, the optical product was not obtained by this method.

4.3.6 Synthesis From L-Tartaric Acid As mentioned previously, in 2012, Hong-Jay Lo et al. described a C2-symmetric poolbased flexible strategy to synthesize (1)-valienamine. With the intermediate 187, (1)-valiolamine could also be obtained by several steps (Scheme 4.40) (Lo et al., 2012). Asymmetric dihydroxylation of 187 with K2OSO4 mediated by (DHQD)2-AQN led to unsaturated dihydroxycarbonimidothioates 318 with complete regioselectivity and good diastereoselectivity. With 318, the epoxy carbonate 319 was obtained by iodine-promoted N-cyclization in the presence of K2CO3. Finally, efficient formation of 274 was completed by reductive

208

Validamycin and Its Derivatives

OH O

OH

OMe OMe

O

181

OH

O

HO

O O

O

O

HO 185a

184

183

O

+

O

O

+

HO

HO 186a

185b

186b

O

O

O

O OH

OH

O O

OH O

O

O

NBn

NBn

MeS

MeS 187

O

O

O O

O

HO

182

O

O

O

O

O

OH O

O

O

HO

NBn O

318

319

OH OH

HO OH HO NH2 274

SCHEME 4.40 Synthesis of valiolamine from D-tartaric acid.

cleavage of the epoxide and the N-benzyl group in carbamate 319 with Li/NH3 followed by LiOH-promoted carbamate hydrolysis and HOAc-assisted acetal deprotection. This sequence required only nine steps to obtain (1)-valiolamine 274 and proceeded in 12.6% overall yield from the common D-tartaric acid.

4.3.7 Synthesis From (2)-Shikimic Acid Total synthesis of (1)-valiolamine 274 from the naturally abundant ()-shikimic acid 208 was described by Na Quan et al. in 2013 (Scheme 4.41) (Quan et al., 2013). Ethyl 3-epi-5-O-methylsulfonyl-shikimate 325, as the key common intermediate, was first synthesized in five steps in 74% overall yield, and then converted into the target compound in seven steps in 48% overall yield.

Chemical Synthesis Chapter | 4 HO

CO2H

HO

CO2Et

Cl

CO2Et

209 CO2Et

O HO

HO

OHCO

OH

OH

208

OCHO

320

OH

321

HO

CO2Et

CO2Et

322

CO2Et

O

O

HO

HO

CO2Et

OMs

OMs

OH 323

324

TBDPSO

325

CO2Et

TBDPSO OAc

HO H

AcO

HO

N3

H

326

H

N3 327

328

OH

OH OH

OH

OH

TBDPSO

OH

HO OAc

AcO

OH

HO H

N3

329

N3

H

N3 330

HO OH

HO H

NH2 274

SCHEME 4.41 Synthesis of valiolamine from (2)-shikimic.

4.4 SYNTHESIS OF VALIDAMINE Since the discovery of validamine, several methods for validamine syntheses have been reported, including racemic and optically active validamine. But in this book, only the optically active validamine, (1)-validamine 331, is discussed.

4.4.1 Synthesis From (2)-7-endo-Oxabicyclo[2.2.1]-Hept-5Ene-2-Carboxylic Acid With (2)-7-endo-oxabicyclo[2.2.1]-hept-5-ene-2-carboxylic acid 332 as the starting material, the optically active validamine was formed (Scheme 4.42) in seven steps with a total yield of ,17.49% (Ogawa et al., 1985a). The synthesis was carried out in the following sequence. Treatment of 332 with 90% formic acid and 35% hydrogen peroxide gave the hydroxy lactone 333 in 66% yield. Compound 333 was reduced with lithium aluminum hydride

210

Validamycin and Its Derivatives

O

HO

O

O

AcO

O

OAc CH2OAc

O

COOH 332

333

BrH2C

334

AcOH2C

AcO

Br

AcO

AcOH2C

AcO

Br

AcO

AcO AcO

OAc

OAc

335

OAc N 3

336

AcOH2C

337

HOH2C

AcO

HO

AcO

OAc

338

HO

OH

NHAc

NH2

331

SCHEME 4.42 Synthesis of validamine from (2)-7-endo-oxabicyclo[2.2.1] hept-5-ene-2-carboxylic acid.

in THF and then acetylated to give the triacetate 334, which, without purification, was directly subjected to acetolysis to give a mixture of fully acetylated derivatives of pseudosugars. Next, treatment of the triacetate 334 with 20% hydrogen bromide at 85 C for 20 h gave the crystalline dibromide 335 in 53% yield. Compound 335 was selectively converted into the bromide 336 by treatment with sodium acetate in 90% aqueous 2-methoxyethanol at 85 C, followed by acetylation. The secondary bromo group was then displaced with an azide ion via an SN2 reaction to give the azide 337, which was hydrogenated in the presence of Raney nickel and acetylated to give the penta-N,O-acetate 338 in 50% yield. Finally, de-O-acetylation of 338 with 10% NaOMe-MeOH (25 C, 3 h), followed by de-N-acetylation with 80% aqueous NH2NH2 in a sealed tube (100 C, 72 h), furnished 331.

4.4.2 Synthesis From D-Glucose With D-glucose as the starting material, validamine was synthesized by Yoshikawa et al. (Scheme 4.43) (Yoshikawa et al., 1994). Treatment of a nitrofuranose derivative, 339, with KF in DMF in the presence of 18-crown6 (23 C, 3 h) yielded a nitroolefin, 340. When 340 was treated with 28% liquid NH3 in THF at 278 C for 2 h and the product was acetylated with Ac2Oand p-TsOH,H2O, 1R-acetamide 341 was obtained. Elimination of the

Chemical Synthesis Chapter | 4 BzO

BzO

NO2

O OH 80%

H

D-glucose

82% AcO

O2NH2C BnO

OH

BnO

339 BzO

BzO NHAc

56% NHAc

AcO OAc

BnO

OAc 340

NO2

AcO

211

BnO

341

88%

OAc 342

AcO

HO 90% NHAc

AcO

AcO

NH2

HO

OAc 338

HO

OH 331

SCHEME 4.43 Synthesis of validamine from D-glucose.

nitro group in 341 with n-Bu3SnH in benzene in the presence of azobisisobutyronitrile (AIBN) (80 C, 3 h) formed 342. After removal of the acetyl groups and the benzoyl group in 342 with 1% NaOH-MeOH, the product was subjected to debenzylation (Na, liquid NH3, 278 C, 30 min) and subsequent acetylation with Ac2O in pyridine, to provide pentaacetylvalidamine (338). Finally, de-O-acetylation of 338 with 10% NaOMe-MeOH (25 C, 3 h), followed by de-N-acetylation with 80% aqueous NH2NH2 in a sealed tube (100 C, 72 h), furnished 331. So, the synthesis of validamine from D-glucose was achieved with an overall yield of 29.09%.

4.4.3 Synthesis From Nitrofuranose Yoshikawa et al. reported the synthesis of validamine from the nitrocyclitol, which is an intermediate in the synthesis of pseudo-D-glucopyranose (Scheme 4.44). The synthesis was accomplished in five steps from 343 to 331 with an overall yield of 23.36% (Yoshikawa et al., 1988).

212

Validamycin and Its Derivatives BzO

BzO

NO2 64%

OH

HO

BzO

73%

OAc

AcO

BzO

OH

BzO

NO2

343

NO2 NHAc

AcO

BzO

OAc

BzO

OAc 345

344

HO

AcO

50%

100%

NHAc

AcO

BzO

OAc

AcO

HO

OAc

346

NH2

HO

NHAc

AcO

OH

338

331

SCHEME 4.44 Synthesis of validamine from Nitrofuranose. OBn OBn

OBn

HO

COOH OBn

O

HO

N3

O

S

O

OBn

98%

OBn

OH OH

OBn

70%

24%

N3

OBn

OBn OAc

OH O

138

347

348

OBn

349

OBn

OBn

OBn OBn

81%

96%

N3

OBn OTf 350

OBn

98%

N3

OBn AcO 351

36%

N3

OBn HO 352

OAc

OAc

AcHN

OAc AcO 338

SCHEME 4.45 Synthesis of validamine from (2)-Quinic Acid.

4.4.4 Synthesis From (2)-Quinic Acid In this synthesis, a regioselective cyclic sulfate opening was the key step (Scheme 4.45), and the penta-N,O,O,O,O-acetate 338 was the product. The preparation of 338 was carried out in 15 steps with a total yield of 1.06%.

Chemical Synthesis Chapter | 4

213

The product, 338, could be deacetylated smoothly to give the free target molecule 331 in quantitative yield (Shing and Tai, 1995).

4.4.5 Synthesis From D-Xylose Compound 103, which was derived from D-xylose, was utilized for the synthesis of validamine by Tatsuta et al. (Scheme 4.46). The synthesis of TrO

O

HO O

D-xylose

MeO O

78.3%

73%

O

OH

OTBS

c

OTBS

103

354

MeO O

OTBS

353

MeO MeO

94% O

OTBS

OH

O

MeO

OTBS

OTBS

OH 70%

92%

SO2Ph

OTBS

SO2Ph

MeO OTBS

OTBS 355

OTBS 356

O

O

OH TBSO

TBSO

SO2Ph

TBSO

OH 84%

OH

80%

82%

TBSO

TBSO

TBSO OTBS

OTBS

OTBS 357

359

358

OMOM

OMOM

OMOM

HO

HO

O OMOM

OMOM

OMOM

90%

81%

100%

HO

HO OH 360

O N3

N3

361

362

OMOM OH

OMOM O OMOM 100%

O

HO OMOM

n

O

o

O NH2 363

OH

100%

HO

NH2

NH2

364

331

SCHEME 4.46 Synthesis of validamine from D-Xylose.

214

Validamycin and Its Derivatives

validamine was achieved in 15 steps with an overall yield of 13.90% (Tatsuta et al., 2000).

4.5 SYNTHESIS OF HYDROXYVALIDAMINE The (1)-hydroxycalidamine was first reported by Satoshi Horii et al. in 1971. It appeared to be one of the degradation products of validamycin B by hydrogenolysis, followed by acid hydrolysis with acid (Horii et al., 1971). DL-(1, 3, 4/2, 5, 6)-4-Amino-6-hydroxymethyl-1, 2, 3, 5-cyclohexanetetrol (DL-hydroxyvalidamine) was first synthesized as the peracetate, which confirmed the proposed structure of the branched-chain aminocyclitol derived from validamycin B by Seiichiro Ogawa et al (Ogawa et al., 1980). The synthesis of DL-hydroxyvalidamine was achieved in seven steps as shown in Scheme 4.47. CH2OBz

CH2Br

CH2OH

CH2 O

Br OAc

OAc OAc

OH

OAc

O

OH OAc

OAc 365

OH

366

368 CH2 OAc

CH2 OAc

OAc

O

O

O

OAc

PhHC

O

370

NHAc OAc

OAc

OAc 369

OAc

PhHC N3

O

O

HO

OAc

367

CH2 O PhHC

OAc

PhHC

371

CH2 OH

OH NH2

OH

OH 372

SCHEME 4.47 Synthesis of Hydroxyvalidamine by Ogawa in 1980.

Later in 1983, Seiichiro Ogawa described another route for the synthesis of the peracetyl racemate 381 of hydroxyvalidamine 372, and the result fully confirmed the proposed structure of 372 (Scheme 4.48). The sequence of

Chemical Synthesis Chapter | 4

215

Br AcO AcO

OAc OAc

OBz

Br

AcO

OAc 373

OH OH

OH HO

374

375 O OAc OAc

OH OH O

O

O

O

376

O

O

Ph

Ph

Ph 377

378

OAc Ph

OAc OAc

OAc

O O AcO

Ph

O O AcO OAc NHAc 380

OH

OAc

AcO AcO

OAc N3 379

OAc

OAc NHAc 381

OH

HO HO OH NH2 372

SCHEME 4.48 Synthesis of Hydroxyvalidamine by Ogawa in 1983.

reactions involves stereospecific peracid oxidation of the protected branchedchain unsaturated cyclitol 377 to give rise to a versatile epoxide 378 and regioselective azidolysis of 378 followed by reduction of the azido group with hydrogen sulfide (Ogawa et al., 1983a).

4.6 SYNTHESIS OF VALIDOXYLAMINES 4.6.1 Synthesis of Validoxylamine A Validoxylamine A, [(1S)-(1, 4, 6/5)-3-hydroxymethyl-4, 5, 6-trihydroxycyclohex-2-enyl] [(1S)-(1, 2, 4/3, 5)-2, 3, 4-trihydroxy-5-hydroxymethylcyclohexyl]amine, was first isolated from the hydrolysate of antibiotic validamycin A with 1M sulfuric acid, and was later found to exist in the fermentation broth of Streptomyces hygroscopicus. Since the discovery of validoxylamine A, several methods for validamine syntheses have been reported. Seiichiro Ogawa reported the first synthesis of the isomers of validoxylamine A in 1981 (Scheme 4.49) (Ogawa et al., 1981). Two racemic isomers of validoxylamine A were synthesized by the condensation reaction of the blocked DL-validamine, the allyl bromide and the precursor of the

OAc

OH

O

Br

O

Br

OAc

Br

OH

OAc

H

OH

O N3

O

O

383

H

O

O

OAc 382

H

O

OH

O

O

384

O

385 CH2OR1

386 OR2

H CH2Br

O

H R2O CH2OR1

OR2

H O

+

OR1

OAc

HN

CH2

+

OR2 NH2

O O

OAc

OAc OH

387

OR1

HN

CH2

OH OR3 OR3

386

NH2

O

OR3 OR3 OR3

OR3

388: R1=-C(CH3)2, R2=-C(CH3)2, R3=Ac 389: R1=Ac, R2=Ac, R3=Ac 390: R1=H, R2=H, R3=H SCHEME 4.49 Synthesis of validoxylamine A by Ogawa in 1981.

OR3 OR3

Chemical Synthesis Chapter | 4

217

unsaturated branched-chain cyclitol moiety. The compound 386 was synthesized and then reacted with 387 to afford 388. Treatment of the isomer mixture 388 with 70% aqueous acetic acid (room temperature, overnight) and successive acetylation gave the corresponding octa-O-acetyl derivatives 389. De-O-acetylation of 389 was effected by treatment with methanolic sodium methoxide in methanol to furnish 390, the isomers of validoxylamine A. Later, in 1982, Seiichiro Ogawa reported the total synthesis of the racemic form of validoxylamine A. The reaction was undertaken with DL-3,4-di-Oacetyl-1,2-anhydro-5,7-O-benzylidene-(1,2,4,6/3,5)-1,2,3,4,5-cyclohexanepentol to construct the pseudodisaccharide, in which two kinds of polyhydroxy(hydroxymethyl)-cyclohexane are combined by way of an imino linkage. The reaction was 12 steps in total (Ogawa et al., 1982a). In 1983, Tatsushi Toyokuni et al. described a method for the preparation of the two racemic isomers of validoxylamine A, by use of coupling reactions of the protected DL-validamine and the allyl bromides, the precursors of the unsaturated branched-chain cyclitol moiety. The racemic diastereomers thus formed can be separated by chromatography on silica gel. It indicated that optically pure validoxylamine A analogs should be obtained if chiral validamine derivative is used in place of the racemate (Toyokuni et al., 1983). In 1984, Seiichiro Ogawa et al. reported another synthesis route to obtain validoxylamine A. For construction of these types of pseudodisaccharides containing an imino linkage, a coupling reaction of the protected anhydro derivative of DL-pentahydroxy(hydroxymethyl)cyclohexane with the DL-trihydroxy(hydroxymethyl) cyclohexylamine or cyclohexenylamine was undertaken (Ogawa et al., 1984). Until 1988, (1)-validoxylamine A was synthesized by selective deoxygenation of (1)-validoxylamine B derivative, which was obtained by the coupling of the partially protected (1)-valienamine and (1R,2S,5R,7R,8R,9R, 10R)-8,9-dibenzyloxy-5-phenyl-4,6,11-trioxatricyclo undecane (Ogawa and Miyamoto, 1988). The general route was shown in Scheme 4.50. In 2004, Zheng Yuguo et al. described a method using resin-catalyzed degradation of validamycin A for production of validoxylamine A (Scheme 4.51). Different cation resins, IR-120 and HZ-001, were used as catalysts to prepare validoxylamine. A through validamycin A degradation. Herein, influences of reaction time, reaction temperature, resin usage, initial validamycin A concentration, as well as external and internal diffusion limitation, on the degradation ratio of validamycin A were studied. Results demonstrated that validamycin A could almost completely be degraded into validoxylamine A under optimized operation conditions of 5.0% (w/v) resin usage, 1.0% (w/v) initial validamycin A concentration, agitation speed of 200 rpm, and heating at 100 C for 5 h (Zheng et al., 2004). As a competitive trehalase inhibitor and lead compound with broad uses, validoxylamine A has been increasingly studied for its many possible

218

Validamycin and Its Derivatives OBn

OBn Ph O BnO

+

BnO BnO

BnO BnO

O

BnO

O

OBn HN OH

OBn NH2

OBn

OBn

391

393

392 OBn

O

OBn O BnO BnO

BnO BnO OBn HN

S O

Ph

BnO OBn N

BnO

CSCH3 OBn OBn O

395

394

O

O

O Ph Ph OH OBn

OBn

BnO BnO OBn HN

HO HO

BnO BnO

BnO

OH HN

BnO OBn HN

HO

H OH

H

S

OBn

OBn

OH 396

O

397

O

398

OH

O

O Ph

Ph

SCHEME 4.50 Synthesis of (1)-validoxylamine A by selective deoxygenation of (1)-validoxylamine B derivative.

applications. The process of acid-catalyzed hydrolysis of validamycin A was investigated for production of validoxylamine A (Scheme 4.52). The effects of various acids and substrate concentrations were explored, and the mechanism of acid-catalyzed hydrolysis of validamycin A was proposed. Results demonstrated that the optimal condition of acid hydrolysis of validamycin A included 1M H2SO4 and a 30% (w/v) initial validamycin A concentration in a 1-h reaction time (Jin et al., 2006).

Chemical Synthesis Chapter | 4 CH2OH

219

OH

OH HOH2C

N

OH

H

OH

Catalysis

+H2O

OH O

OH

OH

OH 399

O CH2OH

CH2OH

OH

OH

OH N

OH

H

OH

+

OH HOH2C OH

OH OH

O CH2OH

OH 398

SCHEME 4.51 Synthesis of (1)-validoxylamine A by resin-catalyzed degradation of validamycin A.

SCHEME 4.52 Synthesis of (1)-validoxylamine A by acid-catalyzed hydrolysis of validamycin A.

4.6.2 Synthesis of Validoxylamine B Validoxylamine B, [(1S)-(1,4,6/5)-4,5,6-trihydroxy-3-hydroxymethyl-2-cyclohexenyl][(1S)-(1,2,4/3,5,6)-2,3,4,6-tetrahydroxy-5-(hydroxymethyl)cyclohexyl]amine, which was first obtained by acid hydrolysis of antibiotic validamycin B, was also isolated from the fermentation broth of Streptomyces hygroscopicus. The synthesis of DL-validoxylamine B was firstly reported by Seiichiro Ogawa in 1982. It was synthesized by the reaction of DL-4,7:5,6-di-O-isopropylidene-(1,4,6/5)-4,5,6-trihydroxy-3-hydroxy-methy 1-2-cyclohexenylamine with DL-3,4-di-O-acetyl-1,2-anhydro-5,7-O-benzylidene-(1,2,4,6/3,5)-6-hydroxymethyl-1,2,3,4,5-cyclohexane-pentol, followed by removal of the

220

Validamycin and Its Derivatives Cl

Cl O

OAc

OAc

OAc

OAc OAc

OAc 400

O

O O

OH N3 OAc

OAc

O N3

O

N3 OH

OH

404

403

402

OH

OAc

OAc

OAc

401

OAc

OAc OAc

OH OAc

OAc

OAc

NH2

O

O

405

O

406

407

O

O

O

OH

O

Ph +

O

O

OAc NH2

O

O

O

AcO N H

O

OAc

407

OAc 409

OAc OH

O Ph

N H

O

O

OH

OAc

OAc

AcO N H

OAc OAc

OAc OAc

411

410 OH

HO

OAc

AcO

O

O

Ph O

O

408

O

+

O

OH

OH

HO N H

OH OH

OH OH

412

SCHEME 4.53 Synthesis of DL-validoxylamine B by Seiichiro Ogawa in 1982.

protecting groups. The reaction was in total 19 steps, and the detailed pathway was shown in Scheme 4.53 (Ogawa et al., 1982b). In 1988, Seiichiro Ogawa et al. reported the synthesis of validoxylamine B coupling of the epoxide 420 with the amine 415 affords compound 421, the structure of which can be established by converting 421 into validoxylamine B nona-O-acetate, and then deprotected to give the validoxylamine B 422. The detailed pathway was shown in Scheme 4.54 (Ogawa et al., 1988).

221

Chemical Synthesis Chapter | 4 OBzl

OAc

AcO

OAc

AcO

BzlO N3

OBzl

BzlO

OBzl N3

BzlO

413

OBzl NH2

BzlO

414

415

OBz

Br

O Ph

AcO AcO

Br

O

OAc OAc

AcO

OH

OH

OAc 416

417

418 O

O

O Ph

Ph OBzl OBzl

O

OBzl OBzl

O

419

420 OBzl BzlO BzlO

O

OBzl

BzlO

O

OBzl HN

OH

+

OBzl Ph

BzlO

OBzl NH2

BzlO

OBzl OBzl

O

O 415

420

421

OH

O Ph

HO HO HO

OH HN

OH OH

422 OH HO

SCHEME 4.54 Synthesis of validoxylamine B by Seiichiro Ogawa in 1988.

4.6.3 Synthesis of Validoxylamine G The first total synthesis of the antibiotic validoxylamine G as its octa-acetate was reported by Seiichiro Ogawa in 1990. There are 12 steps in the synthesis, and the detailed reaction pathway was shown in Scheme 4.55 (Miyamoto and Ogawa, 1990).

222

Validamycin and Its Derivatives OTr Br O

O

AcO AcO

OH

O

O

Br

OAc

OAc

OAc

424

423

425

OTr

O Ph

O

O BnO

OBn

O

Ph

O

O O BnO

OBn

OBn OBn

OBn

OBn 426

428

427 OBn BnO BnO BnO

OBn

OBn HN

OH OBn

428 + BnO

BnO

BnO

OBn

NH2

O 430

429

O Ph

OBn OAc BnO AcO BnO BnO

OBn HN

431

AcO AcO

SAr OBn

OAc HN OAc

432

BnO

HO O AcO

O

OAc

OH Ph

HO HO HO

OH HN OH 433

HO HO

OH

SCHEME 4.55 Synthesis of validoxylamine G by Seiichiro Ogawa in 1990.

In 1992, Hiroshi Fukase and Satoshi Horii described a synthesis route by the application of branched-chain inosose derivatives 434 and 442 to the total synthesis of natural N-substituted valiolamine derivatives validoxylamine G (Scheme 4.56) (Fukase and Horii, 1992).

Chemical Synthesis Chapter | 4 BnOH2C

BnOH2C O

BnO BnO

CH2P(OCH3)2 OBn OH

O

OBn 434

BnOH2C

435 BnOH2C

O OH OH CH2P(OCH3)2 OBn

BnO BnO

O

O

BnO BnO

O

OH

BnO BnO

CH2P(OCH3)2 OBn O 437

436 BnOH2C

BnOH2C

BnO BnO

223

OBn

BnOH2C

BnO BnO

O

BnO BnO

OH OBn

O

439

438

OBn N

O

440

BnOH2C BnO BnO

OBn NH2

OH

441

HO BnOH2C BnO BnO

HO HO

BnOH2C + BnO BnO OBn

OH HN

HO

OH OBn

O

HO

NH2 441

442

433

HO

OH

SCHEME 4.56 Synthesis of validoxylamine G by Hiroshi Fukase in 1992.

Shing Tony and Cheng Hau (2008) reported that a new and stereoselective intramolecular direct aldol reaction of diketones derived from carbohydrates has been developed to construct carbocycles with D-gluco-, D-galacto-, D-manno-, and L-ido-configurations. The stereochemical outcome of the aldol reaction of the diketone was dependent on the base used. Facile conversion of D-gluco-cyclohexanones into validoxylamine G has been achieved in 12 steps with 15.1% overall yield from D-glucose (Scheme 4.57). As described in Section 4.3.1, the amine 284 was protected as TMS-ether 443. Palladium-catalyzed coupling of 443 with chloride 444 smoothly gave blocked validoxylamine G in an excellent yield. Acid hydrolysis then afforded validoxylamine G 433 in 88% yield.

224

Validamycin and Its Derivatives O

OH

O

O

OH

O

six steps

O

D-glucose O MeO

O

O

OMe

MeO

O

O

282

O

O

O

OMe

MeO

O

NH2 O

283

O OMe 284

OTMS NH2

O O

O

MeO

OMe 443

O

443

H N

O O

O

OTMS

Cl

O

+

O

OMe MeO

HO H N

HO HO

OH

O O

O OMe

444

OH

O

O

MeO

HO

OMe

MeO

O

445

OH OH OH

433

SCHEME 4.57 Synthesis of validoxylamine G by Shing in 2008.

4.7 SYNTHESIS OF VALIDAMYCINS 4.7.1 Synthesis of Validamycin A In 1983, Seiichiro Ogawa reported the first synthesis of validamycin A started from validoxylamine A as shown in Scheme 4.58 (Miyamoto and Ogawa, 1989; Ogawa et al., 1983b, 1985b). The antibiotic validamycin A was synthesized for the first time by glycosylation of the partially protected derivative 450 of the aglycone, validoxylamine A, with 2,3,4,6-tetraO-acetyl-α-D-glucopyranosyl chloride 451, followed by deprotection, thereby establishing the structure previously assigned.

Chemical Synthesis Chapter | 4 OH

OH

N H

OH

OAc

O Ph

OH HO

OH

OAc

OAc O

OH

O

N H

O

OAc

OH 398

Ph

446 OAc

AcO

OBn

BnO OAc

OAc

OBn

OBn

O

N H

OAc

O

OBn

OBn

OBn

BnO

OBn HO

OBn N H

OBn AcO

OBn OH

N H

OBn

OBn

OH

OBn

449

450 OBn

BnO

OAc

O

N H

OBn OAc

AcO OAc

OBn AcO

OBn

O OAc

OBn

451

452

OBn

BnO

N H

OBn

OAc

AcO

OBn

O OBn

OAc

O

AcO OAc

Cl

Ph

448

BnO

+

O

OBn

Ph 447

OBn

O

N H

OAc

450

225

O

BnO OBn

OBn BnO

OAc

O

N H

OAc

OBn

OBn

O OAc

OAc

453

OAc

AcO OAc

OAc AcO

454 OH

HO

O OH N H

OH

OH HO

OH

HO OH O OH

OH 455

SCHEME 4.58 Synthesis of validamycin A from validoxylamine A.

4.7.2 Synthesis of Validamycin B The first complete synthesis of the antibiotic validamycin B as its dodeca-Oacetate was reported by Ogawa in 1988. As shown in Scheme 4.59, coupling of the epoxide 457 and the partially protected valienamine derivative 456, followed by deprotection, gave the product 458, which was identified by 1H NMR spectroscopy. Finally, by a normal deprotection reaction, the desired validamycin B was obtained (Ogawa and Miyamoto, 1987; Ogawa et al., 1988).

226

Validamycin and Its Derivatives OBzl

OBzl

BzlO

BzlO BzlO BzlO

OBzl N H

OBzl N H

BzlO BzlO

OH OBzl

O

421

OBzl OBzl

O

455

O

O

Ph

Ph OBzl BzlO BzlO BzlO

OBzl N H

OBzl OBzl

456 OH AcO OAc AcO OAc

456

OAc N H

AcO AcO

O

AcO + AcO

OAc Br

OAc OAc AcO

O

OAc

AcO

OH

OAc

HO HO HO

AcO

458

457

OH

N H

OH OH HO

O

HO

459 OH

HO OH

SCHEME 4.59 Synthesis of validamycin B.

4.7.3 Synthesis of Validamycin C (1)-Validamycin C was first completely synthesized by use of a common blocked derivative 460 of (1)-validoxylamine A (Scheme 4.60). The diols 461, obtained by acid hydrolysis of 460, were appropriately protected to give the aglycone 466, which were condensed with glycosyl donor 463 to afford

Chemical Synthesis Chapter | 4

227

OH O

BnO

BnO

O

Ph

HO

OBn N H

BnO

OBn N H

BnO OBn

O

460

OBn

O

461

O

O Ph

Ph OBn BnO

OBn O

OBn

BnO BnO

463

OAc

BnO

O

O

O OBn NHX

BnO

BnO BnO OBn

464

462

Ph

OAc

BnO

OBn NHX

BnO

O

X=

OAc

463 OAc O

OAc O BnO BnO OBn

BnO BnO O

O

OBn

BnO

BnO

BnO

465

466

OBn

HO

BnO

OBn HN

BnO

OBn HN

BnO

OBn

AcO

HO

HO

OAc O RO RO OAc 466 +

O

OR

O

RO

AcO AcO

OR HN

RO

OAc Br 467

RO

468: R=Bn 469: R=Ac

OR AcO

AcO OH O HO HO

O

O AcO

OAc OAc

OH

O HO HO

OH HN

HO

OH HO

HO 470

O HO

O

OH OH

SCHEME 4.60 Synthesis of validamycin C.

the condensate 468, being convertible, by deprotection and acetylation, to the totally O-acetylated derivative 469 of validamycin C 470 (Miyamoto and Ogawa, 1991).

4.7.4 Synthesis of Validamycin D With the protected derivative 472 of validoxylamine A, (1)-validamycin D was first synthesized by α-glycosylation, following the procedure in Scheme 4.61 (Miyamoto and Ogawa, 1991).

228

Validamycin and Its Derivatives OBn

OBn BnO

BnO BnO

471

BnO

BnO

OBn N H HO

OBn N H

BnO

OBn

472

OH

OBn HO

OBn

OBn

472

+

BnO

BnO

463

OBn N H

BnO

OBn OBn

473

AcO

O O BnO

OAc AcO AcO

OBn OBn OH

AcO

HO

OAc N H

HO

OAc OAc

474

O

OH N H

OH OH

475

AcO

O AcO

HO

OAc OAc

O

HO O

HO

OH OH

SCHEME 4.61 Synthesis of validamycin D.

4.7.5 Synthesis of Validamycin E Miyamoto and Ogawa (1989) synthesized validamycin E from the validoxylamine A derivative 450, which was convertible, by glycosylation followed by deprotection, into validamycin E 479 (Scheme 4.62). The bromide derivative of D-maltose was prepared from the corresponding free disaccharide according to the standard procedures. Condensation of the aglycone 450 with the bromide derivative 476 in dry dichloromethane in the presence of silver trifluoromethanesulfonate (AgOTf) and 1,1,3,3-tetramethylurea (TMU) at room temperature afforded the validamycin E.

229

Chemical Synthesis Chapter | 4

O AcO OAc

O AcO OAc 450

+

OAc

O Br OAc

OAc

476

OBn

BnO

OBn N H

OBn

O AcO OAc

OBn AcO

O AcO OAc

O

OBn

OAc

O OAc

477

OAc

OAc

AcO

OAc N H

OAc

O AcO OAc

OAc AcO

O AcO OAc

O

OAc

OAc

O OAc

478

OAc

OH

HO

O OH N H

OH

OH HO

OH

O HO OH

HO OH O

479

OH

O OH

OH

SCHEME 4.62 Synthesis of validamycin E.

4.7.6 Synthesis of Validamycin F As shown in Scheme 4.63, (1)-validamycin F was synthesized by use of the blocked derivative 472 of (1)-validoxylamine A. The diol 480, obtained by acid hydrolysis of 472, was protected to give the aglycone 481, which was condensed with glycosyl donor 467 to afford the condensate 484, being convertible, by deprotection and acetylation, to the totally O-acetylated derivative 485 of validamycin F (Miyamoto and Ogawa, 1991).

4.7.7 Synthesis of Validamycin G Application of branched-chain inosose derivatives 1 and hepta-O-benzyl-Dcellobiose 486 to the total synthesis of natural N-substituted validamycin G was described by Fukase and Horii (1992) (Scheme 4.64).

230

Validamycin and Its Derivatives OBz O

Ph

BnO

BnO HO

O BnO

OBn N H

O O Ph

460

463

OBn N H

BnO

OBn

OBn O O Ph

480

OBz

OBz BnO

OAcO

O OBnBnO

OBnOBn

BnO O OAc

OBn N H

O OBn BnO

OBn

OBnOBn

O O Ph

481

OBn

N H

OBn OH OH

482

OAc O OBn

BnO BnO

OBz

O BnO

BnO OAcO

O OBn BnO

OBnOBn

OBn

N H

467

BnO

OBn

HN

OH OAc

483

OBz

384 OBn

AcO O AcO

OAc AcO OAcO

OAc

O AcO

OAc

O OAc

N H

AcO

OAc AcO

OAcOAc

OBn

AcO 485

O AcO

O

OAc OAc

OAc

SCHEME 4.63 Synthesis of validamycin F.

4.7.8 Synthesis of Validamycin H Yasunobu Miyamoto and Seiichiro Ogawa reported that the first total synthesis of 499 was by coupling of the aglycon 495 and the glycosyl donor 496, followed by deblocking (Scheme 4.65). The synthesis of 7-O-acetyl2,3,40 ,50 ,60 ,T-hexa-O-benzylvalidoxylamine A 496 has been described

231

Chemical Synthesis Chapter | 4 OBn

OBn BnO

OBn

O CH 2 OBn

BnO O HO

OBn O

BnO

OBn

CH 2OBn

O O

486

OBn O

O CH 2 OBn

BnO

CH 2 OBn

487

OBn OBn

O CH 2OBn

BnO O

Cl 2HC OH

OBn

BnO

OBn O

BnO

OBn

CH 2OBn

HO OH

Cl 2HC

OBn OBn

O CH 2OBn

BnO O

CHCl 2 O

OBn

BnO

Cl

OBn O

CH 2OBn

489

H

488

OBn

O CH 2OBn

BnO

O

CH 2OBn

OBn

BnO

OBn O

O CH 2OBn

BnO

CH 2OBn

Cl

O

OH 490

491

BnOH 2 C BnO BnO OBn

BnO

OBn

OBn

O CH 2OBn

BnO

BnO

HN

OBn O

CH 2OBn

442

OBn

O

BnOH 2 C OH BnO 492

493

O

OBn O OBn OBn

OH

HO HOH 2 C OH OH HO

N H

OH O

O

HO

OH

HO HO 494

SCHEME 4.64 Synthesis of validamycin G.

previously in this chapter and 2,3,4,20 ,30 ,40 ,60 -hepta-O-acetyl-α-gentiobiosyl bromide 495 was prepared from gentiobiose hepta-acetate by conventional treatment with 30% hydrobromic acid in acetic acid. Condensation of 495 and 496 in the presence of silver trifluoromethanesulfonate and 1,1,3,3-tetramethylurea in boiling dichloromethane gave, after chromatography, 497. The protecting groups of 497 were removed by treatment with sodium in liquid ammonia at 278 to give 499, which was isolated as the tetradecaacetate 498 obtained by treatment of 499 with acetic anhydride and pyridine. This synthesis provided an easy route to validamycin H (Miyamoto and Ogawa, 1992).

232

Validamycin and Its Derivatives OBn OAc BnO

AcO AcO

BnO OBn N H

BnO

+

O O O AcO

OBn

AcO

OAc

OAc AcO

Br OH 496

495 OBn BnO BnO BnO

OBn N H

OAc OBn

AcO

AcO

O

O AcO

OAc O

O

AcO

OAc OAc

497

OAc AcO AcO AcO

OAc N H

OAc OAc

AcO

AcO

O

O AcO

OH

OAc O

O

AcO

OAc OAc

498 HO

HO HO

OH

N H

OH OH

HO O

HO

O

O HO

OH

OH O HO

OH 499

SCHEME 4.65 Synthesis of validamycin H.

REFERENCES Chang, Y.-K., Lee, B.-Y., Kim Dong, J., Lee Gwan, S., Jeon Heung, B., Kim Kwan, S., 2005. An efficient synthesis of valienamine via ring-closing metathesis. J. Org. Chem. 70 (8), 32993302. Chang, Y.-K., Lo, H.-J., Yan, T.-H., 2009. A flexible strategy based on a C2-symmetric pool of chiral substrates: concise synthesis of (1)-valienamine, key intermediate of (1)-pancratistatin, and conduramines A-1 and E. Org. Lett. 11 (19), 42784281.

Chemical Synthesis Chapter | 4

233

Chen, X., Fan, Y., Zheng, Y., 2003. Properties and production of valienamine and its related analogues. Chem. Rev. 103 (5), 19551977. Cumpstey, I., 2005. Formal synthesis of valienamine using ring-closing metathesis. Tetrahedron. Lett. 46 (37), 62576259. Cumpstey, I., Gehrke, S., Erfan, S., Cribiu, R., 2008. Studies on the synthesis of valienamine and 1-epi-valienamine starting from D-glucose or L-sorbose. Carbohydr. Res. 343 (1011), 16751692. Fukase, H., Horii, S., 1992. Synthesis of valiolamine and its N-substituted derivatives AO-128, validoxylamine G, and validamycin G via branched-chain inosose derivatives. J. Org. Chem. 57 (13), 36513658. Horii, S., Iwasa, T., Mizuta, E., Kameda, Y., 1971. Validamycins, new antibiotics. VI. Validamine, hydroxyvalidamine, and validatol, new cyclitols. J. Antibiot 24 (1), 5963. Horii, S., Fukase, H., Kameda, Y., 1985. Stereoselective conversion of valienamine and validamine into valiolamine. Carbohydr. Res. 140 (2), 185200. Hur Y, Oh J-H, Park Y-I; (B T Gin., Inc., S. Korea). assignee. 2004 20031205. Preparation method of valienamine via selective hydrolysis of acarbose, validamycin, and validoxylamine derivatives using exchange resins or zeolite as catalysts. Application: WO, WO patent 2003-KR2657, 2004108657. Jagdhane, R.C., Shashidhar, M.S., 2011. A formal synthesis of valiolamine from myo-inositol. Tetrahedron 67 (41), 79637970. Ji, L., Zhang D-f, Zhao, Q., Hu S-m, Qian, C., Chen, X.-Z., 2013. Diastereospecific epoxidation and highly regioselective ring-opening of ( 1 )-valienamine: practical synthesis of ( 1 )-valiolamine. Tetrahedron 69 (34), 70317037. Jin, L.-Q., Xue, Y.-P., Zheng, Y.-G., Shen, Y.-C., 2006. Production of trehalase inhibitor validoxylamine A using acid-catalyzed hydrolysis of validamycin A. Catal. Commun. 7 (3), 157161. Kameda, Y., Horii, S., 1972. The unsaturated cyclitol part of the new antibiotics, the validamycins. J. Chem. Soc., Chem. Commun. 12, 746747. Kapferer, P., Sarabia, F., Vasella, A., 1999. Carbasaccharides via ring-closing alkene metathesis. A synthesis of (1)-valienamine from D-glucose. Helv. Chim. Acta 82 (5), 645656. Knapp, S., Naughton, A.B.J., Dhar, T.G.M., 1992. Intramolecular amino delivery reactions for the synthesis of valienamine and analogs. Tetrahedron. Lett. 33 (8), 10251028. Kok, S.H., Lee, C.C., Shing, T.K., 2001. A new synthesis of valienamine. J. Org. Chem. 66 (21), 71847190. Krishna, P.R., Reddy, P.S., 2009. Stereoselective total synthesis of (1)-valienamine and (1)-4-epi-valienamine via a ring-closing enyne metathesis protocol. Synlett. 2, 209212. Li, Q.R., Kim, S.I., Park, S.J., Yang, H.R., Baek, A.R., Kim, I.S., et al., 2013. Total synthesis of (1)-valienamine and (2)-1-epi-valienamine via a highly diastereoselective allylic amination of cyclic polybenzyl ether using chlorosulfonyl isocyanate. Tetrahedron 69 (48), 1038410390. Lo, H.-J., Chen, C.-Y., Zheng, W.-L., Yeh, S.-M., Yan, T.-H., 2012. A C2-symmetric pool based flexible strategy: an enantio-convergent synthesis of (1)-valiolamine and (1)-valienamine. Eur. J. Org. Chem. 2012 (14), 27802785, S2780/1-S2780/28. Miyamoto, Y., Ogawa, S., 1989. Synthetic studies on antibiotic validamycins. Part 13. Total synthesis of (1)-validamycins A and E, and related compounds. J. Chem. Soc., Perkin Trans. 1 (5), 10131018.

234

Validamycin and Its Derivatives

Miyamoto, Y., Ogawa, S., 1990. Total synthesis of (1)-validoxylamine G. J. Chem. Soc., Chem. Commun. 14 (5), 889890. Miyamoto, Y., Ogawa, S., 1991. Synthetic studies on antibiotic validamycins. Part 14. Total synthesis of (1)-validamycins C, D and F. J. Chem. Soc., Perkin Trans. 1 (9), 21212128. Miyamoto, Y., Ogawa, S., 1992. Total synthesis of (1)-validamycin H. Carbohydr. Res. 223, 299301. Nicotra, F., Panza, L., Ronchetti, F., Russo, G., 1989. Stereocontrolled synthesis of (1)-valienamine. Gazz. Chim. Ital. 119 (11), 577579. Ogawa, S., Kanto, M., 2007. Synthesis of valiolamine and some precursors for bioactive carbaglycosylamines from (2)-vibo-quercitol produced by biogenesis of myo-inositol. J. Nat. Prod. 70 (3), 493497. Ogawa, S., Miyamoto, Y., 1987. Total synthesis of (1)-validamycin B. J. Chem. Soc., Chem. Commun. 24, 18431844. Ogawa, S., Miyamoto, Y., 1988. Total synthesis of (1)-validoxylamine A. Chem. Lett. 14 (5), 889890. Ogawa, S., Chida, N., Suami, T., 1980. Synthesis of hexa-N,O-acetyl-DL-hydroxyvalidamine. Chem. Lett. 12, 15591562. Ogawa, S., Toyokuni, T., Suami, T., 1981. Synthesis of some racemic isomers of validoxylamine A. Chem. Lett. 328 (7), 947950. Ogawa, S., Ogawa, T., Chida, N., Toyokuni, T., Suami, T., 1982a. Synthesis of DL-validoxylamine A. Chem. Lett. 5, 749752. Ogawa, S., Toyokuni, T., Iwasawa, Y., Abe, Y., Suami, T., 1982b. Synthesis of DL-validoxylamine B. Chem. Lett. 3, 279282. Ogawa, S., Chida, N., Suami, T., 1983a. Synthetic studies on the validamycins. 5. Synthesis of DL-hydroxyvalidamine and DL-valienamine. J. Org. Chem. 48 (8), 12031207. Ogawa, S., Ogawa, T., Nose, T., Toyokuni, T., Iwasawa, Y., Suami, T., 1983b. A formal total synthesis of validamycin A. Chem. Lett. 25 (6), 921922. Ogawa, S., Shibata, Y., Chida, N., Suami, T., 1983c. Synthetic studies on the validamycins. I. Synthesis of β-D-glucopyranosylvalidamine: 1L-2-O-(β-D-glucopyranosyl)-(1,3,4/2,6)-4amino-6-(hydroxymethyl)-1,2,3-cyclohexanetriol. Bull. Chem. Soc. Jpn. 56 (2), 494498. Ogawa, S., Ogawa, T., Iwasawa, Y., Toyokuni, T., Chida, N., Suami, T., 1984. Synthetic studies on the validamycins. 10. Total synthesis of DL-validoxylamines A and B. J. Org. Chem. 49 (14), 25942599. Ogawa, S., Iwasawa, Y., Nose, T., Suami, T., Ohba, S., Ito, M., et al., 1985a. Total synthesis of (1)-(1,2,3/4,5)-2,3,4,5-tetrahydroxycyclohexane-1-methanol and (1)-(1,3/2,4,5)-5-amino2,3,4-trihydroxycyclohexane-1-methanol [(1)-validamine]. X-ray crystal structure of (3S)(1)-2-exo-bromo-4,8-dioxatricyclo[4.2.1.03,7]nonan-5-one. J. Chem. Soc., Perkin Trans. 1 (4), 903906. Ogawa, S., Nose, T., Ogawa, T., Toyokuni, T., Iwasawa, Y., Suami, T., 1985b. Synthetic studies on antibiotic validamycins. Part 11. Synthesis of validamycin A. J. Chem. Soc., Perkin Trans. 1 (11), 23692374. Ogawa, S., Shibata, Y., Nose, T., Suami, T., 1985c. Synthetic studies on the validamycins. XII. Synthesis of optically active valienamine and validatol. Bull. Chem. Soc. Jpn. 58 (11), 33873388. Ogawa, S., Miyamoto, Y., Nose, T., 1988. Synthetic studies on antibiotic validamycins. Part 12. Total synthesis of (1)-validamycin B and (1)-validoxylamine B. J. Chem. Soc., Perkin Trans. 1 (9), 26752680.

Chemical Synthesis Chapter | 4

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Ogawa, S., Ohishi, Y., Asada, M., Tomoda, A., Takahashi, A., Ooki, Y., et al., 2004a. Convenient synthesis of (1)-valiolamine and (2)-1-epi-valiolamine from (2)-vibo-quercitol. Org. Biomol. Chem. 2 (6), 884889. Ogawa, S., Sakata, Y., Ito, N., Watanabe, M., Kabayama, K., Itoh, M., et al., 2004b. Convenient synthesis and evaluation of glycosidase inhibitory activity of alpha- and beta-galactose-type valienamines, and some N-alkyl derivatives. Bioorg. Med. Chem. 12 (5), 9951002. Park, T.K., Danishefsky, S.J., 1994. A synthetic route to valienamine: an interesting observation concerning stereoelectronic preferences in the SN20 reaction. Tetrahedron. Lett. 35 (17), 26672670. Paulsen, H., Heiker, F.R., 1981. Synthese von enantiomerenreinem Valienamin aus Quebrachit. Liebigs Ann. Chem. 12, 21802203. Quan, N., Nie, L.-D., Zhu, R.-H., Shi, X.-X., Ding, W., Lu, X., 2013. Total syntheses of (1)-valiolamine and (2)-1-epi-valiolamine from naturally abundant (2)-shikimic acid. Eur. J. Org. Chem. 2013 (28), 63896396. Ramstadius, C., Hekmat, O., Eriksson, L., Stalbrand, H., Cumpstey, I., 2009. β-Mannosidase and β-hexosaminidase inhibitors: synthesis of 1,2-bis-epi-valienamine and 1-epi-2-acetamido-2deoxy-valienamine from D-mannose. Tetrahedron Asymmetry 20 (68), 795807. Schmidt, R.R., Koehn, A., 1987. α-Glucosidase inhibitors. Part 4. Synthesis of valienamine. Angew. Chem. 99 (5), 490491. Shing, T.K.M., Li, T.Y., Kok, S.H.L., 1999. Enantiospecific syntheses of valienamine and 2-epi-valienamine. J. Org. Chem. 64 (6), 19411946. Shing, T.K.M., Tai, V.W.F., 1995. Enantiospecific syntheses of penta-N,O,O,O,O-acetylvalidamine and penta-N,O,O,O,O-acetyl-2-epi-validamine. J. Org. Chem. 60 (16), 53325334. Shing, T.K.M., Wan, L.H., 1996. Facile syntheses of valiolamine and its diastereomers from (2)-quinic acid. Nucleophilic substitution reactions of 5-(hydroxymethyl)cyclohexane1,2,3,4,5-pentol. J. Org. Chem. 61 (24), 84688479. Shing Tony, K.M., Cheng Hau, M., 2008. Intramolecular direct aldol reactions of sugar diketones: syntheses of valiolamine and validoxylamine G. Org Lett 10 (18), 41374139. Tatsuta, K., Mukai, H., Takahashi, M., 2000. Novel synthesis of natural pseudo-aminosugars, (1)-valienamine and (1)-validamine. J. Antibiot. 53 (4), 430435. Toyokuni, T., Ogawa, S., Suami, T., 1983. Synthetic studies on the validamycins. IX. Synthesis of some racemic isomers of validoxylamine A. Bull. Chem. Soc. Jpn. 56 (10), 29993004. Trost, B.M., Chupak, L.S., Luebbers, T., 1998. Total synthesis of (6)- and (1)-valienamine via a strategy derived from new palladium-catalyzed reactions. J. Am. Chem. Soc. 120 (8), 17321740. Wang, Z., Cui, Y.T., Xu, Z.B., Qu, J., 2008. Hot water-promoted ring-opening of epoxides and aziridines by water and other nucleopliles. ChemInform 73 (30), 22702274. Yoshikawa, M., Cha, B.C., Okaichi, Y., Takinami, Y., Yokokawa, Y., Kitagawa, I., 1988. Syntheses of validamine, epi-validamine, and valienamine, three optically active pseudoamino-sugars, from D-glucose. Chem. Pharm. Bull. 36 (10), 42364239. Yoshikawa, M., Murakami, N., Yokokawa, Y., Inoue, Y., Kuroda, Y., Kitagawa, I., 1994. Stereoselective conversion of D-glucuronolactone into pseudo-sugar: syntheses of pseudoα-D-glucopyranose, pseudo-β-D-glucopyranose, and validamine. Tetrahedron 50 (32), 96199628. Zheng, Y.-G., Jin, L.-Q., Shen, Y.-C., 2004. Resin-catalyzed degradation of validamycin A for production of validoxylamine A. Catal. Commun. 5 (9), 519525. Zhou, B., Luo, Z., Lin, S., Li, Y., 2012. A concise synthetic approach to (1)-valienamine starting from Garner’s aldehyde. Synlett 23 (6), 913916.

Chapter 5

Voglibose: An Important Drug for Type 2 Diabetes As of 2017, interest in diabetes is blowing up, as the number of people with diabetes is expected to rise from the current estimated of 150220 million in 2010 and 300 million in 2025 (King et al., 1998). There are two main forms of diabetes. Type 1 diabetes is the most common chronic disease in children and is due to autoimmune-mediated destruction of pancreatic β-cell islets resulting in absolute insulin deficiency. It can be treated by supplying exogenous insulin (Colman and Harrison, 1984). Type 2 diabetes or non-insulin-dependent mellitus diabetes (NIDDM), which represents 90% of cases, is a multifactorial disease and is characterized by insulin resistance in peripheral tissues and/or abnormal insulin secretion from the pancreas and increasing blood glucose levels (Atkinson and Eisenbarth, 2001). NIDDM is increasing in developed and developing countries and is associated with sedentary lifestyle and obesity (Groop, 1997). Many complications such as retino-, neuro- and nephropathies are associated with NIDDM and lowering blood glucose may be an effective mechanism for preventing the development of diabetic complications (Zimmet, 1999). At present, therapies for NIDDM are directed toward the reduction of hyperglycemia itself. Thus, sulfonylureas and related insulin secretagogues (UKPDS Group, 1998) increase insulin release from pancreatic islets; the biguanide metformin (Groop, 1992) delays the absorption of dietary carbohydrates by inhibiting the glucose transporter found on the brush border of the epithelial lining of the intestines and so acts to reduce hepatic glucose production. Thiazolidinediones (Bailey and Turner, 1996), which are peroxisome proliferator-activated receptorγ agonists, enhance insulin action. Insulin itself stops glucose production and increases glucose utilization (Lehmann et al., 1995). Plasma levels of D-glucose and insulin are usually high in diabetics especially after food ingestion, and reducing intestinal carbohydrate absorption is one way to control disorders of carbohydrate metabolism. For example, starch is hydrolyzed by α-amylase, followed by further hydrolysis to monosaccharides by α-D-glucosidase. The resulting monosaccharides are absorbed from the small intestine by active transport and simple diffusion. Therefore, α-D-glucosidase inhibitors are thought to be valuable aids in the treatment of diabetes. They act by delaying the absorption of carbohydrates thereby inhibiting postprandial hyperglycemia (PPG) and hyperinsulinemia. Validamycin and Its Derivatives. DOI: http://dx.doi.org/10.1016/B978-0-08-100999-4.00005-8 © 2017 Elsevier Ltd. All rights reserved.

237

238

Validamycin and Its Derivatives

Since the mid-1970s, quite a few pseudooligosaccharides of microbial origin that exhibit a very pronounced inhibitory effect on intestinal α-D-glucosidase have been reported (Chen et al., 2003, 2006), and some of them have aroused medical interest in the treatment of metabolic disease such as diabetes. In general, these microbial α-D-glucosidase inhibitors have valienamine (1) as their key constituent, which was first found in validamycins. As previously reported, valienamine itself is an inhibitor for α-D-glucosidase, and some N-alkyl- and N-araldylvalienmine derivatives have stronger inhibitory activity against porcine sucrase and maltase than the parent valienamine 16. These results suggest that the 4, 6-dideoxy- and the 4-deoxy-α-Dglucopyranose units of 15a and 15b, found in the naturally occurring pseudooligosaccharide α-D-glucosidase inhibitors, such as acarbose, trestatins, amylostatins, and adiposins, are not essential to sucrase and maltase inhibitory activity and are substitutable by some other structural unit. Later, some researchers also found that the valiolamine (2), (1S)-(1-(OH), 2, 4, 5/1, 3)-5-amino-1-C-(hydroxymethyl)-1, 2, 3, 4-cyclohexanetetrol, has more potent α-D-glucosidase inhibitory activity than the other pseudoaminosugars such as valienamine, validamine, hydroxyvalidamine, and epi-valiolamine against porcine intestinal sucrase and maltase. On the basis of these results, a program of synthesis of N-substituted valiolamine derivatives in order to find a more potent α-D-glucosidase inhibitor than naturally occurring oligosaccharide α-D-glucosidase inhibitors has been embarked upon. Voglibose is one of these N-substituted valiolamine derivatives. Voglibose (code number AO-128, trade name Basen) (Horii 1993; Horii et al., 1986) is an N-substituted derivative of valiolamine, which is a branched-chain aminocyclitol, or pseudoaminosugar, and its N-substituted moiety is derived from glycerol. The structure of voglibose is shown in Fig. 5.1. Voglibose inhibits the intestinal α-D-glucosidases, which are responsible for the digestion of disaccharides such as maltose and sucrose. In animals and healthy volunteers, voglibose significantly reduced postprandial blood glucose concentration (Atkinson et al., 1991a,1991b; Goto et al., 1995; Ikeda and Odaka, 1995; Lina et al., 1991; Matsuo et al., 1992; Morseth and Nakatsu, 1991a, 1991b, 1991c, 1991d; Odaka and Ikeda, 1995; Til et al., 1991). Clinical trials in patients with diabetes mellitus also demonstrated that voglibose improves postprandial blood glucose levels.

5.1 CHEMICAL STRUCTURES OF VARIOUS PSEUDOAMINOSUGAR GLUCOSIDASE INHIBITORS The structures of valienamine 1, valiolamine 2, validamine 3, hydroxyvalidamine 4, epi-valiolamine 5, validoxylamine A 6, validamycin A 7, acarbose 8, amylostatins 9, voglibose (AO-128) 10, acarviosin 11, adiposins 12, trestatins 13, oligostatins 14, are shown in Fig. 5.1.

239

Voglibose: An Important Drug for Type 2 Diabetes Chapter | 5 CH2OH

CH2OH OH

OH

NH2

OH

OH

CH2OH

OH OH

NH2

2

1

OH

OH

OH

NH2

3

OH

CH2OH

CH2OH OH

HO

OH

NH2

OH

4

H

CH2OH OH

O

OH

OH

NH

OH

m

OH

OH

O OH

OH

CH2OH

OH N H

OH

OH

CH2OH O

OH

O

OH

OH

n

8: m=0, n=1 9

OH OH

CH2OH O OH

CH2OH O

O

OR

HOH 2C OH

OH

6: R=H 7: R=Glu

OH

N H

OH

CH2OH O

OH

N H

OH

m

CH3

OH

O

OH

N H

OH

OH

10

O

OH

NH2

CH3

CH2OH

H

OH

5

CH2OH O OH

O

CH2OH HO OH OH OH

OH

OCH3

11 CH2OH O OH

CH2OH O OH

O

OH

OH

CH2OH O O

OH

OH OH

n

12 CH2OH OH

O

H

OH

CH3 N H

CH2OH O

O

OH

OH

O

OH

O OH

OH

CH2OH O O

OH

OH

n

OH

CH2OH O O

OH HOH2C

OH

13 CH2OHOH

CH2OH O H

O

OH

O OH

CH3

OH

N H

OH

m

CH2OH O

O

OH

OH

O

O OH

OH

14

CH2OH OH

CH2R

OH OH 15a 15b

N H

O

OH OH

CH2OH O

OCH3

R=H R=OH

FIGURE 5.1 Chemical structures of various glucosidase inhibitors.

n

OH

OH OH

OH

240

Validamycin and Its Derivatives

5.2 α-D-GLUCOSIDASE INHIBITORY ACTIVITY N-SUBSTITUTED VALIOLAMINE DERIVATIVES AND RELATED COMPOUNDS The porcine maltase and sucrase inhibitory effects of two typical types of simple N-substituted valiolamine derivatives (Ishida et al., 1998; Kleist et al., 1998; Tanaka et al., 1998), including voglibose, were compared to the effects of the corresponding N-substituted valienamine derivatives (Bando et al., 1998; Kawakami et al., 2001) and validamine derivatives (Ishida et al., 1998; Kleist et al., 1998). As shown in Table 5.1, the valiolamine derivatives as well as the parent valiolamine, and the presence and configuration of the hydroxyl group of the aralkyl unit of N-(β-hydroxyphenethyl) valiolamines, also markedly affect the inhibitory activity. Stereochemistry of the hydroxyl group on the cyclohexyl unit of N(hydroxycyclohexyl) valiolamines also influences the activity. The hydroxy group of the N-[(1R, 2R)-2-hydroxycyclohexyl] isomer 23 exerts a positive effect on activity, while the hydroxyl group of the N-[(1S, 2S)-2-hydroxycyclohexyl] isomer 27 exerts a negative effect on activity in comparison with the nonsubstituted cyclohexyl derivative 25. The inhibitory activity tends to increase, especially against porcine maltase, with the introduction of a hydroxyl group into the proper position on the alkyl, cyclohexyl, or aralkyl moiety of the N-substitute group, which is supposed to interact with the aglycone binging subsite of the enzyme. Replacement of the valienamine unit of acarviosin (33) (Nagai et al., 2000) and its 60 -hydroxy derivative 29, the key pseudodisaccharides of naturally occurring oligosaccharide α-D-glucosidase inhibitors, with a valiolamine unit leads to a remarkable increase in porcine maltase and sucrase inhibitory activity, especially maltase inhibitory activity (compounds 24 and 31 in Table 5.2). The pseudodisaccharides 28 and 30, which were formed by coupling two pseudosugar units, valiolamine and 7-deoxy-pseudo-D-glucopyranose, by an aNH- bond, showed undiminished potency as compared to the valiolamine analogue of 33. The enzyme inhibitory activity was not greatly affected by the functional group (hydroxyl or amino group) or the stereochemistry (α or β) of the C-1ʹ carbon, which corresponds to the anomeric carbon atom of the reducing end group of 33. Synthetically, especially for large-scale preparation, the N-[2-hydroxy-1(hydroxymethyl) ethyl] derivative is more attractive than the derivatives that have an asymmetric carbon in their N-substituted moieties and require a stereoresolution. After taking into consideration the ease of preparation and the safety of the possible metabolites of the N-substituted moiety in the living body, voglibose, the N-substituted moiety of which is derived from glycerol, was selected for further biological evaluation over the other N-substituted valiolamine derivatives that showed high α-D-glucosidase inhibitory activity.

TABLE 5.1 Inhibitory Effects (EC50 (mol/L)) of N-substituted Valienamine, Validamine, and Valiolamine Derivatives on Porcine Maltase and Sucrase CH2OH

CH2OH OH

OH OH

N H

R

OH

CH2OH

OH OH

N H

OH

R

OH

OH OH

N H

R

VE-R

VD-R

VO-R

VE (valienamine)

VD (validamine)

VO (valiolamine)

Maltase

Sucrase

Maltase

Sucrase

5.9 3 1026

1.8 3 1027

1.4 3 1026

1.9 3 1027

1.7 3 1026

2.2 3 1028

-R

CH2OH OH

OH OH

OH N H

-R

CH2OH OH

OH

OH

OH

OH

OH

OH

OH

17

5.5 3 1027

1.3 3 1028

CH2OH OH

N H

OH OH

OH

18

16

CH2OH

H2 H C C

N H

OH

N H

H2 H C C OH

19

(Continued )

TABLE 5.1 (Continued) CH2OH

CH2OH OH

OH OH

N H

R

OH

OH

VE-R

CH2OH

N H

R

OH

CH2OH

OH

OH OH

OH

1.5 3 10

29

4.6 3 10

N H

OH

OH OH

OH

N H

21

H2 C

CH2OH OH OH

OH

10

H N H

C H

5.0 3 1028

1.9 3 1028

H2 C C OH

20

OH

R

VO (valiolamine)

OH OH

CH2OH

N H

VO-R

VD (validamine) 28

OH OH

VD-R

VE (valienamine)

OH

OH OH

5.8 3 1029

2.9 3 1029

TABLE 5.2 Inhibitory Effects (IC50 (mol/L)) of N-substituted Valienamine, Validamine, and Valiolamine Derivatives on Porcine Maltase and Sucrase Compounds

Maltase

CH2OH

CH3

OH

OH

OH

N H

26

1.8 3 10

O

Sucrase 27

7.4 3 10

OH OH

Compounds CH2OH OH

OCH3

OH OH

OH

OH

N H

4.9 3 1029

O

1.0 3 1028

OH

OH OH

OH OH

OH

OCH3

OH OH

OH

OH

2.8 3 1028

CH3 N H

26

6.1 3 10

5.2 3 1029

4.1 3 1027

1.5 3 1028

1.6 3 1026

1.6 3 1027

OH

CH2OH

24

CH2OH

Sucrase

29

23

CH3

OH OH

N H

OH

22

CH2OH

Maltase

7.5 3 1029

OH OH

OH

25

OH

CH2OH

OH NH2

N H

OH OH

N H

27

(Continued )

TABLE 5.2 (Continued) Compounds

Maltase

OH

OH

6.8 3 10

CH3

CH2OH OH OH

28

N H

Sucrase 28

3.6 3 10

Compounds CH2OH O

CH2OH

OH

OH

OH

OH

OH OH

28

OH

OH OH

OH

N H

OH

7.0 3 1028

3.5 3 1028

OH

CH2OH OH

OH

OH OH

30

OH

OH OH

OH

N H

32

7.2 3 10

3.2 3 1027

4.4 3 1026

5.8 3 1027

3.02 3 1026

7.0 3 1027

OH OH

OCH3

N H

CH2OH O OH OH

OCH3

31

7.2 3 1028

CH2OH O

CH2OH

N H

Sucrase

26

29

CH3

CH2OH

Maltase

8.0 3 1028

OH OH

OCH3

CH3

CH2OH OH

OH

OH

N H

33

O

OH OH

OCH3

Voglibose: An Important Drug for Type 2 Diabetes Chapter | 5

245

The Ki values of valienamine, validamine, valiolamine, and their N-[2-hydroxy-1- (hydroxymethyl)ethyl] derivatives for rat intestinal disaccharidases are shown in Table 5.3. These N-[2-hydroxy-1-(hydroxymethyl)ethyl] derivatives are also competitive α-D-glucosidase inhibitors, as are the parent pseudosugars. The Ki values of voglibose for sucrase and maltase are about 106 and 105 times smaller than the Km values for sucrose and maltose. However, voglibose showed practically no α-amylase and β-D-glucosidase inhibitory activity in vitro. This strong inhibitory activity of voglibose against the disaccharidases can be explained by assuming that the hydroxyl group of the N-substituent unit (aCH(CH2OH)2 can assume a three-dimensional position that is very similar to that of the C-3 hydroxyl groups in the fructofuranoside portion of sucrose and the reducing glucose portion of maltose by rotation around the pseudoglucosidic linkage bond (-C-NH-C-) between the valiolamine unit and the aCH(CH2OH)2 unit. In the nuclear magnetic resonance (NMR) spectral data on voglibose, nuclear overhauser effect (NOE) was observed between the two protons, the C-1 proton of valiolamine and the α-proton of the N-substituent. This indicates that the geometry of voglibose is similar to that of maltose and its related compound in aqueous solution (Fig. 5.2) and the conformation is stabilized by the formation of an intramolecular hydrogen bond between the two hydroxyl groups in the aC(OH)-C-N-C-C(OH)-part.

5.3 PHYSICOCHEMICAL PROPERTIES OF VOGLIBOSE AND DRUG SUMMARY Voglibose [(1S)-[1(OH),2,4,5/3]-5-[[2-hydroxy-1-(hydroxymethyl) ethyl] amino]-1-C-hydroxymethyl)-1,2,3,4-cyclohexanetetrol], is an N-substituted derivative of valiolamine, which is a branched-chain aminocyclitol, or pseudoaminosugar, and the N-substituted moiety is derived from glycerol. It has the molecular formula C10H21NO7 and molecular weight 267.28; its crystal, m.p. about 166℃, no smell but sweet. It is easily soluble in water and acetic acid, not easily soluble in methanol, difficultly soluble in ethanol and almost insoluble in ether. (Chen et al., 2006). The α-glucosidase inhibitor (AGI) voglibose (Box 5.1) was developed by Takeda Pharmaceutical Co. Ltd and is one of the most commonly used oral antidiabetic agents in Japan. Voglibose is used as first-line treatment for improvement of PPG in diabetic patients with inadequate response to diet and exercise therapy and as add-on treatment to other oral antidiabetic drugs and insulin. In 2009, voglibose was approved in Japan for the management of prediabetes (impaired glucose tolerance, or IGT). Formulations of voglibose approved in Japan for treatment of T2DM include 0.2/0.3 mg tablets (brand name, Basen), 0.2/0.3 mg oral disintegrating tablets (brand name, Basen OD) and a fixed-dose combination of voglibose 0.2 mg and mitiglinide 10 mg (brand name, Glubes).

TABLE 5.3 Ki Values (mol/L) of Valienamine, Validamine, and Valiolamine Derivatives for Rat Small Intestinal Disaccharidase

VE

Sucrase

Maltase

Glucoamylase

Isomaltase

Trehalase

3.0 3 1024

9.6 3 1024

8.9 3 1024

7.6 3 1024

8.8 3 1024

25

24

24

25

VD

3.2 3 10

1.8 3 10

1.6 3 10

8.8 3 10

2.7 3 1024

VO

4.1 3 1027

3.5 3 1026

2.7 3 1026

9.1 3 1027

4.9 3 1025

VE-R

1.6 3 1026

1.6 3 1025

1.2 3 1025

7.6 3 1026

6.8 3 1025

VD-R

1.1 3 1027

1.4 3 1026

9.8 3 1027

8.8 3 1027

3.4 3 1024

VO-R

4.1 3 1028

4.5 3 1028

9.3 3 1028

4.9 3 1027

5.0 3 1024

Substrate

Sucrose

Maltose

Soluble starch

Palatinose

Trehalose

Km(mol/L)

22

3.4 3 10

23

3.0 3 10

OH VE, Valienamine; VD, Validamine; VO, Valiolamine; g/mL.

R=

a

OH

23a

9.5 3 10

23

6.2 3 10

1.1 3 1022

Voglibose: An Important Drug for Type 2 Diabetes Chapter | 5 CH2OH OH

OH OH

OH

CH2 OH O

OH N H

HO

CH2 OH O

OH

OH

OH OH

OH

247

10

OH

34

FIGURE 5.2 Relative disposition of functional groups in maltose and voglibose (10, AO-128).

BOX 5.1 Drug Summary Drug name Indication

Mechanism of action Route of administration Chemical structure

Voglibose Improvement of postprandial hyperglycemia in type 2 diabetes mellitus (T2DM); prevention of T2DM from impaired glucose tolerance Delays glucose absorption by competitively inhibiting α-glucosidase enzymes Oral OH

CH2OH OH

OH

N H

OH

OH

OH

Pivotal studies

Kataoka et al. (2012), Kawamori et al. (2009), Seo et al. (2013)

Pharmaprojects—copyright to Citeline Drug Intelligence (an Informa business). Readers are referred to Pipeline (http://informa-pipeline.citeline.com) and Citeline (http://informa.citeline.com).

5.4 PREPARATION OF VOGLIBOSE 5.4.1 Synthesis From Compound 35 A method 39 was described in Scheme 5.1 about the synthesis of voglibose from compound 35. The total yield was more than 65%.

5.4.2 Synthesis of Voglibose From Valiolamine In this method 40, valiolamine is oxidativedly deamined to produce valiolone 41. Oxidizing agents that are known to be effective in converting amines to imines can be used. These are illustrated by 3,5-di-t-butyl-1,2-benzoquinone (DBQ), nicotinic aldehyde in the presence of a base, benzothiazole-2-aldehyde,

248

Validamycin and Its Derivatives O O PdCl2, dioxane-H2O

OBn OBn

OBn

OMe

OBn

35 OBn

NH2

OH OBn OBn

O

MgBr

OH Toluene

OBn

36 OBn

OH

DMSO, SO3, pyridine, Et3N

37 OBn

OH OH

OH OBn

MeOH, NaBH4

O3, NaBH4

NH OBn

38 OBn

39 OBn

OH OH

OH

OH OH OBn

Pd-black/HCOOH

NH OBn

OH OBn

40 OBn

OH OH

OH OH NH OH

10 OH

OH OH

SCHEME 5.1 Synthesis of voglibose from compound 35.

and the like. The oxidation reaction proceeds easily generally without heat but heat can be used if desired, such as from room temperature up to about 50 C, preferably. The reactions can be conducted with (preferably) or without an appropriate solvent. The solvents can be illustrated by alcohols such as methanol, ethanol, isopropanol and the like. The intermediate can be isolated but preferably it is not isolated and is hydrolyzed in situ in the reaction mixture to produce the desired valiolone. Hydrolysis proceeds using known reagents and conditions for hydrolyzing an imine to a ketone. Mild acidic conditions (about pH 46; preferably about 5) can be used for hydrolysis. Oxalic acid and acids of similar pKa that allow good buffering within the pH range of about 46 and preferably 4.55.5 can be used. Acids, preferably an organic acid, that can be used include oxalic acid, acetic acid, malonic acid, and formic acid, as well as inorganic acids and acidic compounds, such as HCl, H2SO4, wet silica gel, and the like. The type of acid used can be readily selected by one of ordinary skill. The pH range selected can be guided by the qualifications that a significantly more acidic pH can allow for the formation by dehydration of valiolone or the intermediate imine. If the pH is allowed to become too basic, yields can decrease, in theory because hydrolysis becomes more sluggish and the possibility of a retroaldol cleavage as a side reaction increases. It has been found that between these two extremes there is a narrow pH range in which both decomposition reactions are sufficiently slow to allow for effective product recovery. The valiolamine is preferably unprotected when oxidized. While a variety of oxidizing agents can be used, preference is given to DBQ.

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Likewise, a variety of hydrolysis agents can be used to convert the imine to the ketone, but preference is given to oxalic acid. Further, the preferred reaction scheme requires careful control of the pH in the hydrolysis step to about 5.0. After the achievement of valiolone, voglibose can be prepared by reacting valiolone with 2-amino-1, 3-propanediol (serinol) in the presence of a reducing agent, preferably Na(CN)BH3, with or without an acid catalyst (e.g., acetic acid, HCl), according to the reaction (Scheme 5.2).

5.4.3 Synthesis of Voglibose From Glucose In this synthetic method 18, a bis(methylthio) group was chosen as the electron-withdrawing group. As shown in Scheme 5.3, tetra-O-benzyl-D-glucono1,5-lactone, which is readily available from D-glucose, was treated with bis (methylthio)methyllithium to give the 1-C-[bis(methylthio)methyl]-D-glucopyranose derivative 43. The direct oxidation of the D-gluco-2-heptulose derivative 43 to the Dxylo-2,6-heptodiulose derivative 45 was difficult, because the target C-6 hydroxyl group was masked by pyranose ring formation. Therefore, the Dgluco-2-heptulose derivative 43 was reduced with lithium aluminum hydride to yield the acyclic heptitol derivative 44. Although the heptitol derivative 44 was obtained as a mixture of two stereoisomers at the C-2 hydroxyl group in a ratio of approximately 14:1 ((1R)-isomer: (1S)-isomer), the mixture was subjected to the next reaction without resolution of the two isomers, as this chiral center disappears in the next oxidation reaction. The oxidation of the unprotected hydroxyl groups of the heptose dithioacetal derivative 44 with dimethyl sulfoxide (DMSO), trifluoroacetic anhydride and triethylamine (Swern oxidation) gave the 2,6-dioxo-heptose

t-Bu O COOH O

CH2OH

COOH

t-Bu

oxalic acid

DBQ

OH OH

OH OH

NH2

OH

CH2OH O

OH

OH

OH

2

41 CH2OH Na(CN)BH3 HCl

OH

OH OH

OH OH

N H OH

10

SCHEME 5.2 Synthesis of voglibose from valiolamine.

H 2N

CH2 OH CH CH2 OH

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Validamycin and Its Derivatives

HOH2C

BnOH2C

O

HO HO

OH

OH

BnOH2C

O

BnO BnO

O

BnO O

OBn

BnOH2C

43

OH

BnOH2C

SCH3 SCH3

H3CS O

SCH3

45

SCH3 SCH3

BnOH 2 C

SCH3

BnO BnO

SCH3 OBn O

44

BnOH2C

O

BnO BnO

BnO OBn OH

SCH3

OBn OH

42 BnO

SCH3

BnO

BnO BnO

O OBn

46

OH

OBn

47

O

BnOH 2 C BnO BnO

OH

OBn

O

48

CH2OH H 2N

CH CH2OH

BnOH2C BnO BnO

OH OBn NH

49

CH2OH CH CH2OH

SCHEME 5.3 Synthesis of voglibose from glucose.

1,1-dithioacetal derivative 45. The 2,6-dioxo-heptose derivative 45 was too labile to isolate as a pure compound; however, the partially purified compound showed reasonable spectral data. The 2,6-dioxo-heptose derivative 45 underwent intramolecular aldol condensation via the intermediate enolate anion 46 to give the desired α,αbis (methylthio)inosose derivative 47 stereospecifically not only with sodium acetate in the presence of 18-crown-6 ether in toluene but also with silica gel during the silica gel chromatography of the 2, 6-dioxo-heptose derivative 45. Desulfurization of the α,αbis(methylthio)inosose derivative 47 using hydrogen-saturated Raney nickel gave the desulfurized inosose derivative 48. Finally, voglibose was synthesized with high stereoselectivity by direct reductive amination of the branched-chain inosose 48 with 2-amino-1, 3-propanediol to give 49 and then removal of the O-benzyl protecting group. The epimer of 10 was not found in the reaction mixture. The results can be explained on the basis that the attack by the reducing agent occurs on the less-hindered side of the intermediate Schiff base. The branched-chain inosose derivative 48 should be a useful synthon for the branched-chain cyclitol moiety of N-substituted valiolamine derivatives, especially when the synthon for the substituent moiety is advantageously available in the form of an amino compound.

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5.5 PHARMACOLOGY, PHARMACOKINETICS, AND PHARMACODYNAMICS 5.5.1 Mechanism of Action The α-glucosidases are a group of enzymes located in the brush border of enterocytes that hydrolyze nonabsorbable oligosaccharides and polysaccharides into glucose and other absorbable monosaccharides. By competitively inhibiting the action of these enzymes, voglibose delays the digestion and absorption of carbohydrates, thereby attenuating the postprandial spike in plasma glucose levels and insulin secretion. As with other AGIs, voglibose does not stimulate insulin release and therefore does not cause hypoglycemia.

5.5.2 Pharmacokinetic Properties Voglibose is slowly and poorly absorbed after oral administration; plasma concentrations are generally undetectable at therapeutic dosages. After ingestion, the majority of active unchanged drug remains in the lumen of the gastrointestinal tract, where it is metabolized by intestinal enzymes and microbial flora (Derosa and Maffioli, 2012). No active metabolites have been identified to date. Voglibose is excreted rapidly in stools and has only negligible renal excretion (Kaku, 2014). In drug interaction experiments, concomitant administration of voglibose had no effect on the pharmacokinetics of warfarin (Fuder et al., 1997), hydrochlorothiazide (Kleist et al., 1998), digoxin (Kusumoto et al., 1999), glibenclamide (Kleist et al., 1997), dapagliflozin (Imamura et al., 2013), or vildagliptin (Yamaguchi et al., 2013).

5.5.3 Other Glycemic/Metabolic Effects Studies of voglibose in small groups of healthy Japanese volunteers or Japanese patients with T2DM have identified other glycemic/metabolic effects of voglibose. For example, during a 2-h meal tolerance test (MTT), voglibose was found to mobilize the endogenous pool of insulinotropic glucagonlike peptide 1 (GLP-1) (Goeke et al., 1995; Narita et al., 2011), a potent antihyperglycemic gut hormone, and significantly decrease the level of gastric inhibitory polypeptide (Narita et al., 2011), which induces insulin secretion. Through its sparing effect on endogenous insulin secretion, voglibose inhibits the overwork of pancreatic β-cells (Kageyama et al., 1997; Matsumoto et al., 1998) but has a minimal effect on insulin sensitivity (Matsumoto et al., 1998). The insulin-sparing effect of voglibose may account for its neutral effect on body weight (Negishi et al., 2008). In conjunction with reductions in PPG and hyperlipidemia, voglibose was superior to diet alone in reducing oxidative stress generation and soluble intercellular adhesion molecule-1 (sICAM-1) in obese T2DM patients despite similar

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improvements in both groups in body mass index (BMI) and hemoglobin A1c (HbA1c) levels (Satoh et al., 2006). Levels of the inflammatory mediator sICAM-1 are directly associated with BMI, triglycerides, and various inflammatory biomarkers (Shai et al., 2006), and, in a large epidemiological study, baseline sICAM-1 concentrations in the highest quartile were associated with an 80% higher risk of future myocardial infarction in otherwise apparently healthy men (Ridker et al., 1998). Although glucose levels (mean 24-h, mean daytime, before breakfast, before lunch) as measured by continuous glucose monitoring (CGM) were significantly lower with the dipeptidyl peptidase 4 inhibitor sitagliptin (50 mg/day) compared with voglibose (0.9 mg/ day) in a small crossover study of 17 Japanese patients with T2DM, the slopes of glucose elevation after each meal (breakfast, lunch, and dinner) were all significantly lower with voglibose, thus confirming its ability to delay postprandial glucose absorption (Seo et al., 2013).

5.5.4 Voglibose in Combination With Mitiglinide for T2DM The insulin secretagogue mitiglinide also targets postprandial plasma glucose directly with a different but complementary mechanism of action to voglibose. Like sulfonylureas, the glinides stimulate the rapid release of insulin from pancreatic β-cells but, in contrast to sulfonylureas, have a much shorter half-life (about 12 h), thus reducing the risk of hypoglycemia in the late meal phase (Ceriello and Colagiuri, 2008). Using MTTs to evaluate glycemic/metabolic responses, a series of small crossover studies compared the effects of voglibose and mitiglinide alone and in combination in Japanese patients with T2DM. In a recent review of these studies (Konya et al., 2013), it was reported that the combination of voglibose 0.6 mg/day and mitiglinide 30 mg/day achieved significantly better control of postprandial glucose excursions and had an insulin-sparing effect compared with either agent alone under laboratory conditions (Inoue, 2012) and in outpatients who selfmonitored their blood glucose at home before and after meals (Ono et al., 2013). Although the glycemic/metabolic responses differed somewhat between breakfast, lunch, and dinner, the voglibose/mitiglinide combination significantly reduced PPG and increased postprandial C-peptide and active GLP-1 levels compared with baseline after each of three unified meals (Ono et al., 2014). Reductions in glucose excursion and glycoalbumin were also significantly greater with the voglibose/mitiglinide combination than with sitagliptin 50 mg/day (Ohta et al., 2013).

5.6 CLINICAL EFFICACY 5.6.1 Comparative Studies With Other Oral Antidiabetic Agents Voglibose has been compared with a number of oral antidiabetic agents such as nateglinide (Kurebayashi et al., 2006), alogliptin (Seino et al., 2011a,

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2011b), linagliptin (Kawamori et al., 2012a), sitagliptin (Iwamoto et al., 2010b), and vildagliptin (Iwamoto et al., 2010a) in patients with T2DM. Although the glucose-lowering effect of voglibose was generally milder, it showed no propensity to cause hypoglycemia. This property makes voglibose particularly useful for use in patients with IGT or early stage T2DM as well as suitable for use in combination with other agents with complementary mechanisms of action to improve glycemic control without increasing the risk of adverse events, especially hypoglycemia.

5.6.2 Impaired Glucose Tolerance The ability of voglibose to prevent the development of T2DM was investigated in a multicenter, double-blind, parallel group study involving high-risk Japanese individuals with IGT on a standard diet and taking regular exercise (Kawamori et al., 2009). Participants were randomized to receive voglibose 0.6 mg/day or placebo until the development of T2DM (primary end point), normoglycemia, or for a minimum of 3 years. In a planned interim analysis, the cumulative number of cases of T2DM was 40 in the voglibose group and 84 in the placebo group (p 5 .0026), which led to early termination of the study. Final analysis on the full analysis set involved 1780 individuals who had been treated for a mean of 48 weeks and included about 6 months’ additional data since the time of the interim analysis. Individuals in the voglibose group had a significantly lower risk for progression to T2DM than those in the placebo group (50/897 vs 106/881; hazard ratio [HR] 0.595, 95% confidence interval [CI] 0.4330.818; p 5 .0014; Fig. 5.3) and more individuals

FIGURE 5.3 Cumulative probability (KaplanMeier method) of individuals with impaired glucose tolerance developing type 2 diabetes mellitus while receiving treatment with voglibose or placebo.

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Validamycin and Its Derivatives

treated with voglibose achieved normoglycemia compared with their placebotreated counterparts (599/897 vs 454/881; HR 1.539, 95% CI 1.3571.746; p , .0001). This study was the basis for approval of voglibose in Japan for delaying/preventing the progression to T2DM in individuals with IGT. In a retrospective post hoc analysis of this pivotal study, baseline characteristics of each outcome group (individuals who achieved normal glucose tolerance/sustained IGT or developed T2DM), plus follow-up findings from individuals who achieved normal glucose tolerance during the study and were now off treatment, were analyzed to investigate which serum insulin kinetic parameters might predict IGT reversion to normal glucose tolerance and maintenance of normal glucose tolerance once achieved (Kawamori et al., 2012b). Higher insulinogenic indexes at baseline or higher 30-min serum immunoreactive insulin (IRI) levels in a 75-g oral glucose tolerance test (OGTT) were associated with a greater likelihood of achieving and sustaining normal glucose tolerance. In individuals who achieved normal glucose tolerance, voglibose increased the OGTT 30-min IRI level from baseline to a significantly greater extent than the placebo possibly due to improved insulin secretion (30 min) by pancreatic β-cells. Thus, voglibose not only inhibits α-glucosidase but also increases insulin secretion capacity, which makes it useful for preventing the development of T2DM in individuals with IGT.

5.6.3 Add-on Therapy to Insulin As postprandial glucose in patients with T2DM is not always successfully controlled by basal insulin treatment, the add-on effects of AGIs have been investigated in this setting. The efficacy and safety of acarbose and voglibose were compared in T2DM patients whose blood glucose levels were inadequately controlled with basal insulin alone or in combination with metformin or a sulfonylurea (Lee et al., 2014). Participants in this prospective, openlabel, multicenter study were randomized to receive either acarbose 300 mg/ day (n 5 59) or voglibose 0.9 mg/day (n 5 62) for 24 weeks. At study end, mean HbA1c (a0.72% and a0.70%; both p , .001) and fasting plasma glucose (FPG) levels (a16.27 mg/100 mL, p 5 .041 and a10.44 mg/100 mL, p 5 .057) were significantly decreased from baseline to a similar extent in both treatment groups. Self-monitored blood glucose measurements 1 h before and 1 h after each of three meals also showed significant decreases from baseline at all six time points in both groups. Although the change from baseline at 1 h after dinner at week 24 was significantly greater in the acarbose group (a15.88 mg/100 mL; p 5 .001 vs voglibose), acarbose was associated with a higher incidence of treatment-related adverse events (16.7% vs 9.7%). The additive effects of voglibose and miglitol were compared in 36 patients with poorly controlled T2DM who were treated with three daily injections of insulin lispro mix 50/50 to maintain a FPG level , 130 mg/100 mL and a 2-h postprandial plasma glucose level , 180 mg/100 mL (Kimura et al., 2012).

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Twenty patients were randomly assigned to either voglibose 0.3 mg or miglitol 50 mg, which was administered at breakfast every other day. In the remaining 16 patients, the AGI was switched every day during a 6-day crossover study. Compared with insulin lispro mix 50/50 alone, miglitol but not voglibose blunted the postprandial rise and significantly decreased 1-h and 2-h postprandial C-peptide levels. In addition, miglitol significantly decreased the 1-h postprandial triglyceride rise and remnant-like particle-cholesterol rise. In the crossover study, miglitol increased 1-h postprandial high-density lipoprotein cholesterol and apolipoprotein A-I levels.

5.6.4 Combination Therapy in Patients on Hemodialysis Therapeutic choices in patients with T2DM and end-stage kidney disease are limited because of the reduced glomerular filtration rate, which can lead to accumulation of some drugs and/or their metabolites (Yale, 2005). Conventional oral hypoglycemic agents, such as sulfonylureas, are not suitable due to the risk of prolonged hypoglycemia, and metformin is contraindicated. As voglibose has no active metabolites and only negligible renal excretion, it is suitable for use in patients on hemodialysis either as monotherapy or in combination with other appropriate agents. Two similarly designed prospective open-label randomized studies investigated voglibose in combination with pioglitazone (Abe et al., 2007) and mitiglinide (Abe et al., 2010) in T2DM patients on hemodialysis. In the first of these studies, 31 patients with unstable glycemic control receiving constant doses of voglibose were randomized to continue with voglibose or receive add-on pioglitazone 30 mg (Abe et al., 2007). Combination therapy reduced plasma glucose and HbA1c from baseline levels from week 4 onward, and was also effective in reducing triglycerides. Homeostasis model assessment of insulin resistance (HOMA-IR), as a measure of insulin resistance, was significantly decreased at 4 weeks with combination therapy, and the decrease was maintained up to the last measurement at week 12. In the second study, 36 T2DM patients on hemodialysis with poor glycemic control were randomized to continue with voglibose monotherapy (0.9 mg/day) or receive add-on mitiglinide (initial dose 7.515 mg, titrated to 30 mg/day as required) for 24 weeks (Abe et al., 2010). Compared with baseline values, the addition of mitiglinide reduced FPG and glycated albumin levels after 4 weeks and HbA1c levels after 8 weeks; the levels continued to decrease over the 24-week treatment period. Triglyceride levels and HOMA-IR values also decreased significantly with add-on mitiglinide treatment.

5.6.5 Coronary Atherosclerosis To determine whether correction of postprandial glycemic status prevents atherosclerotic changes, the DIANA (diabetes and diffuse coronary narrowing)

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study compared lifestyle intervention, voglibose 0.9 mg/day, and nateglinide 180 mg/day in 302 patients with early stage diabetes mellitus (IGT/diabetes mellitus pattern based on a 75 g OGTT and HbA1c , 6.9%) and coronary artery disease (CAD) (Kataoka et al., 2012). After 1 year of treatment, the incidence of reversion to normal glucose tolerance was significantly higher with voglibose, compared with nateglinide or lifestyle intervention (35% vs 25% vs 22%, respectively; p , .05). Although no significant changes in coronary atherosclerosis were observed on angiography for any treatment, atheroma progression was significantly less in the 137 patients whose glycemic status had improved on treatment compared with those with no improvement (change in total lesion length: 3.5% vs 26.2%, p , .01; change in average lesion length: 0.7% vs 18.6%, p 5 .02). A subsequent post hoc multivariate analysis indicated that a greater increase in systolic blood pressure (p 5 .006), lack of statin treatment (p 5 .03), and baseline total lesion length (p 5 .007) were independent risk factors for atheroma progression even in the presence of optimal glycemic control, thus highlighting the need for aggressive modification of multiple risk factors in early diabetes to prevent progression of CAD (Kataoka et al., 2014).

5.6.6 Inflammation The effects of AGIs on endothelial dysfunction were compared in a study involving 50 Japanese patients with T2DM and CAD who were randomized to receive miglitol 150 mg/day or voglibose 0.6 mg/day for 3 months (Emoto et al., 2012). At study end, HbA1c was decreased to a similar extent in both groups, but improvements from baseline in 1,5-anhydroglucitol, a marker of frequent short-term elevations in glucose, were more pronounced in the miglitol group than in the voglibose group. HOMA-IR, C-reactive protein, and percentage flow-mediated dilatation were also improved with miglitol but not with voglibose. In view of the open design and small sample size, the results need to be interpreted carefully. Concomitant use of sulfonylurea agents was slightly higher in the voglibose group than in the miglitol group and sulfonylureas may contribute to the progression of atherosclerosis via hyperinsulinemia. Fujitaka and coworkers (2011) compared the effect of early intervention with pioglitazone or voglibose on physical and metabolic profiles and serum adiponectin levels in Japanese with newly diagnosed T2DM associated with metabolic syndrome. Patients were randomly assigned to receive pioglitazone 30 mg/day (n 5 30) or voglibose 0.9 mg/day (n 5 30) in addition to conventional diet and exercise training. During the 6-month follow-up period, BMI and waist circumference decreased significantly in the voglibose group but were unchanged in the pioglitazone group. In contrast, changes from baseline in HbA1c, FPG, and HOMA-IR were significantly greater with pioglitazone than with voglibose. Serum adiponectin (especially highmolecular-weight adiponectin) was increased from baseline in both groups,

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albeit more modestly with voglibose. High-sensitive CRP was decreased significantly only by pioglitazone. Although longer-term followup may have unveiled differences between pioglitazone and voglibose with respect to further progression of T2DM associated with metabolic syndrome, results at 6 months suggest that benefits might be achieved by combining antidiabetic agents with complementary mechanisms of action above those observed with single-agent therapy.

5.7 SAFETY AND TOLERABILITY Since AGIs prevent the degradation of complex carbohydrates to glucose, carbohydrate remains in the intestine and is delivered to the colon, where it is digested by the microbial flora (Derosa and Maffioli, 2012). This can result in the development of gastrointestinal side effects, although these tend to subside with continued treatment and can be minimized by use of an appropriate stepwise dosing regimen and careful choice of diet. In a large T2DM prevention study of 1780 Japanese individuals with IGT, the incidence of possible treatment-related adverse events was 48% with voglibose and 29% with placebo (Kawamori et al., 2009). Adverse events with an incidence significantly higher than that of placebo included flatulence, abdominal distension, diarrhea, and abnormal bowel sounds (all p , .0001), but all events were considered to be mild to moderate in severity. The proportion of individuals who discontinued treatment due to possible treatment-related adverse events did not differ markedly between voglibose and placebo (5% vs 3%; p 5 .009). No serious adverse effects such as hypoglycemia or liver impairment were observed when voglibose was administered in combination with pioglitazone or mitiglinide in T2DM patients on hemodialysis (Abe et al., 2007, 2010). Rare cases of pneumatosis cystoides intestinalis (gas cysts) associated with AGIs have been reported in the clinical literature (2012; Tsujimoto et al., 2008). Patients were reported as having nonspecific abdominal symptoms such as abdominal pain, abdominal distension, rectal bleeding, and loss of appetite, although these resolved after treatment withdrawal (2012). In view of the gastrointestinal symptoms commonly associated with this drug class, clinicians are advised to be vigilant of this potential diagnostic complication in patients receiving AGIs, including voglibose, who present with gastrointestinal symptoms.

5.8 COMPARISON WITH OTHER DRUGS OF ACARBOSE AND MIGLITOL At present, AGIs available for clinical use are acarbose (approved by FDA in 1999), voglibose (in Japan, Korea), and miglitol (approved by FDA in 1999). They have chemical structures similar to those of oligosaccharides derived from the digestion of starch. These structural configurations allow

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them to inhibit α-glucosidases competitively and reversibly. For example, acarbose potently inhibits brush-border enzymes (e.g., glucoamylase, dextrinase, maltase, sucrase) in vitro and in vivo and pancreatic α-amylase. The inhibition of α-glucosidases delays digestion of carbohydrates, which reduces the rate of absorption of simple sugars and reduces postprandial blood glucose levels. As a consequence of the reductions in average postprandial glucose levels over time, AGI significantly reduce levels of glycated HbA1c in patients with NIDDM.

5.8.1 Drug Summary of Acarbose and Miglitol Acarbose (O-4,6-dideoxy-4-[[(1S,4R,5S,6S)-4-5,6-trihydroxy-3-(hydroxymethyl)-2-cyclohexen-1-yl]amino]-α-D-glucopyranosyl-(1-4)-O-α-D-glucopyranosyl-(1-4) glucopyranose, BAY g 5421) (trade name GlucoBay) (Fig. 5.1) is one of the most famous hypoglycemic agents. Owing to its inhibiting effects on α-glucosidases, it is frequently used for the treatment of NIDDM. Acarbose history began in 1975 with its isolation from Actynomycetals species. Since then, numerous projects have been carried out on the basis of both its biological properties and its particular chemical structure, so that more than 1000 related studies have been published in the last 10 years. Miglitol (1, 5-dideoxy-1, 5-[2-hydroxyethylimino]-D-glucitol, BAY m 1099) (trade name Glyset) (Fig. 5.4) is derived from 1-deoxynojirimycin. Studies by Dimitriadis using the artificial endocrine pancreas established that miglitol reduces blood glucose fluctuations over 24 h and improves glycemic control in patients with type 1 diabetes, while long-term administration did not induce any appreciable degree of carbohydrate malabsorption. The efficacy of miglitol in glycemic control has been shown to be comparable with that of sulphonylureas in NIDDM inadequately controlled by diet alone.

5.8.2 Mechanism of Action Voglibose, acarbose, and miglitol are pseudocarbohydrates that competitively inhibit α-glucosidase enzymes located in the brush border of enterocytes that HO

FIGURE miglitol.

HO

N

HO

OH HO

Miglitol

5.4 Chemical

structure

of

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hydrolyze nonabsorbable oligosaccharides and polysaccharides into absorbable monosaccharides. Acarbose is the most used drug of this family. It is a pseudotetrasaccharide with a nitrogen bond between the first and second glucose units, which is obtained from fermentation processes of a microorganism, A. utahensis. This modification of the tetrasaccharide is important for its high affinity for active centers of α-glucosidases of the brush border of the small intestine and for its stability (Puls, 1996). Acarbose is the most effective against glucoamylase, followed by sucrase, maltase, and dextranase (Krause and Ahr, 1996). It also inhibits α-amylase, but does not inhibit β-glucosidases such as lactase. Acarbose is poorly absorbed and is excreted in the feces, mostly intact, but with up to 30% undergoing metabolism predominantly via fermentation by colonic microbiota (Derosa and Maffioli, 2012). Similarly, voglibose is slowly and poorly absorbed and rapidly excreted in stools, with no metabolites identified to date (Goeke et al., 1995). In contrast, miglitol is fully absorbed in the gut and cleared unchanged by the kidneys (Standl et al., 2001). Since AGIs prevent the digestion of complex carbohydrates, they should be taken at the start of main meals, taken with the first bite of a meal. Moreover, the amount of complex carbohydrates in the meal will determine the effectiveness of AGIs in decreasing PPG. Minor variations in the mechanism of action of the three molecules are listed in Fig. 5.5 (Joshi et al., 2015).

5.8.3 Pharmacokinetic Properties Comprehensive data on preclinical pharmacokinetics are only available for a few AGIs either on the market or in late phases of development (acarbose, voglibose, miglitol, emiglitate). Although similar in their extrasystemic mode of action toward the carbohydrate-digesting enzymes at the surface of the intestinal mucosa, the AGIs differ in their chemical structure, their

FIGURE 5.5 Mechanism of action of acarbose, miglitol, and voglibose.

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intrinsic activity, and also in their pharmacokinetic properties (Table 5.4) (Joshi et al., 2015). The most striking difference is observed for absorption of the different drugs. Neither acarbose nor voglibose are absorbed in their active forms to a relevant extent, thus both drugs being confined to their desired site of action. In contrast, the second-generation miglitol is readily and almost completely absorbed at low doses. Miglitol shows a concentration/dose-dependent saturation of absorption based on an active transport process across the intestinal mucosa. It is tempting to speculate that these drugs are mainly active in the upper parts of the small intestine, where most of the carbohydrate digestion takes place. In addition, for miglitol, a long-lasting presence of substancerelated radioactivity at or in the mucosa of the small intestine has been observed in autoradiographic studies, which may be based on the high affinity to the digestive enzymes localized there. Once absorbed, the four drugs exhibit a similar pharmacokinetic behavior. Their tissue affinity is relatively low, as shown by the low volumes of distribution and a predominantly extracellular distribution pattern in wholebody autoradiography. High concentrations are mainly found in the kidney, according to the very predominant renal excretion. The drugs do not penetrate the bloodbrain barrier. Protein binding of acarbose, voglibose, and miglitol is negligible at high concentrations. In some animal species, higher and saturable binding for acarbose and voglibose was observed at low concentrations. The elimination of these drugs occurs very rapidly. Acarbose, voglibose and miglitol are stable against systemic metabolic attack. They are excreted unchanged via the renal route, probably only via glomerular filtration as shown by the similarity of their clearances to the glomerular filtration rate. The excretion of acarbose and voglibose was mainly fecal according to their low absorbed fraction. Miglitol, as well as the absorbed fraction of acarbose and voglibose, are excreted rapidly and predominantly via the renal route.

5.8.4 Clinical Recommendations AGIs can be used as first-line drugs in newly diagnosed type 2 diabetes insufficiently treated with diet and exercise alone, as well as in combination with all oral antidiabetics and insulin if monotherapy with these drugs fails to achieve the targets for HbA1c and postprandial blood glucose. As firstline drugs, they are particularly useful in newly diagnosed type 2 diabetes with excessive PPG, because of their unique mode of action in controlling the release of glucose from complex carbohydrates and disaccharides. AGIs may also be used in combination with a sulfonylurea, insulin or metformin (Tseng, 2014). AGIs are contraindicated in patients with known hypersensitivity to the drug, in patients with diabetic ketoacidosis or inflammatory bowel disease,

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TABLE 5.4 Summary of Pharmacokinetic Properties of α-glucosidase Inhibitors Drug

Acarbose (BAY g 5421)

Voglibose (AO-128)

Miglitol (BAY m 1099)

Year of launch (global)

1990

1994

1996

Initial

25 mg t.i.d., orally at the start of each meal

0.2 mg t.i.d., orally before/with each meal

25 mg t.i.d., orally at the start of each meal

Maintenance

25-50 mg t.i.d. after 48 weeks intervals 50-100 mg t.i.d.

May be increased up to 0.3 mg t.i.d.

25-50 mg t.i.d. after 48 weeks intervals 50-100 mg t.i.d.

Maximum dose

Up to 100 mg t.i.d.

Not mentioned

100 mg t.i.d.

Extent of absorption

Low

Low, dose dependent

High, dose dependent

Unchanged drug

,2%

,6%

.96%

Metabolites

,35%





Bioavailability

2%

,6%

.96%

Clearance

Mainly renal by glomerular filtration

Mainly renal

Mainly renal by glomerular filtration

Protein binding

Low to high speciesdependent saturable

Distribution

Extracellular low tissue affinity

Low tissue affinity

Extracellular low tissue affinity

Metabolism

Extrasystemic in the intestine

None

None

Fecal

51%

98%



Renal

34%

5%

.95%

Biliary

,5%



,0.2%

Plasma elimination half-life

2h



2h

Dose

Low to high speciesdependent saturable

Excretion

(Continued )

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TABLE 5.4 (Continued) Drug

Acarbose (BAY g 5421)

Voglibose (AO-128)

Miglitol (BAY m 1099)

Therapeutic indications

Adjunct to diet and exercise to improve glycemic control in adults with T2DM; Adjunct to diet and insulin in T1DM; Prevention of onset of T2DM in individuals with IGT in combination with diet 1 exercise

Adjunct to diet and exercise to improve glycemic control in adults with T2DM

Adjunct to diet and exercise to improve glycemic control in adults with T2DM

Use in special populations Elderly patients

Can be used

Should be initiated at a lower dose and carefully administered under close observation

Can be used

Pregnancy

Category B

No data

No data

Nursing mother

Not to be administered

Not to be administered

Not to be administered

Pediatric use

Safety and efficacy not established

Safety and efficacy in children not established

Safety and efficacy not established

Hepatic impairment

To be used with caution

To be used with caution

Not metabolized, hence no influence of hepatic function on its kinetics

Renal impairment

Can be used in mildto-moderate renal impairment. Not recommended in patients with Clcr , 25 ml/min

Administered with caution. Poorly absorbed and renal excretion is negligible; no dose adjustment is required

Not recommended in patients with Clcr , 25 mL/min

US FDA

Yes

No

Yes

Japan

Yes

Yes

Yes

EMA

Yes

No data

No data

Regulatory status

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colonic ulceration, partial intestinal obstruction, or in patients predisposed to intestinal obstruction. Furthermore, they are contraindicated in patients who have chronic intestinal diseases associated with marked disorders of digestion or absorption and in patients who have conditions that may deteriorate as a result of increased gas formation in the intestine. The recommended starting dose of acarbose is 25 mg three times daily, increasing to 50 mg three times daily, until a maximum dose of 100 mg three times a day. Voglibose should be orally administered in a single dose of 0.2 mg three times a day, just before each meal; if not sufficient, the dose can be uptitrated to 0.3 mg three times a day. Miglitol should be started at 25 mg three times daily and then increased after four to eight weeks to 50100 mg three times daily.

5.8.5 Adverse Events Since AGIs prevent the degradation of complex carbohydrates into glucose, some carbohydrate will remain in the intestine and be delivered to the colon. In the colon, bacteria digest the complex carbohydrates, causing gastrointestinal side effects, such as flatulence (78% of patients) and diarrhea (14% of patients). Since these effects are dose-related, in general it is advised to start with a low dose and gradually increase the dose to the desired amount. A few cases of hepatitis have been reported with acarbose use, which regressed when the medicine was stopped; therefore, liver enzymes should be checked before and during use of this medicine. As stated above, AGIs should be started at a low dose, both to reduce gastrointestinal side effects and to permit identification of the minimum dose required for adequate glycemic control of the patient. If the prescribed diet is not observed, the intestinal side effects may be intensified.

5.8.6 Cost-effectiveness Ratio Pin˜ol et al. (2007) conducted a cost-effectiveness analysis of the addition of acarbose to existing treatment in patients with type 2 diabetes mellitus in Spain. Acarbose treatment was associated with improved life expectancy (0.23 years) and quality-adjusted life years (QALY) (0.21 years). Direct costs were on average h468 per patient more expensive with acarbose than with placebo. The incremental cost-effectiveness ratios were h2002 per life year gained and h2199 per QALY gained. An acceptability curve showed that with a willingness to pay h20,000, which is generally accepted to represent very good value for money, acarbose treatment was associated with a 93.5% probability of being cost effective. Similar results were observed by Roze et al. in Germany (2006): acarbose treatment was associated with improvements in discounted life expectancy (0.21 years) and quality-adjusted life expectancy (QALE) (0.19 QALYs), but was marginally on average more

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expensive than treatment in the placebo arm (h135 per patient). This led to incremental cost-effectiveness ratios of h633 per life year and h692 per QALY gained. For comparison, the incremental cost-effectiveness ratio for pioglitazone/metformin was h47,636 per life year gained versus sulfonylurea/metformin, and h19,745 per life year gained for pioglitazone/sulfonylurea versus metformin/sulfonylurea (Neeser et al., 2004). These studies showed that the addition of acarbose to existing treatment was associated with improvements in life expectancy and QALE, and provided excellent value for money over patient lifetimes.

5.8.7 Glycemic Control in Type 2 Diabetes Mellitus Derosa et al. (2009) compared acarbose and repaglinide in type 2 diabetic patients treated with a sulfonylurea-metformin combination therapy. One hundred and three patients were randomized to receive repaglinide, 2 mg three times a day or acarbose, 100 mg three times a day with forced titration for 15 weeks. The treatment was then crossed over for a further 12 weeks until the 27th week. After 15 weeks of therapy, the repaglinide-treated patients experienced a significant decrease in HbA1c (a1.1%, p , .05), FPG (a9.5%, p , .05), and PPG (a14.9%, p , .05), with a significant increase in body weight ( 1 2.3%, p , .05), BMI ( 1 3.3%, p , .05), and fasting plasma insulin (FPI) ( 1 22.5%, p , .05); the increase was reversed during the crossover phase. After 15 weeks of therapy, the acarbose-treated patients experienced a significant decrease in HbA 1c (a1.4%, p , .05), FPG (a10.7%, p , .05), PPG (a16.2%, p , .05), body weight (a1.9%, p , .05), BMI (a4.1%, p , .05), FPI (a16.1%, p , .05), postprandial insulin (PPI) (a26.9%, p , .05), and HOMA index (a30.1%, p , .05), when compared to the baseline values. All these changes were reversed during the crossover study phase, except those relating to HbA1c, FPG, and PPG. The only changes that significantly differed when directly comparing acarbose and repaglinide treated patients were those relating to FPI (a16.1% vs 1 22.5%, respectively, p , .05) and HOMA index (a30.1% vs 1 2.7%, p , .05). Based on the evidence that basal insulin treatment is frequently unsuccessful in controlling PPG, Kim et al. (2011) conducted a study where 58 type 2 diabetic patients, after FPG was optimized by insulin glargine, were randomized to take nateglinide 120 mg three times a day just before meals or acarbose 100 mg three times a day together with meals and then crossed over after the second wash-out period. Both drugs effectively reduced PPG levels compared with the insulin glargine monotherapy. No significant differences were found between nateglinide and acarbose in terms of mean glucose level, standard deviation of glucose levels, mean average glucose excursion, and average daily risk range. There was no episode of severe hypoglycemia, and no serious adverse events were recorded.

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Kimura et al. (2012) investigated the additive effect of AGIs in 36 type 2 diabetic patients taking lispro mix 50/50 by three times daily injection to maintain FPG , 130 mg/dL and 2-h PPG , 180 mg/dL. Twenty patients were randomly assigned to either 0.3 mg of voglibose or 50 mg of miglitol, which was administered at breakfast every other day. Another group of 16 patients was assigned to a crossover study, in which each AGI was switched every day during the 6-day study. The addition of voglibose had no effect on PPG, but miglitol blunted the PPG rise and significantly decreased 1-h and 2-h postprandial C-peptide levels compared with Mix50 alone. In addition, miglitol significantly decreased the 1-h postprandial triglyceride rise and the remnant-like particle-cholesterol rise, while it increased the 1-h postprandial high-density lipoprotein cholesterol and apolipoprotein A-I levels in the crossover study.

5.8.8 Glycemic Excursions Mori et al. (2011) investigated using CGM to assess mean amplitude of glycemic excursions (MAGE) with acarbose. Five of the patients were randomized to acarbose at 300 mg/day on days 1 and 2, but not on days 3 and 4; the remaining five patients were not administered acarbose on days 1 and 2, but were given 300 mg/day on days 3 and 4. During CGM, insulin was administered at the same time and the same dose. When acarbose was administered, the average CGM profile was decreased in almost all patients regardless of the current insulin regimen. The 24-h mean blood glucose level when acarbose was not administered was 158.03 6 32.78 mg/dL, the 24-h blood glucose fluctuation was 677.05 mgh/dL, and MAGE was 97.09. The 24-h mean blood glucose level when acarbose was administered was 131.19 6 22.48 mg/dL (p 5 .004), the 24-h blood glucose fluctuation was 453.27 mg/ dL (p 5 .002), and MAGE was 65.00 (p 5 .010). The mean proportion of time spent in the hyperglycemic range (defined as $180 mg/dL) during CGM was 29.5 6 24.4% when acarbose was not administered and 16.2 6 25.4% when it was administered. The mean proportion of time spent in the hyperglycemic range (defined as $140 mg/dL) during CGM was 58.7 6 29.4% and 40.4 6 36.3%, respectively. The mean proportion of time spent in the hypoglycemic range (defined as ,70 mg/dL) during CGM was 0.31 6 0.63% when acarbose was not administered and 0.02 6 0.5% when it was administered. These data show that hypoglycemia was not increased by concomitant treatment targeting PPG. A similar study conducted by Wang et al. (Lin et al., 2011; Wang et al., 2011) evaluated the effects of acarbose versus glibenclamide on MAGE and oxidative stress in type 2 diabetic patients not well controlled by metformin. Patients treated with metformin monotherapy (1500 mg daily) were randomized to either acarbose (50 mg three times a day for the 1 month, then 100 mg three times a day), or glibenclamide (2.5 mg three times a day for

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the 1 month, then 5 mg three times a day) for 16 weeks. CGM for 72 h and a MTT after a 10-hr overnight fast were conducted before randomization and at the end of the study. HbA1c significantly decreased in both treatment groups (from 8.2 6 0.8% to 7.5 6 0.8%, p , .001 with acarbose, and from 8.6 6 1.6% to 7.4 6 1.2%, p , .001 with glibenclamide). The MAGE did not change significantly with glibenclamide, whereas oxidized low-density lipoprotein (ox-LDL) increased significantly (from 242.4 6 180.9 ng/mL to 470.7 6 247.3 ng/mL, p , .004). Acarbose decreased MAGE (5.6 6 1.5 mmol/L to 4.0 6 1.4 mmol/L, p , .001) without significant change in ox-LDL levels (from 254.4 6 269.1 ng/mL to 298.5 6 249.8 ng/mL, p , .62). Body weight and serum triglycerides decreased (all p , .01) and serum adiponectin increased (p , .05) after treatment with acarbose, whereas HDL-C decreased (p , .01) after treatment with glibenclamide. β-Cell response to PPG increments was negatively correlated with MAGE (r 5 .570, p , .001) and improved significantly with acarbose (35.6 6 32.2 pmol/mmol to 56.4 6 43.7 pmol/mmol, p , .001), but not with glibenclamide (27.9 6 17.6 pmol/mol to 36.5 6 24.2 pmol/ mmol, p , .12).

5.8.9 Inflammation Derosa et al. (2011a, 2011b) evaluated effects of acarbose 100 mg three times a day compared to placebo on glycemic control, lipid profile, insulin resistance, and inflammatory parameters in diabetic patients before and after a standardized oral fat load (OFL). As expected, acarbose better reduced HbA1c (p , .01), FPG (p , .05), PPG (p , .05), and HOMA-IR (p , .05) compared to placebo after 7 months. Regarding lipid profile, acarbose significantly reduced total cholesterol (TC), triglycerides (Tg), and low-density lipoprotein cholesterol (LDL-C) after 7 months compared with the control group (p , .05 for all). Acarbose also improved adiponectin (ADN) and retinol binding protein-4 compared to placebo (p , .05) in a fasting condition. After the OFL, acarbose was more effective in reducing the post-OFL peaks of all the various parameters including the insulin resistance and the inflammatory markers, after 7 months of therapy. Shimazu et al. (2009) investigated the effect of acarbose on circulating levels of platelet-derived microparticles (PDMP), selectins, and ADN in patients with type 2 diabetes. Expression of cell adhesion molecules is increased in diabetes, and these molecules have been suggested to have a role in the microvascular complication of this disease. Patients were instructed to take acarbose 300 mg/day for 3 months. Acarbose therapy significantly decreased the plasma PDMP level relative to baseline (0 vs 3 months, 53.3 6 56.7 U/mL vs 32.5 6 30.1 U/mL, p , .05). Acarbose also caused a significant decrease of sP-selectin (0 vs 3 months, 235 6 70 U/mL vs 174 6 39 U/mL, p , .05), and sL-selectin (0 vs 3 months, 805 6 146 U/ mL vs 710 6 107 U/mL, p , .05). On the other hand, acarbose therapy led to

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a significant increase of AND levels after 3 months compared with baseline (0 vs 3 months, 3.61 6 1.23 μg/mL vs 4.36 6 1.35 μg/mL, p , .05). The authors investigated the effect of acarbose in diabetic patients with or without thrombosis, since 12 of the 30 diabetic patients had a history of thrombotic complications, too. The decrease of PDMP and selectin levels during acarbose therapy was significantly greater in the thrombotic group than in the nonthrombotic group (p , .05). On the other hand, ADN did not show such a difference. These data suggest that acarbose may be beneficial for primary prevention of atherothrombosis in patients with type 2 diabetes. Osonoi et al. (2010) examined the effects of switching from acarbose or voglibose to miglitol in type 2 diabetes mellitus patients for 3 months on gene expression of inflammatory cytokines/cytokine-like factors in peripheral leukocytes and on glucose fluctuations. Forty-seven Japanese patients with HbA1c levels of 6.5%7.9% were treated with acarbose (100 mg three times a day) or voglibose (0.3 mg three times a day) in combination with insulin or sulfonylurea. The current AGIs were switched to miglitol (50 mg three times a day), and the new treatments were maintained for 3 months. The switch to miglitol for 3 months did not affect hemoglobin HbA1c, FPG, or lipid profile. On the other hand, hypoglycemia symptoms and glucose fluctuations were improved significantly by the switch. The expression of interleukin-1β, TNF-α, and inflammatory cytokines that are expressed predominantly in monocytes and neutrophils were suppressed by switching to miglitol. Emoto et al. (2012) studied the effect of 3-month repeated administration of miglitol on endothelial dysfunction: 50 patients with type 2 diabetes and CAD were assigned randomly to miglitol 150 mg/day or voglibose 0.6 mg/ day for 3 months. At the end of the trial, HbA1c decreased in two groups, but the improvements in 1,5-anhydroglucitol, a marker of frequent shortterm elevations in glucose, in the miglitol group were significantly higher than in voglibose group. Insulin resistance index, C-reactive protein, and percentage flow-mediated dilatation were also improved in the miglitol group, but not in the voglibose group. Fujitaka et al. (2011) studied the effect of early intervention with pioglitazone versus voglibose on physical and metabolic profiles and serum ADN level in type 2 diabetic patients associated with metabolic syndrome. Sixty patients were analyzed for insulin sensitivity, lipid profile, serum and, and systemic inflammation. Those patients were assigned randomly to pioglitazone or voglibose in addition to conventional diet and exercise training. BMI and waist circumference did not change in the pioglitazone group, whereas these physical parameters significantly decreased in the voglibose group during a 6-month follow-up period. However, HbA1c, FPG, and HOMA-IR decreased more significantly in the pioglitazone group; the level of serum ADN, especially high-molecular-weight ADN, increased markedly in the pioglitazone group, and hs-CRP decreased significantly only in the pioglitazone group.

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5.8.10 Carotid Plaque A study (Hirano et al., 2012) evaluated whether acarbose may stabilize unstable atherosclerotic plaques rapidly in patients with acute coronary syndrome and type 2 diabetes mellitus. Patients were assigned randomly to acarbose (150 or 300 mg/day) or to placebo. Acarbose treatment was initiated within 5 days after the onset of acute coronary syndromes (ACS). Unstable carotid plaques were assessed by measuring plaque echolucency using carotid ultrasound with integrated backscatter (IBS) before, and at 2 weeks and 1 and 6 months after, the initiation of treatment. An increase in the IBS value reflected an increase in carotid plaque echogenicity. In the results, the IBS value of echolucent carotid plaques showed a significant increase at 1 month and a further increase at 6 months after treatment in the acarbose group, but there was minimal change in the control group. The increase in IBS values was correlated with a decrease in C-reactive protein levels significantly, showing that acarbose improved carotid plaque echolucency rapidly. A similar study was investigated by Koyasu et al. (2010), where patients with established CAD (B50% stenosis on quantitative coronary angiography), recently diagnosed with IGT or mild type 2 diabetes, were randomized to receive acarbose 150 mg/day or placebo to evaluate the absolute change from baseline to 12 months in the largest measured intima-media thickness (IMT) value in the right and left common carotid arteries. After 12 months in the acarbose group, IMT increased from a mean of 1.28 6 0.53 mm to 1.30 6 0.52 mm (mean change 0.02 6 0.29 mm, p not significant), whereas in the control group, it increased from a mean of 1.15 6 0.37 mm to 1.32 6 0.046 mm (mean change: 0.17 6 0.25 mm; p , .001). The difference between the acarbose and control groups was statistically significant (p 5 .01). On the other hand, voglibose was evaluated in the DIANA study (Kataoka et al., 2012): in this trial 302 patients with CAD, IGT/diabetes mellitus pattern according to 75-g OGTT and HbA1c , 6.9% were assigned randomly to lifestyle intervention, voglibose (0.9 mg/day), or nateglinide treatment (180 mg/ day). One-year coronary atherosclerotic changes were evaluated by quantitative coronary arteriography. Although voglibose significantly increased the number of patients with normal glucose tolerance at 1 year, there were no significant differences in coronary atherosclerotic changes at 1 year. However, overall, less atheroma progression was observed in patients in whom glycemic status was improved at 1 year (% change in total lesion length: 3.5% vs 26.2%, p , .01, % change in average lesion length: 0.7% vs 18.6%, p 5 .02).

5.8.11 Impaired Glucose Tolerance Kawamori et al. (2010) conducted a study to assess whether voglibose could prevent type 2 diabetes developing in high-risk Japanese subjects with IGT.

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Voglibose was administered in 897 patients, while 883 received placebo; the study was designed for treatment to be continued until participants developed type 2 diabetes or for a minimum of 3 years. An interim analysis significantly favored voglibose; subjects treated with voglibose had a significantly lower risk for progression to type 2 diabetes than placebo (50/897 vs 106/ 881: HR 0.595). Also, significantly more subjects in the voglibose group achieved normoglycemia compared with those in the placebo group (599/897 vs 454/881:HR 1.539). Acarbose also proved to be safe and effective in patients with IGT; in the STOP-NIDDM (Study To Prevent Non-Insulin Dependent Diabetes Mellitus) trial (Chiasson et al., 2002), 714 patients with IGT were randomized to acarbose 100 mg three times daily and 715 to placebo. Acarbose significantly increased reversion of IGT to normal glucose tolerance (p , .0001); the risk of progression to diabetes over 3.3 years was reduced by 25%. At the end of the study, treatment with placebo was associated with an increase in conversion of IGT to diabetes. Also the same study showed that decreasing PPG with acarbose was associated with a 49% relative risk reduction in the development of cardiovascular events (p 5 .03) and a 2.5% absolute risk reduction (Chiasson et al., 2003). Among cardiovascular events, the major reduction was in the risk of myocardial infarction (p 5 .02). Acarbose was associated with a 34% relative risk reduction in the incidence of new cases of hypertension (p 5 .006) and a 5.3% absolute risk reduction. Even after adjusting for major risk factors, the reduction in the risk of cardiovascular events (p 5 .02) and hypertension (p 5 .004) associated with acarbose treatment was still statistically significant.

5.8.12 Conclusion for Comparison of α-Glucosidase Inhibitors Of all AGIs, acarbose remains the most widely studied drug of the class. From the preceding studies, AGIs were superior to placebo in reducing HbA1c, FPG, and PPG. There is evidence that AGIs more effectively reduced intraday and interday glucose variability compared to other antidiabetic drugs (Derosa et al., 2011a). Regarding the effects on inflammatory markers, miglitol seemed more effective than voglibose or acarbose in suppressing glucose fluctuations and the gene expression of inflammatory cytokines/cytokine-like factors in peripheral leukocytes, with fewer adverse effects (Osonoi et al., 2010). Moreover, acarbose showed some additive action compared to voglibose and miglitol: acarbose improved echolucency in carotid plaque after 1 month of treatment, continuing during the next 5 months (Hirano et al., 2012). These results suggest that early treatment of hyperglycemia with acarbose may potentially stabilize vulnerable carotid plaques in acute coronary syndrome type 2 diabetic patients. The mechanism of that can be sought in PPG: hyperglycemia induces oxidative stress, endothelial dysfunction, and proinflammatory cytokines through oxidative

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stress-induced activation of nuclear factor κB (Zarich and Nesto, 2007). Reducing hyperglycemia, acarbose also reduced proinflammatory cytokines and stabilized carotid plaque. This positive action on carotid plaque was not confirmed by voglibose, suggesting that the effect was peculiar to acarbose (Kataoka et al., 2012). Finally, both voglibose and acarbose proved to significantly increase reversion of IGT to normal glucose tolerance (Chiasson et al., 2002, 2003; Kawamori et al., 2009), and to give a 49% relative risk reduction in the development of cardiovascular events in patients with IGT (Chiasson et al., 2003). Also from the cost-effectiveness ratio point of view, acarbose improved life expectancy and QALE, and provided excellent value for money over patient lifetimes (Pin˜ol et al., 2007; Roze et al., 2006). From all these reported considerations, we can safely conclude that AGIs proved to be safe and effective in improving glycemic control and PPG, and in particular acarbose proved to have a lot of additive effects that can help in reducing the macro- and microvascular complications related to type 2 diabetes.

5.9 MARKET AND DEVELOPMENT The α-glucosidase inhibitors (acarbose, miglitol, and voglibose) have been in clinical use for about 20 years and, particularly in Asian countries, are among the most commonly prescribed oral antidiabetic agents (Standl and Schnell, 2012). The AGIs delay the absorption of glucose, thereby preventing both PPG and hyperinsulinemia (Chen et al., 2006; Mooradian and Thurman, 1999; Scheen, 2003), with little or no risk of hypoglycemia (Derosa and Maffioli, 2012; Scheen, 2003). In contrast to some other classes of oral antidiabetic agents, no signals of potential cardiovascular harm have emerged in relation to their use (Standl and Schnell, 2012). Voglibose improves PPG with the effect that is generally milder than that of other antidiabetic agents but with little or no risk of hypoglycemia. This is potentially advantageous in patients with IGT or early T2DM and makes voglibose particularly useful for use in combination regimens to improve glycemic control without increasing the risk of adverse events such as hypoglycemia. Voglibose increases the reversion of IGT to normal glucose tolerance and may therefore be used, either as an alternative or in addition to changes in lifestyle, to delay the development of T2DM in patients with IGT. The ability of voglibose to improve cardiovascular outcomes by targeting PPG in patients with IGT or early stage T2DM appears to be worthy of further research. For quick reference, the advantages and disadvantages of voglibose are summarized in Table 5.5. Many studies of voglibose had small sample sizes and were conducted almost exclusively in Japanese patients, thus restricting generalizability of the results to other ethnic groups. Although Japanese guidelines for treatment of newly diagnosed T2DM recommend AGIs among the first-line

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TABLE 5.5 Advantages and Aisadvantages of Voglibose Advantages

Disadvantages

Specifically targets postprandial hyperglycemia, with little or no risk of hypoglycemia

Milder antihyperglycemic effect compared with other antidiabetic agents

Available in different formulations / dosages to facilitate individualized titration

Effects on cardiovascular risk yet to be established

No active metabolites; negligible renal excretion

Associated with gastrointestinal symptoms, which may limit patient acceptability

Can be used as monotherapy or in combination with other antidiabetic agents

Rare cases of pneumatosis cystoides intestinalis (gas cysts) have been reported

Additional glycemic/metabolic effects

Studies to date have been conducted almost exclusively in Japanese patients

Strong evidence for the prevention of type 2 diabetes mellitus from impaired glucose tolerance

pharmacotherapeutic options, this drug class does not feature in the American Diabetes Association and European Association for the Study of Diabetes guidelines (Nathan et al., 2008), possibly because of the lack of clinical trials in populations outside of Japan. In view of incontrovertible evidence for voglibose to delay the progression to T2DM from IGT (Kawamori et al., 2009), and the burgeoning incidence of IGT worldwide, opportunity exists for other populations to benefit from targeted treatment of PPG with voglibose through the design and conduct of appropriate clinical trials. The relationship between increasing levels of plasma glucose and risk of micro- and macrovascular complications is well documented. Traditionally, the predominant focus of therapy in patients with T2DM has been on lowering HbA1c, with a strong emphasis on FPG levels (Ceriello and Colagiuri, 2008; Shimabukuro et al., 2012). Even if adequate HbA1c is reached, however, PPG can still occur (Derosa and Maffioli, 2012). As the progressive relationship between glucose levels and cardiovascular risk is stronger with postmeal glucose than with fasting glucose (DECODE Study Group, the European Diabetes Epidemiology Group, 2001) and extends below the diabetic threshold (Coutinho et al., 1999), PPG is now recognized as a key therapeutic target to reduce the excess morbidity and mortality associated with diabetes. The large-scale STOP-NIDDM study of acarbose in patients with IGT provided clear evidence of a reduction in cardiovascular

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events (p 5 .02) and new cases of hypertension (p 5 .03) by targeting PPG specifically (Chiasson et al., 2003). Lifestyle modifications such as weight loss and increased physical activity continue to be core interventions for reducing the cumulative incidence of IGT and T2DM but success rates are generally disappointing. Availability of agents including AGIs that preferentially reduce postprandial blood glucose excursions has made it possible to achieve glycemic goals in a larger proportion of individuals (Mooradian and Thurman, 1999). Moreover, in view of the exponential increase in the numbers of people with IGT, effective strategies to prevent the progression of prediabetes to overt T2DM have enormous potential to reduce the individual and societal burden of disease (Choudhary et al., 2012). Voglibose was the first oral antidiabetic agent to be approved for this indication in Japan on the basis of a large study showing that voglibose reduced the risk of progression to T2DM from IGT by 40.5% over placebo (Kawamori et al., 2009). In this respect, voglibose joins acarbose, which was first approved in China in 2002, for the treatment of prediabetes after having demonstrated a 25% decrease in the risk of conversion of IGT to T2DM in the STOP-NIDDM trial (Chiasson et al., 2002). There is tantalizing early evidence to suggest that targeting PPG may reduce cardiovascular risk factors in patients with IGT and early stage T2DM, but this needs to be demonstrated conclusively in appropriately powered randomized clinical studies (Shimabukuro et al., 2012). In addition, reports of voglibose reducing postprandial hyperlipidemia either alone (Satoh et al., 2006) or in combination (e.g., with pioglitazone (Abe et al., 2007) or mitiglinide (Ono et al., 2014) in patients on hemodialysis) are intriguing on the basis that elevated postprandial triglycerides are also a recognized risk factor for cardiovascular disease (Kolovou et al., 2011). Based on this description, voglibose appears to be most appropriate for the prevention of T2DM in patients with IGT and, either as monotherapy or in combination with other suitable antidiabetic agents with complementary modes of action including insulin, for the treatment of early stage T2DM. Specific therapeutic situations where voglibose may be particularly well suited include individuals with elevated postprandial glucose levels but normal HbA1c levels, persons at greatest risk of hypoglycemic episodes, and patients receiving hemodialysis.

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Chapter 6

N-Octyl-β-Valienamine and N-Octyl-4-epi-β-Valienamine: Two Highly Potent Drug Candidates for Chemical Chaperone Therapy 6.1 CHAPERONE THERAPY A large number of genetic diseases (currently .7000) listed in a genetic disease catalog are caused by functional defect of enzymes (enzyme deficiency), resulting in diverse metabolic derangements in human somatic cells. The metabolic defect is expressed generally in various tissues and organs, but most prominently in the central nervous system (neurogenetic diseases). Among them the diseases involving the lysosome, one of the important cellular organelles, digesting various high molecular endogenous or exogenous compounds under the acidic condition (Duve and Wattiaux, 1966) have been well recognized as classic neurogenetic diseases caused by specific enzyme deficiency affecting mainly infants and young children. Cellular dysfunction caused by an excessive storage of substrates ensues, and a genetic metabolic disease (lysosomal disease) develops in humans and other animals with neurological and other somatic manifestations. Severity of enzyme deficiency is variable in individual patients. In general, severe enzyme deficiency tends to cause severe clinical manifestations in early life (Suzuki et al., 2009). Since the mid-1960s, attempts have been made to treat patients with lysosomal diseases. Enzyme replacement therapy was the most successful approach by intravenous administration of the functional human recombinant enzyme. First, purified β-glucosidase was shown to be effective for Gaucher disease, the most prevalent lysosomal storage disorder in humans (Brady, 2006). This approach has been extended to other lysosomal diseases, including Fabry disease, mucopolysaccharidoses, and Pompe disease. However, the effect has not been confirmed on brain pathology in patients with neurological manifestations.

Validamycin and Its Derivatives. DOI: http://dx.doi.org/10.1016/B978-0-08-100999-4.00006-X © 2017 Elsevier Ltd. All rights reserved.

279

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Validamycin and Its Derivatives

TABLE 6.1 Effect of Protease Inhibitors on Exogenous β-Galactosidase in Human Fibroblasts Protease Inhibitor Added

Target Protease

None

Relative β-galactosidase Activity 100

E-64

Cysteine protease

229

Leupeptin

Serinecysteine protease

222

Antipain

Papain, trypsin, cathepsin A/B

224

Chymostatin

Chymotrypsin

188

Elastatina 1

Elastinase

103

Bestatin

Aminopeptidase

97

Phosphoramidon

Metalloprotease

88

Pepstatin

Aminopeptidase

90

Human GM1-gangliosidosis fibroblasts were cultured with exogenous β-galactosidase purified from Aspergillus oryzae, and incubated with one of the protease inhibitors for 24 h. E-64, leupeptin, antipain, and chymostatin are thiol protease inhibitors.

In the early 1980s, it was found that thiol (cysteine) protease inhibitors protected degradation of endogenous human or exogenous fungal β-galactosidase (Ko et al., 1983; Sakuraba et al., 1982; Suzuki et al., 1981), which was responsible for GM1-gangliosidosis in humans (Table 6.1). These results encouraged us to search for a new way to rescue apparently inactive mutant enzyme proteins for a new molecular therapy of enzyme deficiency disorders. In the connection, a correlation between residual β-galactosidase activity and clinical onset in GM1-gangliosidosis patients (Suzuki et al., 2009) (Fig. 6.1) was discovered. The amount of residual enzyme activity showed positive parabolic correlation with the age of onset in various phenotypic forms of β-galactosidase deficiency disorders. The enzyme activity was generally less than 3% of the control in infantile GM1-gangliosidosis, 3%6% in juvenile GM1-gangliosidosis, and more than 6% in late onset (adult/chronic) GM1-gangliosidosis patients. Furthermore, Morquio B disease, another nonneurological phenotype of β-galactosidase deficiency, showed relatively high residual enzyme activity. Based on these results, that at least 10% of normal enzyme activity is necessary for washout of the storage substrate in somatic cells was anticipated, particularly in neuronal cells. The age of onset in patients expressing enzyme activity above this level would be theoretically beyond the human life span. However, it should be kept in mind that this theoretical curve was drawn on the basis of enzyme assay results using

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FIGURE 6.1 Correlation between residual β-galactosidase activity and clinical onset. The amount of residual enzyme activity shows positive parabolic correlation with the age of onset in various phenotypic forms of β-galactosidase deficiency disorders. The age of onset in patients expressing enzyme activity above this level is theoretically beyond the human life span. See text for details.

cultured skin fibroblasts (not neuronal cells) and a synthetic fluorogenic substrate 4-methylumbelliferyl β-galactopyranoside (not natural substrates like ganglioside GM1 or keratan sulfate). In the calculation, for technical reasons, substrate specificity was not taken into account, although mutant enzymes show different spectrum in GM1-gangliosidosis and Morquio B disease (Suzuki, 2013). During the last decade of the 20th century, extensive gene and protein molecular analyses of β-galactosidase deficiency disorders (GM1-gangliosidosis and Morquio B disease) (Oshima et al., 1991; Yoshida et al., 1991, 1992) and α-galactosidase A deficiency disorders (Fabry disease) (Ishii et al., 1995; Okumiya et al., 1995a,b) were carried out. During this period, some mutant α-galactosidase A proteins were found to be unstable and unable to express catalytic activities (Ishii et al., 1993; Okumiya et al., 1995a,b). Galactose and a galactose analog compound 1-deoxygalactonojirimycin (DGJ) were effective to restore the mutant α-galactosidase A activity in Fabry cells and tissues. After subsequent extensive molecular analysis, the following hypothesis was proposed (Suzuki, 2006; Suzuki et al., 2009). A substrate analog inhibitor binds to a mutant lysosomal misfolding protein as a kind of molecular chaperone (chemical chaperone), to achieve normal molecular folding at the endoplasmic reticulum (ER)/Golgi compartment in somatic cells, resulting in the formation of a stable molecular complex at neutral pH. The proteinchaperone complex is safely transported to the lysosome, where it dissociates under the acidic condition, the mutant enzyme remains stabilized in its normal folding structure, and its catalytic function is expressed (Fig. 6.2).

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FIGURE 6.2 Postulated molecular events between mutant enzyme molecules and chaperone compounds. Mutant enzyme protein is unstable in the ER/Golgi compartment at neutral pH, and rapidly degraded or aggregated possibly to cause ER stress. An appropriate substrate analog inhibitor binds to mutant misfolding protein as chemical chaperone at the ER/Golgi compartment in somatic cells, resulting in normal folding and formation of a stable complex at neutral pH. The proteinchaperone complex is safely transported to the lysosome. The complex is dissociated under the acidic condition and possibly in the presence of an excessive amount of the substrate stored as substrateprotein complex (membranous cytoplasmic body, MCB). The mutant enzyme remains stabilized, and expresses catalytic function. However, the enzyme may not be able to utilize all substrate molecules within the MCB complex. The released chaperone is either secreted from the cell or recycled to interact with another mutant protein molecule. These molecular events have been partially clarified by analytical and morphological analyses, and computer-assisted prediction of molecular interactions.

Molecular pathology of inherited metabolic diseases can be generally classified into the following three major conditions related to the structure and function of mutant proteins (Suzuki, 2006). (1) Biosynthetic defect of the protein in question. Mutant enzyme is not synthesized, and accordingly rescue of the protein is not possible. (2) Defect of biological activity. In spite of normal biosynthesis, the protein does not maintain biological activity because of its drastic structural abnormality (misfolding). There is no possibility to restore the biological activity of this molecule. (3) Unstable mutant protein with normal or near-normal biological activity. The mutant protein has normal biological function in its mature form under normal folding. However, it is unstable because of misfolding and rapidly degraded immediately after biosynthesis. In the third case, the protein function is expected to be restored if the molecule is somehow stabilized and transported to the cellular compartment where it is expected to exhibit biological activity, e.g., the

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lysosome in the case of lysosomal enzymes. This is the principle of chaperone therapy to restore the enzyme activity by low molecular compounds with appropriate molecular structure fitting in the enzyme molecule. They are particularly important for correction of brain pathology if they are delivered to the central nervous system through the blood brain barrier. This principle is applied to at least 30%50% of mutant genes in Fabry disease and GM1-gangliosidosis patients using a single chaperone compound.

6.2 N-OCTYL-β-VALIENAMINE FOR CHAPERONE THERAPY OF GAUCHER DISEASE Gaucher disease is a group of diverse clinical manifestations involving both the central nervous system and extraneural visceral organs, caused by β-glucosidase deficiency, resulting in massive storage of glucosylceramide. Clinically it is classified into three major phenotypes: chronic nonneuronopathic (adult), acute neuronopathic (infantile), and subacute neuronopathic (juvenile). Enzyme replacement therapy is available for nonneuronopathic patients, and the clinical effect has been well confirmed. However, neurological manifestations have not been controlled by this therapeutic approach. Attempts were made to develop chaperone compounds for Gaucher disease. N-Octyl-β-valienamine (NOV) is one of the most important chaperone compounds for Gaucher disease.

6.2.1 Synthesis and Screening of N-Alkyl-β-Valienamine as β-Glucocerebrosidase Inhibitors In the findings of potent and specific β-glucocerebrosidase inhibitors, a similar type of inhibitor was developed by transforming β-valienamine 1 into some derivatives with simple structures (Ogawa et al., 1996). They have strong inhibitory activity against β-glucocerebrosidase. In order to investigate the inhibitory potency of these glucosidase inhibitors (Legler, 1990) N-alkyl glucopyranosylamines, six homologous N-alkyl-β-valienamines 2a2f

FIGURE 6.3 Chemical structures of β-valienamine and N-alkyl-β-valienamines.

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SCHEME 6.1 Synthesis of N-alkyl β-valienamines from a protected β-valienamine.

(Fig. 6.3) were synthesized by the conventional procedure: conversion of the protected β-valienamine 3 into the corresponding amides, reduction with lithium aluminum hydride (LAH), and subsequent deprotection (Scheme 6.1). Acylation of di-O-isopropylidene-β-valienamine (Ogawa et al., 1992) (3) with n-octanoyl chloride (1.2 mol/L equiv) in pyridine at room temperature produced the amide 4c, quantitatively. Treatment of 4c with an excess of LAH(15 mol/L equiv.) in THF for 2 h at reflux temperature gave the protected amine 5c in 85% yield. O-Deisopropylidenation was carried out with aqueous 80% acetic acid at 80 C for 4 h to give, after chromatography on silica gel with chloroform/methanol (3:1) as an eluent, the amine 2c as acetate in quantitative yield. Other homologous series of compounds 2a, 2b, 2d, 2e, and 2f were similarly prepared as in the preparation of 2c. Inhibitory activity of 1, 2af, α-valienamine 6, against β-glucocerebrosidase is listed in Table 6.2. Inhibitory activity against β-glucocerebrosidase seems to largely depend on the length of N-alkyl chain. The activity of the most potent N-octyl compound is 3c. Since 1 and 6 have no activity against β-glucocerebrosidase, the N-alkyl chain with an appropriate length should be needed for exhibiting inhibitory potency. Therefore, it is interesting to know the structure and inhibitory activity relationship of this kind of inhibitor by modifying the alkyl chains. Moreover, N-alkyl-β-valienamines are expected to act as specific competitive inhibitors, since valienamine moiety seems to mimic the transition state of the substrate for β-glucosidase reaction. On the other hand, compounds 2a2f completely lack inhibitory activity against glucocerebroside synthase.

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TABLE 6.2 Inhibitory Activity of Six N-Alkyl-β-Valienamines and their Related Compounds Against Three Enzymes Compounds

Inhibitory Activity (IC50, mol/L) β-Glucocerebrosidase

Glucocerebroside Synthase

α-Glucosidase

1

NI

NI

1.0 3 1024

2a

1.1 3 1025

NI

a

2b

3.0 3 1027

NI

5.0 3 1025

2c

3.0 3 1028

NI

1.7 3 1025

2d

7.0 3 1028

NT

NT

2e

27

1.2 3 10

NT

NT

2f

3.0 3 1027

NI

a

6

NI

NI

1.0 3 1024

NI, No inhibitory activity observed at 1.0 3 1024 mol/L; NT, Not tested. a Activity less than IC50 1.0 3 1024 mol/L.

6.2.2 Chemical Modification of N-Octyl-β-Valienamine In order to elucidate the structureinhibitory activity relationship of the hydrophobic moiety, chemical modification of NOV 3c was carried out (Ogawa et al., 1998, 2002). The hydrophobic portion of 3c was modified by introducing a double-chain structure by converting it into N-alkanoyl and N-alkyl derivatives (Scheme 6.2). Thus, the N-alkanoyl 8ad and N-alkyl derivatives 9ag were prepared conventionally by treatment of N-octyl 2,3:4,6-di-O-isopropylidene-β-valienamine (Ogawa et al., 1996) (5c) with the corresponding acid chloride in pyridine (5c-6ag) and subsequent reduction with LAH in tetrahydrofuran (THF) (6ag-7ag). Removal of the protecting groups of 6ad and 7ag afforded 8ad and 9ag, respectively. These compounds were subjected to assay for β-glucocerebrosidase (mouse liver) in vitro and, especially, four compounds 8a, 8d, 9a, and 9d were prescribed into mouse-derived B16 melanoma cells and the amount of glucosylceramide and GM3 in cells after treatment was evaluated. Another two derivatives, 4-epimer 10, β-galacto-type N-octyl-valienamine, and 4-O-(β-D-galactopyranosyl) derivative 11 (Fig. 6.4), a carbalactosylceramide analogue, were synthesized and evaluated biologically (Ogawa et al., 2002). The secondary amino function of N-octyl-2,3:4,6-di-O-isopropylideneβ-valienamine1 (5c) was first protected to give the N-tert-butoxycarbonyl

SCHEME 6.2 Chemical modification of N-octyl-β-valienamine.

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FIGURE 6.4 Chemical structures of N-octyl-valienamine and its derivatives.

derivative (12), the isopropylidene groups of which were then removed with aqueous acetic acid (Scheme 6.3). The crude tetrol was then treated with α,α-dimethoxytoluene in N,N0 -dimethylformamide (DMF) to give the 4,6-Obenzylidene derivative (14), which was further protected with methoxymethyl ether groups (-15). The benzylidene group was then removed and the resulting 6-hydroxyl group was selectively silylated to give the tertbutyldimethylsilyl derivative (16). The remaining free 4-hydroxyl group was then oxidized with pyridinium chlorochromate in CH2Cl2 and the resulting α,β-unsaturated ketone was reduced with 1 mol/L lithium tri-sec-butyl borohydride in THF at 78 C to give almost selectively the epimeric alcohol 17 in 66% yield. This compound was finally deprotected by treatment with a mixture of 4 mol/L hydrochloric acid and THF at 65 C to afford, after purification over a column of Dowex 50 W 3 2 (H1) resin with 1% ammonia, N-octyl β-galacto-valienamine 10 in 91% yield. The N-trifluoroacetyl derivative 6 was obtained quantitatively by treatment with trifluoroacetic anhydride in pyridine (Scheme 6.4). Compound 18, obtained by similar deprotection of 11 followed by benzylidenation, was first treated with 11 molar equivalent of benzoyl chloride in pyridine at room temperature to yield the dibenzoate 19, which, after removal of the benzylidene group, was subjected to a selective benzoylation of the primary hydroxyl group with 1.5 molar equivalent of the reagent at -15 C, affording the 2,3,6-tribenzoate 20 in 97% overall yield. Incorporation of a β-galactose residue at C-4 was effected, however, in a poor yield by coupling 20 with 3 molar equivalent of 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl trichloroacetimidate in dichloromethane in the presence of BF3 etherate at 0 C to room temperature, affording a carbadisaccharide derivative 21 in 19% yield, together with 20 (58%) recovered. The product was treated with methanolic potassium carbonate for 1 h at room temperature to give, after purification

(c)

(a) (b)

(h)

(e)

(d)

(f )

(g)

SCHEME 6.3 Chemical synthesis of 4-epimer 10. Reagents and conditions: (a) (t-BuOCO)2O, Et3N, CH2Cl2, rt; (b) (CF3CO)2O, pyridine, rt; (c) 60% aq AcOH, 60 C; PhCH(OMe)2, TsOH, DMF, 45 C; (d) MeOCH2Cl, ClCH2CH2Cl, i-Pr2EtN, 60 C; (e) 60% aq AcOH, 60 C; t-BuMe2SiCl, DMF, rt; (f) PCC, CH2Cl2, rt; (g) s-Bu3LiBH, THF, 78 C-0 C; (h) 4 mol/L HCl, THF, 65 C; Dowex 50 3 2 (H1) resin, 1% NH3/MeOH.

(a)

(c)

(b)

(d)

(h)

SCHEME 6.4 Chemical synthesis of 4-O-(β-D-galactopyranosyl) derivative 11. Reagents and conditions: (a) 80% aq AcOH, 80 C; PhCH(OMe)2, TsOH, DMF, 45 C; (b) BzCl (6 mol/L equiv), pyridine, rt; (c) 80% aq AcOH, 80 C; BzCl (1.5 mol/L equiv), pyridine, 15 C; (d) 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl trichloroacetimidate (3 mol/L equiv), BF3/diethyl ether, molecular sieves 4 A, CH2Cl2, 0 C, 0.5 h; (e) aq K2CO3, 1 H, rt; Dowex 50 3 2 (H1) resin, 1% NH3/MeOH.

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by a resin column as in the preparation of 10, free N-octyl-5α0 -carba-β-lactosylamine 11 in 73% yield.

6.2.3 Biological Activities of N-Octyl-β-Valienamine Derivatives Inhibitory activity of 8a-d and 9a-g against β-glucocerebrosidase (mouse liver) is listed in Table 6.3. All N-alkanoyl derivatives 8a-d completely lack the activity, whereas N-alkyl derivatives 9a-g were shown to possess strong potential comparable to the parent 2c. These results demonstrated that the basic cationic property of the alkyl amino functions should be very important, and the structures of the hydrophobic portions seem to be not so strictly recognized at its binding to the enzyme, although the appropriate combination of chain lengths of the N-alkyl functions, e.g., C8 and C10, should be preferred for exhibiting optimum activity. Biological assays of compound 10 (Table 6.1) showed medium inhibitory activity (IC50 5 5.0 3 1026 mol/L) toward β-galactocerebrosidase (mouse liver). Interestingly, it was also demonstrated to possess strong activity (IC50 5 3 3 1027 mol/L) against β-galactosidase (human). Therefore, chemical modification of 10 as a lead compound has been carried out extensively in our laboratory. Compound 11 was found to possess inhibitory activity TABLE 6.3 Inhibitory Activity (IC50, mol/L) of N-Alkanoyl-N-Octyl 8a-d and N-Alkyl-N-Octyl-β-Valienamines 9a-g Against β-glucocerebrosidase Mouse Liver Compound

Inhibitory Activity Against β-glucocerebrosidase (IC50, mol/L)

8a

a

8b



8c



8d



9a

1.4 3 1026

9b

3.5 3 1027

9c

3.5 3 1027

9d

1.4 3 1027

9e

3.2 3 1027

9f

3.5 3 1027

9g

4.2 3 1027

Inhibitory activity IC50 less than 1.0 3 1024 mol/L.

a

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(IC50 5 2 3 1025 mol/L) only against β-glucocerebrosidase (mouse liver). The results might be understood in terms of its original potential and/or formation of the strong inhibitor, NOV, generated by partial enzymatic hydrolysis of 11. However, the experiments in this direction with NOV remain at the level of culture cells, since appropriate animal models have not been produced. Its effectiveness has been extensively studied in the cell culture system (Jo et al., 2010; Lei et al., 2007; Lin et al., 2004; Luan et al., 2010a,b; Suzuki, 2013; Suzuki et al., 2009). However, its application will be achieved if animal models become available in the near future.

6.3 N-OCTYL-4-EPI-β-VALIENAMINE FOR CHAPERONE THERAPY OF GM1-GANGLIOSIDOSIS GM1-gagliosidosis is a relatively rare lysosomal disease caused by β-galactosidase deficiency. The incidence was estimated at 1:100,000200,000 live births (Meikle et al., 1999). It is the fourth common sphingolipidosis in Turkey (Ozkara and Topc¸u, 2004). Major storage compounds are ganglioside GM1 and its derivative GA1, keratan sulfate, and glycoprotein-derived oligosaccharides. It presents clinically with progressive neurological deterioration mainly in infancy and childhood. This disease has been the major target for many researchers. The correlation of phenotypic manifestations with storage compounds (Suzuki et al., 1971, 1978), enzyme activities (Suzuki et al., 1974, 1977; Suzuki and Suzuki, 1974a,b,c), and protection of enzyme molecules as stated above (Ko et al., 1983; Sakuraba et al., 1982), and enzyme molecular analysis (Nanba et al., 1988) was analyzed. After β-galactosidase cDNA cloning (Oshima et al., 1988), extensive mutation data have been accumulated in GM1-gagliosidosis and Morquio B patients (Oshima et al., 1991; Yoshida et al., 1991), and currently more than 160 mutations have been identified in the Human Gene Mutation Database (HGMD). Mutations in GM1-gangliosidosis patients are heterogeneous and complex. No ethnic prevalence is known although some common mutations have been identified: p.R208C in American patients with infantile GM1-gangliosidosis; p.R482H in Italian patients with infantile GM1-gangliosidosis; and p.I51T in Japanese patients with adult GM1-gangliosidosis; p.R201C in Japanese patients with juvenile GM1-gangliosidosis. Another mutation, p.W273L, is known to cause Morquio B disease, the second β-galactosidase deficiency phenotype (generalized skeletal dysplasia) (Oshima et al., 1991). At present, only symptomatic therapy is clinically available for the brain lesion in human GM1-gangliosidosis patients. Various experimental therapeutic approaches were reported toward GM1-gangliosidosis. Thiol (cysteine) protease inhibitors prolonged the effect of exogenous β-galactosidase in human GM1-gangliosidosis fibroblasts (Ko et al., 1983). The effect was enhanced when the enzyme was supplied as liposomes. Enzyme replacement

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of cultured cells from cats with GM1-gangliosidosis was tried with the liposome-entrapped enzyme, and the storage of glycopeptides decreased (Reynolds et al., 1978). Allogeneic bone marrow transplantation was performed in a Portuguese water dog affected with GM1-gangliosidosis using a dog leukocyte antigen-identical sibling as donor (O’Brien et al., 1990). β-Galactosidase activity in leukocytes of the transplanted dog was similar to that in the donor. However, neither the subsequent clinical course nor the enzyme activity was modified. Beneficial effects of substrate reduction therapy have been reported in a mouse model of GM1-gangliosidosis (Elliotsmith et al., 2008). The first report about the chaperone experiment on β-galactosidase was published in 2003 (Matsuda et al., 2003), with a newly synthesized organic compound, valienamine derivative, N-octyl-4-epi-β-valienamine (NOEV), as a chemical chaperone for β-galactosidase toward a genetically engineered GM1-gangliosidosis model mouse expressing the p.R201C mutant (R201C mouse) (Matsuda et al., 1997).

6.3.1 Synthesis and Screening of NOEV as β-Galactosidase Inhibitor N-Octyl-4-epi-valienamine (NOEV) (10) was first synthesized by epimerization at C-4 of N-octyl-valienamine (NOV) (2c) via multistep reactions (Scheme 6.3) (Ogawa et al., 2002). Therefore, selective reduction of the 4-keto derivative, provided by oxidation of the 4-OH unprotected derivative of 10, could be carried out under careful conditions to improve acceptable selectivity for the 4-epimer. Production of a large quantity of NOEVs is now needed for further development of possible oral medicines applicable for chaperone therapy of genetic diseases caused by lysosomal accumulation. Versatile key compounds can be envisaged for combinatorial preparation of a homologous series of N-alkyl-4-epi-valienamines (Ogawa et al., 2004) (Scheme 6.5). So the 3-epimeric alkadiene 25 was designed and synthesized by conventional dehydrobromination of the dibromide 24 derived from the tribromide 23. The 2,3-O-isopropylidene acetate 26 was converted into the dibromides, which were subjected without isolation to selective acetolysis at the primary site to give an isomeric mixture 27 of the reactive allylic bromides. The mixture was found to offer convenient precursors for preparation of a number of N-alkyl-4-epi-β-valienamine homologues. Thus, the α-allyl bromide was considered to be attacked by alkylamine in a SN2 fashion to mainly give β-amino compound, while, on the other hand, the β-allyl bromide might produce a similar mixture of products through neighboring participation with the 3-acetoxyl group at C-4 to form a 3,4-acetoxonium ion, followed by upside attack of nucleophiles. The mixture 27 readily undergoes substitution reactions with nucleophiles, such as azide anions, alkyl and phenylalkyl amines, etc., to afford various N-substituted β-epivalienamines selectively.

(c)

(b)

(a)

(f) (e)

(h)

(g)

SCHEME 6.5 Convenient synthesis of 5α-carbaglycopyranosylamines with β-galacto configuration, starting from the intermediate 22 synthesized from the DielsAlder endoadduct of furan and acrylic acid. Reagents and conditions: (a) 15% HBr/AcOH, 80 C; (b) NaOMe/MeOH; 1% H2SO4/aq. acetone; Ac2O/Pyr; (c) DBU/toluene, 60 C; (d) NaOMe/MeOH; DMP, p-TsOH/DMF; Ac2O/Pyr; (e) Br2, AIBN, toluene; AcONa/MCS; Ac2O/Pyr; (f) NaN3/DMF; (g) RNH2/i-PrOH; aq. AcOH; 4 mol/L HCl; acidic resin treatment; (h) NaOMe/MeOH; H2S or Ph3P/aq. p-dioxane; acidic resin treatment.

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Validamycin and Its Derivatives

(b)

(a)

(c)

(d)

(f)

(e)

SCHEME 6.6 Convenient synthesis of 5α-carbaglycopyranosylamines starting from optically active deoxyinositol produced by bioconversion of myo-inositol. Reagents and conditions: (a) Bioconversion; (b) Biooxidation; (c) CH3N2/MeOH, Et2O; (d) HI, AcOH; (e) Zn, AcOH; (f) Br2/AcOH; Zn/toluene.

Later, biooxidation of (2)-vibo-quercitol derived by bioconversion (Ogawa et al., 2007) of myo-inositol gave a quantity of (2)-2-deoxy-scylloinosose (33) (Ogawa et al., 2004). This has already been employed to allow establishment of a new convenient route for carbaglycosylamines through crystalline spiro epoxy 34, methylene compounds 35, and the alkadiene 36 (Aich and Loganathan, 2005) (Scheme 6.6). Recently, Kuno et al. (2011, 2013) described a synthesis route for NOEV starting from ( 1 )-proto-quercitol (37), which is readily provided by the bioconversion of myo-inositol. Acetonation of 37 with 2,2-dimethoxypropane in the presence of (6)-10-camphorsulfonic acid in acetone, followed by oxidation with a pyridinesulfur trioxide complex and triethylamine, afforded crystalline ketone 38 in a 53% yield (Scheme 6.7). Next, the trans-isopropylidene group of 38 was selectively removed with a catalytic amount of pyridinium-p-toluene-sulfonate in methanol to give a dihydroxyketone that was, after treatment with benzoyl chloride in pyridine, converted into the single enol-benzoate 39 in 60% yield. It is noteworthy that the enol-benzoate 39 could be readily prepared from 37 on a 10-g scale without column purification. A similar acylation with acetic anhydride produced a syrupy mixture of products, most likely composed of unsaturated ketones and enol-acetates. Direct treatment of 39 under Wittig reaction conditions with an excess of methyltriphenylphosphonium bromide (6 mol/L equiv)/n-BuLi (4 mol/L equiv) in THF smoothly resulted in the desired conjugated alkadiene 40 in 66% yield after purification on a silica gel column. The alkadiene 40 was treated with a slight excess of bromine in carbon tetrachloride to give a 68% yield of an epimeric mixture of the 1,4-addition

N-Octyl-β-Valienamine and N-Octyl-4-epi-β-Valienamine Chapter | 6

(b)

(a)

295

(c)

(h)

(d)

(f)

(e) (i)

(g)

SCHEME 6.7 Synthesis of NOEV from ( 1 )-proto-quercitol. Reagents and conditions: (a) 2,2-dimethoxypropane (10 mol/L equiv), (6)-10-camphorsulfonic acid (0.2 mol/L equiv), acetone, rt, 19 h; pyridinesulfur trioxide complex (3 mol/L equiv), Et3N (2 mol/L equiv), DMSO, 0 C to rt, 4.5 h, 53% based on 2; (b) pyridinium-p-toluenesulfonate (0.2 mol/L equiv), MeOH, 4 C, 23 h; benzoyl chloride (8 mol/L equiv), pyridine, 0 C to rt, 21 h, 60% based on 38; (c) methyltriphenylphosphonium bromide (6 mol/L equiv), n-BuLi (4 mol/L equiv), THF, a78 C to 4 C, 21 h, 66%; (d) bromine (1.2 mol/L equiv), NaHCO3 (1.1 mol/L equiv), CCl4, rt, 0.5 h, 68%; (e) anhydrous sodium benzoate (1.2 mol/L equiv), DMF, rt, 22 h, 75% [42α (48%) and 42β (27%)]; (f) sodium methoxide (0.56 mol/L equiv), MeOH, rt, 2.5 h, 65%; (g) sodium methoxide (1.1 mol/L equiv), MeOH, rt, 2 h, 44%; (h) n-octylamine (2.5 mol/L equiv), K2CO3 (1.5 mol/L equiv), MeCN, 6065 C, 22 h; 80% aqueous acetic acid, 80 C, 4 h, 52%; (i) n-octylamine (2.5 mol/L equiv), MeCN, 6070 C, 22 h; 80% aqueous acetic acid, 80 C, 4 h, 48%.

products 41 (Scheme 6.7). The ratio of 41α and 41β was estimated to be approximately 1:1 based on the integrals of the CHBr signals. Because separation of the epimers was rather difficult, the intact mixture was used directly in the next reaction. The primary bromo group was easily replaced with a benzoate anion to give the monobrominated compounds. Thus, a selective nucleophilic substitution of 41α and 41β was conducted with sodium benzoate (1.2 mol/L equiv) in DMF at room temperature affording, after separation on a silica gel column (95:5 hexane/ethyl acetate), the monobromo compounds 42a (48%) and 42b (27%). An NOE correlation was observed between H-1 and H-2 from 42b, whereas its correlation was not shown from 42b. These results suggest the stereo configuration of the bromine atom at C-1 position in 42a and 42b. Zemple´n deacylation of 42a and 42b with sodium methoxide (using 0.56 and 1.1 mol/L equiv, respectively) in methanol gave diol 43α (65%) and epoxide 44 (44%), respectively. Under these conditions, the expected β-bromodiol could not be obtained, but instead, α-bromodiol 43 and epoxide 44 were isolated. First, the β-bromodiol was formed, followed by a possible neighboring group participation reaction where the transhydroxyl moiety at the adjacent carbon atom displaced the bromine to give 44. In studies on amination of the corresponding diacetyl derivatives, the

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β-amino was displaced by the nucleophilic amine to give the bamino compound through the neighboring participation of the acetoxyl group. Therefore, in this case, the reaction course was controlled by the nucleophilicity and the stereochemical preference of the neighboring hydroxyl group under the basic conditions. Incorporation of an alkyl amino functionality at C-1 of α-bromodiol 43a was accomplished by direct nucleophilic substitution with n-octylamine (2.5 mol/L equiv) in acetonitrile in the presence of potassium carbonate (1.5 mol/L equiv) at approximately 60 C. The coupling products were treated with aqueous acetic acid and the resulting aminoalcohol was purified by a silica gel column with a solvent system of acetic acid/chloroform/methanol followed by a Doulite C20 (H1) resin column with a gradient elution of 80% aqueous methanol to methanol/conc. aqueous ammonia (4:1). This afforded free NOEV at a 52% yield. This compound was unambiguously identified by comparing its spectral data, specific rotation, and biological activity, including its inhibitory activity toward β-galactosidase and chemical chaperone activity. Likewise, epoxide 44 underwent regioselective cleavage with n-octylamine (2.5 mol/L equiv) to afford, after a similar purification process, the free base NOEV (48%). Thus, both isomeric intermediates formed by bromination could be successfully transformed into the target compound.

6.3.2 Chemical Modification of NOEV The chemical structure of the lead chaperone compound was modified to investigate the requirements for moderate inhibitory activity while improving the enzyme enhancement activity (Kuno et al., 2015). It is interesting to modify the structure of NOEV to generate a novel pharmacological chaperone based on the strategy shown in Fig. 6.5. First, the pseudo-β-galactose moiety of NOEV was simplified, which was expected to lower its enzyme inhibitory activity and streamline its preparation. Therefore, the C-5 function of NOEV was modified to form the dehydroxy (44) derivative, which is a previously reported 6-deoxy derivative of NOEV (Kuno et al., 2013), and the dehydroxymethyl (45) derivative, which is N-octyl(1)-conduramine F-4. Some conduramines and their derivatives have shown moderate inhibitory activity toward glycosidases (Bellomo et al., 2007; Herna´ndez Daranas et al., 2012; Łysek et al., 2006, 2007). Moreover, to increase the enzyme enhancement activity, the N-substituent of 45 was modified. Because the space adjacent to the active site of β-galactosidase is hydrophobic

FIGURE 6.5 Strategy of modification of NOEV (10) to decrease the inhibitory activity while increasing the enzyme enhancement activity.

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(Suzuki et al., 2014), a functional group such as an alkyl or aryl group is desirable for interactions in this region. The synthesis of various N-substituted (1)-conduramine F-4 derivatives is shown in detail in Scheme 6.8. A concise route toward (1)-conduramine F-4 derivatives starting from (1)-proto-quercitol was report by Kuno et al. (2015). First, an O-isopropylidenation (Wacharasindhu et al., 2009) of (1)-proto-quercitol 37 with 2,2-dimethoxypropane in the presence of an acid catalyst in DMF afforded diacetonide 46 at 90% yield. Afterwards, the remaining hydroxyl group on 46 was sulfonylated, providing mesylate 47 (97%). Treating 47 with excess DBU in refluxing toluene produced cyclohexene 48 at 76% yield. The appearance of two ring protons at d 5.78 and 6.16 in the 1H-NMR spectrum of 48 supported the assigned alkene structure. Next, the trans-isopropylidene group of 48 could be removed selectively by using catalytic pyridinium-p-toluenesulfonate (PPTS) in methanol, giving diol 49 in high yield (91%). The subsequent epoxidation of 49 was accomplished under conditions described by Martin et al. (1974); treatment with a slight excess of Martin sulfurane generated epoxide 50 at 69% yield. Alternatively, 50 was also obtained in a moderate yield (59%) under Mitsunobu conditions with PPh3 and diisopropyl azodicarboxylate (DIAD). Incorporation of various amine functionalities into the C-1 position of 50 was performed via simple addition reactions with alkylamines. Because the allylic C-1 carbon atom of the epoxide was more reactive, the amination reaction occurred in a regio- and stereoselective fashion. After considering the solubility of these compounds for successive biological assays, we intended to isolate the pure amine hydrochlorides directly. Silica gel column chromatography was followed by treatment with hydrochloric acid/aqueous THF to afford the N-substituted (1)-conduramine F-4 derivatives as the HCl salts (45an) at 64%100% yields.

SCHEME 6.8 Synthesis of the N-substituted ( 1 )-conduramine F-4 derivatives from ( 1 )-proto-quercitol.

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The inhibitory and enhancement activities of the compounds for normal human β-galactosidase and R201C mutated human β-galactosidase, respectively, are shown in Table 6.4. Regarding the enzyme enhancement activity, R201C is often used to select pharmacological chaperone candidates for mutant human β-galactosidase. First, NOEV·HCl (10) has an IC50 5 1.7 mmol/L, while 6-deoxy-NOEV·HCl (44) and N-octyl-(1)-conduramine F-4·HCl (45a) have IC50 5 46 mmol/L and 120 mmol/L, respectively. Therefore, the inhibitory activity was decreased based on the functional groups at C-5, and the inhibitory activity of 45a could be remarkably diminished to 1.4% compared to 10. The enhancement activity of the mutant R201C β-galactosidase induced by treatment with both 10 (2 mmol/L) and 44 or 45a (20 mmol/L) was almost identical, producing an approximately fivefold increase compared to the additive-free system.

6.3.3 Physicochemical and Biological Characteristics of NOEV NOEV is a potent competitive inhibitor of β-galactosidase in vitro, and a chemical chaperone to restore mutant enzyme activities in somatic cells from patients with GM1-gangliosidosis (Suzuki, 2006; Suzuki et al., 2009; Matsuda et al., 2003; Iwasaki et al., 2006; Suzuki, 2008; Suzuki et al., 2007, 2012). Its structure has been fully assigned by a combination of correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), and heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy (Matsuda et al., 2003). It is stable at room temperature, and freely soluble in methanol or dimethyl sulfoxide (DMSO). Solubility in water is limited up to 35 μmol/L at room temperature, but the amine hydrochloride is easily soluble in water. The molecular weight is 287.40. The IC50 is 0.125 μmol/L toward human β-galactosidase (Iwasaki et al., 2006). Addition of NOEV to the culture medium was found to restore mutant enzyme activity in cultured human or murine fibroblasts at low intracellular concentrations, resulting in a marked decrease of intracellular substrate storage (Matsuda et al., 2003). It is 50-fold more active than DGJ in chaperone effect on mutant human β-galactosidase in GM1-gangliosidosis (Matsuda et al., 2003; Tominaga et al., 2001). The calculation suggests that at least 10% of normal enzyme activity is necessary for washout of the storage substrate in GM1-gangliosidosis (Suzuki et al., 2009) and lysosomal diseases in general. The age of onset in patients expressing enzyme activity above this level is theoretically beyond the human life span. It is anticipated that the effective NOEV concentrations in human cells and animal tissues are much lower than the IC50 calculated in vitro, on the basis of culture cell experiments and results of tissue concentration after oral NOEV administration in experimental model mice. In fact, NOEV is effective at the IC50 concentration in the culture medium for enhancement of mutant enzyme activity (Iwasaki et al., 2006). This molecular interaction is gene mutationspecific (Iwasaki et al., 2006; Li et al., 2010; Okumiya et al., 1995b).

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TABLE 6.4 StructureActivity Relationships for NOEV · HCl (10), 6-DeoxyNOEV · HCl (44), and N-Substituted (1)-Conduramine Derivatives (45an)

Compound

R

IC50 (μmol/L)a

Enhancement (fold)b

10



1.7

3.6 (4.6)c

44



46

5.2

45a

120

5.4

45b

50

2.0

45c

19

4.0

45d

56

4.6

45e

43

1.6

45f

.1000

0.9

45g

41

4.6

45h

15

7.4

45i

60

8.5

(Continued )

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Validamycin and Its Derivatives

TABLE 6.4 (Continued) Compound

R

IC50 (μmol/L)a

Enhancement (fold)b

45j

180

1.5

45k

. 1000

1.2

45l

490

1.4

45m

28

4.6

45n

86

4.2

IC50 for normal human β-galactosidase determined at pH 4.5. Comparison between the addition of 20 μmol/L of compounds and the additive-free system. For the addition of 2 μmol/L of 10.

a

b c

Pharmacokinetic analysis revealed rapid intestinal absorption and renal excretion after oral administration of NOEV (Suzuki et al., 2012) (Table 6.5). The hydrochloride salt was absorbed more efficiently than the free NOEV. Accordingly, subsequent experiments were conducted using the hydrochloride salt. Intracellular accumulation was not observed after long-term treatment. NOEV was delivered to the central nervous system through the bloodbrain barrier to achieve high expression of the apparently deficient β-galactosidase activity (Suzuki 2006; Suzuki et al., 2012) in R201C Tg mice. NOEV treatment starting at the early stage of disease resulted in remarkable arrest of neurological progression within a few months (Suzuki et al., 2007, 2012) (Fig. 6.6). Survival time was significantly prolonged (Table 6.6). This result suggests that NOEV chaperone therapy will be clinically effective for prevention of neuronal damage if started early in life, hopefully also in human patients with GM1-gangliosidosis (Suzuki et al., 2012).

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TABLE 6.5 Pharmacokinetics of NOEV Administration Cmax (ng/mL)

Tmax (min)

T1/2 (min)

AUC02N (ng min/mL)

AUC/D (ng min kg/mL/ng)

MRT02N (min)

Free NOEV (3 mg/ kg, N 5 2)

501.5

60.0

86.5

94,703

0.03

149.0

HCl salt (3 mg/ kg, N 5 4)

664.0

60.0

112.3

144,037

0.05

186.5

The inhibitory effect of NOEV is much higher toward galactocerebrosidase than β-galactosidase (Fig. 6.7) (Ogawa et al., 2007). Therefore, chaperone experiments on cultured fibroblasts from patients with Krabbe disease, caused by galactocerebrosidase deficiency, were tried (Suzuki and Suzuki, 1971). However, enhancement of the deficient enzyme activity was not achieved under the same culture conditions as for β-galactosidase deficiency (GM1gangliosidosis). Since galactocerebrosidase is known to be unique for its physicochemical characteristics, intracellular transport, and expression of catalytic activity in somatic cells, a more sophisticated strategy may be necessary for realizing chaperone effects with this disease.

6.3.4 NOEV Effects on Cultured Human and Mouse Fibroblasts Expressing Mutant Human Genes Heterogeneous responses to NOEV in human cells expressing mutant β-galactosidase (Iwasaki et al., 2006) in line with results for mouse fibroblasts were observed (Matsuda et al., 2003). However, the degree of enhancement differed for some mutations between human and mouse cells. A common observation was a 5- to 10-fold increase for the R427Q mutation at 0.2 mol/L of NOEV in the culture medium; and a higher concentration (2 mol/L) was required for the R201C or R201H mutation for enhancement to the same degree (Iwasaki et al., 2006). About one-third of the cells from patients with GM1-gangliosidosis responded to NOEV treatment. Almost all patients with juvenile GM1-gangliosidosis, and some with infantile GM1-gangliosidosis, responded to a significantly greater extent. Equivalent or greater effects were achieved with NOEV at a 50-fold lower concentration than with DGJ or N-butyl-DGJ (Tominaga et al., 2001). Addition of a ganglioside mixture to the culture medium resulted in a remarkable increase of intracellular GM1 in the cells expressing the mutation R201C causing juvenile

FIGURE 6.6 Neurological assessment of NOEV-treated R201C Tg mice at different doses. After starting oral NOEV administration ad libitum at 12 months after birth, the three assessment tests (Suzuki et al., 2012) were performed every month in individual mice, and total scores were recorded. The scores for each treatment group were compared with those for the nontreatment (water) group of the same age. Each value indicates mean 6 SEM. Statistical analysis (two-way ANOVA) revealed per mice (N 5 16) versus 0.1 mmol/L (low dose) NOEV mice (N 5 11) (A); pN 5 16) vs. 0.3 mmol/L (medium dose) NOEV mice (N 5 12) (B); and pN 5 16) vs. 1 mmol/L NOEV (high dose) mice (N 5 6) (C). NOEV dose was estimated on the basis of body weight and mean daily water intake: 6.5 mg/kg/day in 0.1 mmol/L NOEV mice, 20 mg/kg/ day in 0.3 mmol/L NOEV mice, and 65 mg/kg/day in 1 mmol/L NOEV mice.

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TABLE 6.6 Survival Time NOEV Concentration (mmol/L)

Daily Intake (mg/kg/day)

N

Survival Time (m)

0

0

26

516



0.1

6.5

11

1122

S(0.1) . S(0) (p , 01)

0.3

20

12

1118

S(0.3) . S(0) (p , 01)

1.0

65

7

816

S(1)  S(0) (p , 01)

3.0

200

3

810

S(3) # S(0)b

10.0

650

2

23

S(10) , S(0)b

Effecta

R201C Tg mice were fed with various concentrations of NOEV, and survival time was calculated. S(0) 5 survival on water, S(0.1) 5 survival on 0.1 mmol/L NOEV, S(0.3) 5 survival on 0.3 mmol/L NOEV, S(1) 5 survival on 1 mmol/L NOEV, S(3) 5 survival on 3 mmol/L NOEV, S(10) 5 survival on 10 mmol/L NOEV. a GehanBreslowWilcoxon test. b No statistical analysis because of small sample numbers.

GM1-gangliosidosis and only a slight increase in the cells expressing the normal human gene. Incubation with NOEV significantly reduced GM1 storage in these cells (Matsuda et al., 2003).

6.3.5 Chaperone Therapy in Genetically Engineered GM1-Gangliosidosis Model Mice A transgenic (Tg) mouse, expressing the human R201C mutation that causes a mild-type GM1-gangliosidosis (R201C mouse) based on the KO background (Matsuda et al., 2003), was found to have very low β-galactosidase activity in the brain (about 4% of the wild-type activity). It exhibited an apparently normal clinical course for the first 7 months after birth, followed by slowly progressive neurological deterioration, with tremors and gait disturbance and death at 1118 months of age due to malnutrition and emaciation (life span of normal mice 2436 months). Neuropathology revealed vacuolated or ballooned neurons, less abundant than in the KO mouse brain (Itoh et al., 2001; Matsuda et al., 1997). Cytoplasmic storage materials were present in pyramidal neurons and brainstem motor neurons, but not in neurons in the other areas of the brain. Short-term oral administration of NOEV to the R201C model mouse (Matsuda et al., 2003) resulted in significant enhancement of enzyme activity

FIGURE 6.7 NOEV effects on three human galactosidases. Potent inhibitory activity was observed for galactocerebrosidase and (GM1) β-galactosidase, but not for α-galactosidase A. ’-’: control fibroblasts, K-K: GM1-gangliosidosis fibroblasts.

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in all the tissues examined, including the central nervous system. Immunohistochemical staining revealed an increase in β-galactosidase activity and decrease in GM1 and GA1 storage. However, mass biochemical analysis did not show substrate reduction in the brain, probably because of the brief duration of treatment and only localized substrate accumulation at the early stage of the disease in this experiment. The compound NOEV was found in a significant amount in the central nervous system by mass spectrometric analysis, at 10% of the level in liver tissue after oral administration of the NOEV solution for 816 weeks.

6.3.6 NOEV Effect on Model Mice: Clinical Assessment An assessment system for brain function in GM1-gangliosidosis mice has been established (Ichinomiya et al., 2007). This is a simple modification of neurological tests for human infants and young children, consisting of 11 test items mainly concerning spontaneous motor and reflex functions. A four-grade scoring system was introduced for each test, and individual and total scores were recorded for each mouse. This clinical test method is useful and sufficiently sensitive to detect early brain dysfunction in disease model mice. NOEV treatment definitely prevented, albeit partially, disease progression. This provided the first evidence that oral medication can prevent an inherited brain disease in model mice, and it is proposed that NOEV chaperone therapy should be introduced as a new approach to human GM1-gangliosidosis in the near future. Any clear adverse effects on experimental animals during the course of NOEV therapy for up to 6 months have not been observed, although analytical studies have yet to be completed for pathological, biochemical, and pharmacological parameters with this compound. NOEV is an in vitro competitive inhibitor of both β-galactosidase and galactocerebrosidase, and a mutation-specific enhancer of β-galactosidase in human and mouse fibroblasts. Thus exogenous substrates are digested by the R201C mutant β-galactosidase in mouse fibroblasts in the presence of NOEV. After oral administration, NOEV is not digested in the mouse gastrointestinal system, goes directly into the bloodstream, and is delivered to the mouse brain through the bloodbrain barrier. It enhances the mutant β-galactosidase activity in the brain and liver, and substrates abnormally stored in the brain are digested. Clinically NOEV prevents brain damage, to some extent, in mouse GM1-gangliosidosis and is rapidly disposed of after uptake in neural and hepatic cells. Definite adverse effects have not been observed in the R201C mutant mouse after up to 6 months of continuous oral administration.

6.4 FUTURE PERSPECTIVES AND CONCLUSION Chaperone therapy has been proposed mainly as a new therapeutic approach to lysosomal diseases, particularly those with central nervous system involvement.

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Currently enzyme replacement therapy is widely used for extraneural tissue pathology, with successful achievements (Ohashi, 2012). However, therapeutic effectiveness has not been confirmed at present for neural tissue damage, even if general pathology has been successfully treated. A second clinical approach has been proposed to reduce the storage substrates by inhibition of glucosyltransferase: substrate reduction therapy (Aerts et al., 2006). This new approach is meant to diminish glucosylceramide synthesis, the first step of a large number of glycosphingolipid syntheses. In fact, this trial has been reported not only for Gaucher disease with glucosylceramide storage but also for NiemannPick C disease (Wraith et al., 2010), Sandhoff disease (Masciullo et al., 2010), and other diseases with substrate storage. In Table 6.7, the pros and cons are compared for enzyme replacement therapy, substrate reduction therapy, and chaperone therapy. Enzyme replacement therapy has been well confirmed for its clinical effectiveness, but two major disadvantages have been pointed out: regular intravenous administration for life and poor effect to the central nervous system. Substrate reduction therapy has been proposed for oral administration and possible delivery to the central nervous system. However, this approach inevitably deprives somatic cells of biologically active various glycosphingolipids to some degree, possibly ensuing dysfunction of various types of somatic cells. In fact, clinical side effects have been recorded at therapeutic dose levels even in healthy individuals, particularly headache and diarrhea. This is the most important issue when this therapeutic approach is discussed for future clinical practice. TABLE 6.7 Comparison of Three Therapeutic Approaches to Lysosomal Diseases Enzyme Replacement

Substrate Reduction

Chaperone

Principle

Enzyme supplementation

Inhibition of substrate synthesis

Enzyme restoration

Administration

Intravenous

Oral

Oral

Target tissue

Extraneural

Neural, extraneural

Neural, extraneural

Clinical efficacy

Yes

Yes

Yes

a

b

Adverse effect (effective dose)

Yes

Yes

Disease specificity

Specific

Nonspecific

Specific

Mutation specificity

Nonspecific

Nonspecific

Specific

a

Immune response. Diarrhea, headache, etc.

b

Yes

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Chaperone therapy, originally proposed as chemical chaperone therapy, has also been called pharmacological chaperone therapy or enzyme enhancement therapy at present. Advantage of this new trial is noninvasive drug administration to achieve normal metabolic turnover and enhancement of missing enzyme activity in somatic cells and tissues. It is a mutation-specific drug therapy, and it is admitted that not all patients under diagnosis of a single genetic disease can be treated by one chemical chaperone drug, although at least one-third to half of patients can be the candidates of the therapeutic trial. In addition, combination of two or more chaperone compounds will reach a broader chaperone spectrum—at least to two-thirds of patients. Clinical effectiveness has been confirmed for GM1-gangliosidosis model mice (NOEV) and for a few human patients (ambroxol). No clinically recognizable adverse effects have been observed at the effective doses in mice and humans. Further experimental confirmation will be possible for this new therapeutic concept.

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Suzuki, H., Ohto, U., Higaki, K., Mena-Barraga´n, T., Aguilar-Moncayo, M., Ortiz Mellet, C., et al., 2014. Structural basis of pharmacological chaperoning for human β-galactosidase. J. Biol. Chem. 289 (21), 1456014568. Suzuki, Y., 2006. β-Galactosidase deficiency: an approach to chaperone therapy. J. Inherit. Metab. Dis. 29 (2/3), 471476. Suzuki, Y., 2008. Chemical chaperone therapy for GM1-gangliosidosis. Cell. Mol. Life Sci. 65 (3), 351353. Suzuki, Y., 2013. Chaperone therapy update: fabry disease, GM1-gangliosidosis and Gaucher disease. Brain Dev. 35 (6), 515523. Suzuki, Y., Crocker, A.C., Suzuki, K., 1971. G-M1-gangliosidosis. Correlation of clinical and biochemical data. Arch. Neurol. 24 (1), 5864. Suzuki, Y., Ichinomiya, S., Kurosawa, M., Matsuda, J., Ogawa, S., Iida, M., et al., 2012. Therapeutic chaperone effect of N-Octyl 4-Epi-β-valienamine on murine GM1gangliosidosis. Mol. Genet. Metab. 106 (1), 9298. Suzuki, Y., Ichinomiya, S., Kurosawa, M., Ohkubo, M., Watanabe, H., Iwasaki, H., et al., 2007. Chemical chaperone therapy: clinical effect in murine G(M1)-gangliosidosis. Ann Neurol 62 (6), 671675. Suzuki, Y., Miyatake, T., Fletcher, T.F., Suzuki, K., 1974. Glycosphingolipid betagalactosidases. 3. Canine form of globoid cell leukodystrophy; comparison with the human disease. J. Biol. Chem. 249 (7), 21092112. Suzuki, Y., Nakamura, N., Fukuoka, K., 1978. G M1 -gangliosidosis: Accumulation of ganglioside G M1 in cultured skin fibroblasts and correlation with clinical types. Hum. Genet. 43 (2), 127131. Suzuki, Y., Nakamura, N., Fukuoka, K., Shimada, Y., Uono, M., 1977. beta-Galactosidase deficiency in juvenile and adult patients. Report of six Japanese cases and review of literature. Hum. Genet. 36 (2), 219229. Suzuki, Y., Ogawa, S., Sakakibara, Y., 2009. Chaperone therapy for neuronopathic lysosomal diseases: competitive inhibitors as chemical chaperones for enhancement of mutant enzyme activities. Perspect. Med. Chem. 3, 719. Suzuki, Y., Sakuraba, H., Hayashi, K., Suzuki, K., Imahori, K., 1981. Beta-galactosidaseneuraminidase deficiency: restoration of beta-galactosidase activity by protease inhibitors. J. Biochem. 90 (1), 271273. Suzuki, Y., Suzuki, K., 1971. Krabbe’s globoid cell leukodystrophy: deficiency of glactocerebrosidase in serum, leukocytes, and fibroblasts. Science 171 (3966), 7375. Suzuki, Y., Suzuki, K., 1974a. Glycosphingolipid beta-galactosidases. I. Standard assay procedures and characterization by electrofocusing and gel filtration of the enzymes in normal human liver. J. Biol. Chem. 249 (7), 20982104. Suzuki, Y., Suzuki, K., 1974b. Glycosphingolipid beta-galactosidases. II. Electrofocusing characterization of the enzymes in human globoid cell leukodystrophy (Krabbe’s disease). J. Biol. Chem. 249 (7), 21052108. Suzuki, Y., Suzuki, K., 1974c. Glycosphingolipid beta-galactosidases. IV. Electrofocusing characterization in G-M-1-gangliosides. J. Biol. Chem. 249 (7), 21132117. Tominaga, L., Ogawa, Y., Taniguchi, M., Ohno, K., Matsuda, J., Oshima, A., et al., 2001. Galactonojirimycin derivatives restore mutant human β-galactosidase activities expressed in fibroblasts from enzyme-deficient knockout mouse. Brain Dev. 23 (5), 284287. Wacharasindhu, S., Worawalai, W., Rungprom, W., Phuwapraisirisan, P., 2009. ( 1 )-protoQuercitol, a natural versatile chiral building block for the synthesis of the α-glucosidase inhibitors, 5-amino-1,2,3,4-cyclohexanetetrols. Tetrahedron Lett. 50 (19), 21892192.

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Chapter 7

Prospects and Concluding Remarks Validamycins have been used to efficiently prevent and treat sheath blight disease of crops, including rice and wheat, and their current market in China and East Asia is around 30,000 50,000 tons (in 5% preparation) per year, with annual revenue of billions of Chinese dollars. Recently this antibiotic has also been recognized as an important raw material for production of an antidiabetic drug, voglibose, and drugs for chemical chaperone therapy, N-octyl-β-valienamine and N-octyl-4-epi-β-valienamine. Based on the preceding chapters in this book, we conclude with the following important remarks. First, although validamycin has outstanding merits such as excellent control effect, low price, no drug resistance, low toxicity, etc., the product of validamycin is a mixture of validamycins and validoxylamines, including validamycins A H, and validoxylamines A, B, and G. This is due to the cost of purification. Validamycin A is the main component of all, more than 60% in the best product. Its biological activity against Rhizoctonia solani is also the best. Thus, validamycin A content was used as the key index for the product. Most research has focused on validamycin A, while other components in the product have received less attention. Because of the mixture, it is hard for the product to enter the market in countries outside of Southeast Asia, such as the European countries and the United States. If validamycin A can be purified at low cost, the product will contain only one component, which can establish markets in the European countries and United States. Second, validamycin A can be used as an important raw material for production of three drugs, i.e., voglibose, N-octyl-β-valienamine, and N-octyl-4epi-β-valienamine. Due to its wide range of therapeutic and pharmacological properties, including its excellent inhibitory activity against α-glucosidases and its action against hyperglycemia and various disorders caused by hyperglycemia, voglibose has become a new, potent glucosidase inhibitor and is a drug used for NIDDM (non-insulin-dependent diabetes mellitus) in Japan, China, and Korea. N-octyl-β-valienamine and N-octyl-4-epi-β-valienamine are promising as therapeutic agents for human β-galactosidase deficiency disorders (GM1-gangliosidosis and Morquio B disease) and β-glucosidase deficiency disorders, respectively. They are the potent drugs for chemical Validamycin and Its Derivatives. DOI: http://dx.doi.org/10.1016/B978-0-08-100999-4.00007-1 © 2017 Elsevier Ltd. All rights reserved.

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chaperone therapy. In this respect, these three drugs would pull the market of validamycins. Third, structure modification of validamycin A or validoxylamine A is an important way to invent new products for validamycins. Validamycins can be acid- or resin-hydrolyzed to produce validoxylamines, especially validoxylamine A. Validoxylamine A is one of the best trehalase inhibitors. Its IC50 for trehalase in porcine intestine is 1.4 3 1028 mol/L. It also has good insecticidal activity in vitro. However, validoxylamine A is water soluble, which makes it hard to permeate the skin of insects. Thus, the insecticidal activity of validoxylamine A is poor in vivo. How to modify the structure of validoxylamine A to screen compounds with insecticidal activity in vivo is one of the most important issues for the validamycin industry. Fourth, valienamine and validamine are the products from degradation of validamycin A, and are strong enzymatic inhibitors against α-glucosidases. They also have no toxicity. Obesity, a growing global health problem, is a condition where a person has accumulated so much body fat that it might have a negative effect on his or her health. Obesity typically results from overeating and lack of enough exercise. Carbohydrates constitute the largest component of the human diet; however, only monosaccharides (e.g., glucose and fructose) can be absorbed by the small intestine. Polysaccharides and disaccharides, such as starch and sucrose, undergo rapid enzymatic degradation to monosaccharides in the upper intestine through the action of pancreatic α-glucosidases. As a consequence of this digestion, polysaccharides and disaccharides are absorbed by our body, and would change into fat in the body. If α-glucosidase inhibitors are taken before a meal, polysaccharides and disaccharides would not be hydrolyzed to monosaccharides and would not be absorbed by our bodies. Valienamine and validamine, unlike drugs such as voglibose and acarbose, can be employed and developed as a health food supplement that can be eaten before meal. In this way, validamycins will be needed more, to be degraded into valienamine and validamine. Based on these remarks, validamycins will have a much bigger market and will bring huge economic benefits in the future.

Index Note: Page numbers followed by “f” and “t” refer to figures and tables, respectively.

A Acarbose, 238, 239f, 254, 268269 biosynthetic pathway, 47 synthesis from, 192 therapy, 266267 voglibose comparison with drugs of, 257270 adverse events, 263 carotid plaque, 268 clinical recommendations, 260263 comparison of α-glucosidase inhibitors, 269270 cost-effectiveness ratio, 263264 drug summary, 258 glycemic control in T2DM, 264265 glycemic excursions, 265266 impaired glucose tolerance, 268269 inflammation, 266267 mechanism of action, 258259, 258f, 259f pharmacokinetic properties, 259260, 261t Acarviosin, 238, 239f, 240, 243t AcbM homolog, 45 Acetoxymethyl derivative, 203205, 204f N-Acetyl derivatives, 200201, 200f 7-O-Acetyl-2,3,40 ,50 ,60 ,T-hexa-Obenzylvalidoxylamine A, 230231, 232f Acid chloride, 285, 286f Acquired immune deficiency syndrome (AIDS), 37 Acute neuronopathic phenotype, 283 Acyclic heptitol derivative, 249, 250f Adiponectin (ADN), 266 Adiposins, 238, 239f ADN. See Adiponectin (ADN) AGIs. See α-glucosidase inhibitor (AGIs) Aglycone, 226227, 227f, 240

AgOTf. See Silver trifluoromethanesulfonate (AgOTf) Agricultural antibiotics, 60. See also Validamycin A magic, 25 for plant pathogens, 12 AIBN. See Azobisisobutyronitrile (AIBN) AIDS. See Acquired immune deficiency syndrome (AIDS) Alkadiene, 294296, 294f, 295f N-Alkanoyl derivatives, 285, 286f, 290, 290t Alkene diol, 194195, 194f Alkyl amino functions, 290, 296 N-Alkyl chain, 284 N-Alkyl derivatives, 238, 285, 286f, 290, 290t N-Alkyl-β-valienamine, 283284, 283f as β-glucocerebrosidase inhibitors, 283284, 283f, 284f, 285t Allylic alcohol, 180181, 180f Allylic amine, 173174, 174f α,αbis(methylthio)inosose derivative, 250, 250f α,β-unsaturated ketone, 285287 α-alcohol, 177179, 178f α-allyl bromide, 292 α-azide, 177179, 178f α-bromodiol, 295f, 296 α-D-glucopyranoside, 167168, 168f α-D-glucopyranosylvalidamycin A, 115 α-D-glucosidase, 238 α-D-glucosidase inhibitory activity Nsubstituted valiolamine derivatives, 240245, 241t, 243t, 246t, 247f α-glucosidase inhibitor (AGIs), 245, 254263, 269270 α-glucosidases, 37, 251, 257258, 314 enzymes, 258259 α-hydroxymethyl-cyclohexenone, 177179, 178f

315

316

Index

α-ketoglutarate/Fe(II)-dependent dioxygenase, 59 α,α-trehalase, 145146 α-valienamine, 284, 285t Amide, 284, 284f Amination reaction, 297, 297f Amine, 284, 284f Amino function, 285287 (1D)-(1/2,3)-3-Amino-1,2-di-O-benzyl-5(trityloxymethyl)-6-cyclohexene-1,2diol, 200201, 200f (1S)-(1-(OH),2,4,5/1,3)-5-Amino-1-C(hydroxymethyl)-1, 2,3,4cyclohexanetetrol, 238 [(1S)-(1(OH), 2, 4, 5/1, 3)-5-Amino-1hydroxymethyl-1, 2, 3,4cyclohexanetetrol]. See Valiolamine 2-Amino-1,3-propanediol, 249250, 250f [1-Amino-5-hydroxymethyl-2,3, 4, 6cyclohexanetetrol]. See Hydroxyvalidamine [(1S, 2S, 3S, 4R)-1-Amino-5(hydroxymethyl)-cyclohex-5-ene-2, 3, 4-triol]. See Valienamine [(1S)-(1, 2, 4/3, 5)-1-Amino-5hydroxymethyl-2,3, 4cyclohexanetriol]. See Validamine Amylostatins, 238, 239f Anomeric carbon atom, 240, 243t 1,2-Anti-amino alcohol, 196, 196f 1,2-Anti-polybenzyl ether, 196, 196f Antibiotics, 1 Antidiabetic agents, 256257 Antifungal activities. See also Enzyme inhibitory activities; Insecticidal activity antimicrobial activity against C. albicans, 142 against microorganisms, 142145 against F. oxysporum, 135136 mechanism against R. solani, 115135 against P. solanacearum, 136140 against R. solani, 115, 116t against X. campestris pv. campestris, 141142 Antimicrobial activity against C. albicans, 142 against microorganisms, 142145 APCI. See Atmospheric pressure chemical ionization (APCI) APCIMS/MS product, 7273, 72f N-Araldylvalienmine derivatives, 238

Aralkyl unit, 240 Ascomycetes strains, 127128 Atmospheric pressure chemical ionization (APCI), 7172 (1L)-(1,3,4/2)-4-Azido-1,2,3-tri-O-benzyl-6(trityloxymethyl)-5-cyclo-hexene1,2,3-triol, 200201, 200f 1,4-type Azido alcohol, 184185, 186f Azidoacetate, 182183, 182f Azobisisobutyronitrile (AIBN), 210211

B B16 melanoma cells, 285 Background electrolyte (BGE), 67 Basen. See Voglibose Benzoyl chloride, 195196, 287290 Benzoyl cyanide (BzCN), 167168, 168f Benzyl carbamate, 172173, 173f N-Benzyl group in carbamate, 207208, 208f 4,6-O-Benzylidene derivative, 285287, 288f N-Benzyloxazolidinone, 186187 N-Benzyloxycarbonyl derivatives, 200201, 200f Benzyltriethylammonium chloride, 167168 β-1,3-Glucanase in rice plants, induction of validamycins for, 134135 β-bromodiol, 294296 β-Cell, 265266 β-D-glucan-degrading enzymes, 129131, 133f β-D-Glucosidase, 131132, 132t β-galacto-type N-octyl-valienamine, 285, 287f β-galactosidase activity, 280281, 281f β-galactosidase inhibitor, synthesis and screening of NOEV as, 288f, 289f, 292296, 293f, 294f, 295f β-glucocerebrosidase, 290, 290t β-glucosidase, 279 β-glucosidation, 7879 β-valienamine, 283284, 283f, 284f BGE. See Background electrolyte (BGE) Biguanide met-formin, 237 Biooxidation, 294 Biosynthesis of validamycins, 3959. See also Microbes for validamycins biosynthesis pathway to validamycin A, 43f cloning, expression, and deficiency of genes GBL receptor genes, 8890 inactivation of valN gene, 8688, 87f knocking out valG gene, 8486 of ugp gene, 7984 of valG gene, 7679

Index electron density map quality and active site structure, 54f genes in S. hygroscopicus 5008, 49t genes in val, vld, and acb clusters, 42t genetic organization, 42f inactivation of regulatory gene glnR in 5008, 58f inactivation of valL and complementation of mutant, 53f optimization of corn powder and soybean powder, 52t regulatory model of GlnR involved in validamycin A biosynthesis, 59f sequence alignment of valA with representative related enzymes, 57f structure and topology of valA, 56f transcriptomic analysis of strain 5008, 44f 1,2-Bis-epi-valienamine, 192, 193f synthesis of, 199, 200f Bis(methylthio) group, 249 1-C-[Bis(methylthio)methyl]-D-glucopyranose derivative, 249, 250f BLASTCLUST analysis, 1619 Blood glucose fluctuation, 265 measurements, 254 Bloodbrainbarrier, 300 BMI. See Body mass index (BMI) Body mass index (BMI), 251252, 256257, 267 Bombyx mori, 153154 Brain function, 305 Brainstem motor neurons, 303 Branched-chain inosose, 250, 250f Bromine, 294296 (Bromomethyl)cyclitol cyclic carbamate, 203205, 204f N-Bromosuccinimide (NBS), 192 Broth, isolation of validamycins from, 2428 chromatogram of authentic mixture, 27f N-tert-Butoxycarbonyl derivative, 285287, 288f BzCN. See Benzoyl cyanide (BzCN)

C

13 C NMR spectral data, 31, 32t C-10 carbon, 240 C-6-epimeric allylic alcohol, 194195, 194f CAD. See Coronary artery disease (CAD) Candida albicans, 142 antimicrobial activity against, 142

317

Capillary zone electrophoresis (CZE), 60, 6671 chromatogram of sample I, 69f electropherogram of sample I, 68f, 70f, 71f typical electropherograms of dilution buffer, validamycin A standard, 67f Carbadisaccharide, 287290, 289f Carbaglycosylamines, 294 Carbocycle, 197198, 198f Carbohydrate absorption, 237, 314 Carbon sources, 1016, 24 by T-7545 and TH82, 17t Carbonimidothioate, 186187, 187f Carboxymethylcellulase (CMCase), 133134 Cardiovascular events, 269 Cardiovascular risk, 271272 Carotid plaque, 268 CDSs. See Coding sequences (CDSs) Cell adhesion, 266267 Cellobiase, 133134 Cellular compartment, 282283 Cellular dysfunction, 279 Cellulase activity, 160 Central nervous system, 283, 300, 303305 CGM. See Continuous glucose monitoring (CGM); Corn gluten meal (CGM) (CH(CH2OH)2) unit, 245 Chaetomium bostrychoides, 142144 Chaetomium globosum, 142144 Chaperone therapy, 279283 comparison of therapeutic approaches to lysosomal diseases, 306t correlation between residual β-galactosidase activity and clinical onset, 281f NOEV of GM1-gangliosidosis, 291305 chemical modification of NOEV, 296298, 296f, 297f, 299t genetically engineered GM1gangliosidosis model mice, 303305 NOEV effect on model mice, 305 NOEV effects on cultured human and mouse fibroblasts, 301303 physicochemical and biological characteristics, 298301, 301t, 302f, 303t synthesis and screening as β-galactosidase inhibitor, 288f, 289f, 292296, 293f, 294f, 295f NOV of Gaucher disease, 283291 biological activities of NOV, 290291, 290t

318

Index

Chaperone therapy (Continued) chemical modification of, 285290, 286f, 288f, 289f synthesis and screening of N-alkylβ-valienamine as β-glucocerebrosidase inhibitors, 283284, 283f, 284f postulated molecular events, 282f effect of protease inhibitors, 280t Chemical synthesis, 165 m-Chloroperbenzoic acid (m-CPBA), 179 Chlorosulfonyl isocyanate, 176177, 177f Chronic intestinal diseases, 260263 Chronic nonneuronopathic phenotype, 283 Citrate lyase (CitE), 48 CMCase. See Carboxymethylcellulase (CMCase) Coding sequences (CDSs), 1619 Colorimetric method, 61 Combination therapy, 255 Continuous glucose monitoring (CGM), 251252 Conventional oral hypoglycemic agents, 255 Corn gluten meal (CGM), 2324 Corn steep liquor (CSL), 2324 Corncob hydrolysate, 9697 Corncob molasses. See Corncob hydrolysate Coronary artery disease (CAD), 255256 Coronary atherosclerosis, 255256 Correlation spectroscopy (COSY), 298 COSY. See Correlation spectroscopy (COSY) Crystalline ketone, 294 CSL. See Corn steep liquor (CSL) Cultural condition optimization, 93106 application of fermentation strategies, 9698 fermentation medium, 9395 regression coefficients and significance for response surface model, 95t response surface and contour plot of validamycin A production, 96f stimulator addition, 98103 addition of exogenous 1,4-butyrolactone, 102103, 103f addition of external ethanol, 98100, 99f, 100f addition of external H2O2, 100102, 101f Cyclic diol, 184185, 186f Cyclohexane skeleton synthesis from, 179181, 179f, 180f valienamine from cyclohexane skeleton by Trost, 181f

Cyclohexanones, 202, 202f Cyclohexene, 172173, 173f, 297, 297f Cyclohexenone, 177179, 178f Cycloheximide, 2 Cyclohexyl unit, 240 Cytoplasmic storage materials, 303 CZE. See Capillary zone electrophoresis (CZE)

D DBQ. See 3,5-Di-t-butyl-1,2-benzoquinone (DBQ) DBU. See 1,8-Diazabicyclo undec-7-ene (DBU) De-O-acetate, 203205, 204f De-O-acetylation, 215217, 216f O-Deacetylation, 193, 194f DEAD. See Diethyl azodicarboxylate (DEAD) Debenzylation, 176177, 177f DEGs. See Differentially expressed genes (DEGs) Dehydroxy derivative, 296297, 296f O-Deisopropylidenation, 284 7-Deoxy-7-iodo derivative, 203205, 204f 1-Deoxy-epi-myo-inositol, 205206 6-Deoxy-NOEV  HCl, 298, 299t 7-Deoxy-pseudo-D-glucopyranose, 240 (2)-2-Deoxy-scylloinosose, 294, 294f 4-Deoxy-α-D-glucopyranose units, 238, 239f 1-Deoxygalactonojirimycin (DGJ), 281, 301303 Desulfurized inosose derivative, 250, 250f Deuteromycetes strains, 127128 DGJ. See 1-Deoxygalactonojirimycin (DGJ) DL-3,4-di-O-acetyl-1,2-anhydro-5,7-Obenzylidene-(1,2,4,6/3,5)-1,2,3,4,5cyclohexanepentol, 217 Di-O-Isopropylidene-β-valienamine, 284, 284f 3,5-Di-t-butyl-1,2-benzoquinone (DBQ), 247248 Diabetes and diffuse coronary narrowing (DIANA), 255256, 268 Diabetes mellitus, 255256 Diacetonide, 297, 297f DIANA. See Diabetes and diffuse coronary narrowing (DIANA) 1,8-Diazabicyclo undec-7-ene (DBU), 179 DIBAL-H. See Diisobutylaluminum hydride (DIBAL-H) Dibenzoate, 287290, 289f Dibromide, 292, 293f

Index 1,5-Dideoxy-1,5-[2-hydroxyethylimino]-Dglucitol, 258, 258f (O-4,6-Dideoxy-4-[[(1S,4R,5S,6S)-4-5,6trihydroxy-3-(hydroxymethyl)-2cyclohexen-1-yl]amino]-α-Dglucopyranosyl-(1-4)-O-α-Dglucopyranosyl-(1-4) glucopyranose, 258 4,6-Dideoxy-glucopyranose units, 238, 239f Diethyl azodicarboxylate (DEAD), 167168 Diethyl dithioacetal, 173174, 174f Differentially expressed genes (DEGs), 48 Dihydrovalidoxylamines A, 145146 Diisobutylaluminum hydride (DIBAL-H), 190192 3,4-Dimethoxybenzyl chloride (DMBCl), 175176 2-O-Dimethoxybenzyl derivative, 175176, 175f Dimethyl 2, 3-O-isopropylidene-D-tartrate, 186187, 187f Dimethyl sulfoxide (DMSO), 3031, 249250 N,N-Dimethylaminopyridine (DMAP), 190192 Dimethylformamide, 3031 Dimethyloxosulfonium methylide, 165167, 166f 2,6-Dioxo-heptose 1,1-dithioacetal derivative, 249250, 250f 2,6-Dioxo-heptose derivative, 249250, 250f Diphenylphosphoryl azide (DPPA), 173174, 174f Disaccharides, 238, 314 DL-(1, 3, 4/2, 5, 6)-4-amino-6hydroxymethyl-1, 2, 3, 5cyclohexanetetrol (DLhydroxyvalidamine), 214 DL-validoxylamine B, 219220, 220f N,N-DMAP. See N,N-Dimethylaminopyridine (DMAP) DMBCl. See 3,4-Dimethoxybenzyl chloride (DMBCl) DMSO. See Dimethyl sulfoxide (DMSO) Dodeca-O-acetate, 225 L-DOPA, 150153 DPPA. See Diphenylphosphoryl azide (DPPA)

E Electrophoretic mobilityshift assay (EMSA), 89f

319

EMSA. See Electrophoretic mobilityshift assay (EMSA) ()-7-Endo-oxabicyclo[2.2.1]-hept-5-ene-2carboxylic acid, 209210, 210f Endochitinase in rice plants, induction of validamycins for, 134135 Endoplasmic reticulum (ER), 281 Endothelial dysfunction, 255256, 267 Enol-benzoate, 294, 295f Enolate anion, 250, 250f Enone, 169170, 169f Enterocytes, 251 Environmental Protection Agency (EPA), 36 Enzyme activity, 280281, 298 deficiency, 279 enhancement activity, 296297 replacement, 291292 therapy, 283 Enzyme inhibitory activities. See also Antifungal activities; Insecticidal activity against D-glucose hydrolases, 148150, 149t, 151t, 152t against trehalase, 145148, 147t, 148t, 149f against tyrosinase, 150153 EPA. See Environmental Protection Agency (EPA) 2-Epi-5-epi-valiolone, 3941 3-Epi-5-O-methylsulfonyl-shikimate, 208, 209f 4v-Epi-validamycin A, 7678, 78f Epi-valienamine, 165, 166f 1-epi-valienamine, synthesis of, 193196 1,2-bis-epi-valienamine, synthesis of, 199, 200f 2-epi-valienamine, synthesis of, 196198, 197f 4-epi-valienamine, synthesis of, 198199 ( 1 )-1-Epi-valienamine, 196, 196f 1-Epi-valienamine, 192, 193f synthesis of, 193196, 194f, 195f 2-Epi-valienamine, 192, 193f synthesis, 196198, 197f from D-mannose, 198f 4-Epi-valienamine, 192, 193f synthesis of, 198199 from L-serine, 199f Epi-valiolamine, 238, 239f 4-Epimer, 285, 287f 3-Epimeric alkadiene, 292, 293f

320

Index

Epoxidation, 297, 297f Epoxide, 294296, 295f ER. See Endoplasmic reticulum (ER) Ethanol, 98100, 99f, 100f Exogenous 1,4-butyrolactone, 102103, 103f, 104f precursor supply, 103106 Extensive molecular analysis, 281 Extracellular enzymes, 133134, 134t Extraneural visceral organs, 283 Extrasystemic mode, 259260

F Fabry disease, 282283 Fasting plasma glucose (FPG), 254 Fermentation medium composition effect on validamycin production, 2324 effects of NH4Cl and NaCl, 26t by T-7545 and growth on media, 25t process for production of validamycins, 90106, 93f optimization of cultural conditions, 93106 screening and breeding high-yield strains, 9092 of validamycins, 2224 effect of medium volume on validamycin production, 23, 23t temperature of submerged culture, 2223, 23t 50% lethal dose (LD50), 36 Filter paper (FPase), 133134 Flagellate-free termites, 160 Flavobacterium saccharophilum, 37 degradation of validamycin A by, 76, 77f Fluoroquinolones (FQ), 1 FPase. See Filter paper (FPase) FPG. See Fasting plasma glucose (FPG) FQ. See Fluoroquinolones (FQ) Fructofuranoside, 245 Fusarium culmorum, 142144 Fusarium oxysporum, 135 antifungal activity against, 135136, 136t accumulation of total salicylic acid, 138f control of late blight of tomato by foliar spray of validamycin A, 137t control of tomato powdery mildew days, 139t RNA blot analysis, 138f

G Galactocerebrosidase, 301, 305 4-O-(β-D-Galactopyranosyl) derivative, 285, 287290, 287f, 289f Galactose, 281 γ-butyrolactone receptor genes (GBL receptor genes), 8890 effects of afsA homologs, 89f ShbR1 negatively regulated transcription of adpA, 89f Gap. See Glyceraldehyde-3-phosphate dehydrogenase (Gap) Garner’s aldehyde, 189190, 189f Gas chromatography (GC), 6063, 62f, 63f Gastric inhibitory polypeptide, 251252 Gastrointestinal side effects, 263 Gastrointestinal symptoms, 257 Gastrointestinal tract, 251 Gaucher disease, 279 NOV for chaperone therapy, 283291 biological activities, 290291, 290t chemical modification, 285290, 286f, 288f, 289f synthesis and screening of N-alkylβ-valienamine as β-glucocerebrosidase inhibitors, 283284, 283f, 284f, 285t GBL receptor genes. See γ-Butyrolactone receptor genes (GBL receptor genes) GC. See Gas chromatography (GC) Gelatin, 10 Genes, 4143 expression, 269270 genetic diseases, 279 Genomes for two microbes, 1622 S. hygroscopicus subsp. limoneus KCTC 1717 genome, 2122, 22t S. hygroscopicus var. jinggangensis 5008 genome, 1620 Glc. See Glucopyranosyl (Glc) Glnr mutant, 58 Glomerular filtration rate, 255 GLP-1. See Glucagonlike peptide 1 (GLP-1) Glucagonlike peptide 1 (GLP-1), 251252 Glucan synthetase, 129131, 131t D-Gluco configuration, 145146 D-Gluco-2-heptulose derivative, 249, 250f D-Gluco-validoxylamine A, 146 Gluconeogenesis pathway, 20 Gluconokinase (GntK), 48 Glucopyranosyl (Glc), 73 Glucosamine, 124, 124t

Index Glucose, 2324, 124, 124t, 145146, 237. See also Insulin excursion, 252 fluctuations, 267, 269270 glucose-lowering effect, 252253 levels, 271272 synthesis from, 167173, 168f, 169f, 200202, 200f, 210211, 211f valiolamine from D-glucose by Shing, 202f synthesis of voglibose from, 249250, 250f D-Glucose hydrolases, inhibitory activities against, 148150, 149t, 151t, 152t Glucosidase inhibitory, 37, 38f Glycemic control, 255 in type 2 diabetes mellitus, 264265 excursions, 265266 glycemic/metabolic effects, 251252 glycemic/metabolic responses, 252 Glyceraldehyde-3-phosphate dehydrogenase (Gap), 48 Glycerol, 2324 Glycoalbumin, 252 Glycosidases, 37 Glycosyl donor, 229, 230f N-Glycosyltransferase enzymes, 4748 Glycosyltransferases catalyze, 7678 O-Glycosyltransferases, 4748 GM1-gangliosidosis, 279283 NOEV for chaperone therapy, 291305 chemical modification, 296298, 296f, 297f, 299t effect on model mice, 305 effects on cultured human and mouse fibroblasts, 301303 genetically engineered GM1gangliosidosis model mice, 303305 physicochemical and biological characteristics of NOEV, 298301, 301t, 302f, 303t synthesis and screening as β-Galactosidase inhibitor, 288f, 289f, 292296, 293f, 294f, 295f GntK. See Gluconokinase (GntK) Griseofulvin, 2

H

1 H NMR spectral data, 31, 34t HbA1c, 255, 267 HbA1c. See Hemoglobin A1c (HbA1c)

321

HCl salts, 297, 299t Hemicellulosic biomass, 9697 Hemo-lymph trehalose, 157 Hemodialysis, 255 Hemoglobin A1c (HbA1c), 251252 2,3,4,20 ,30 ,40 ,60 -Hepta-O-acetyl-cr-gentiobiosyl bromide, 230231, 232f Hepta-O-benzyl-D-cellobiose, 229, 231f Heptose dithioacetal derivative, 249250, 250f Heteronuclear single quantum coherence NMR spectroscopy (HSQC NMR spectroscopy), 298 HGMD. See Human Gene Mutation Database (HGMD) High-performance liquid chromatography (HPLC), 4344 with reverse phase chromatography, 63 High-performance liquid chromatography with ultraviolet detector (HPLC-UV), 60 HIV. See Human immunodeficiency virus (HIV) HOMA-IR. See Homeostasis model assessment of insulin resistance (HOMA-IR) Homeostasis model assessment of insulin resistance (HOMA-IR), 255 HPLC. See High-performance liquid chromatography (HPLC) HPLC-UV. See High-performance liquid chromatography with ultraviolet detector (HPLC-UV) HSQC NMR spectroscopy. See Heteronuclear single quantum coherence NMR spectroscopy (HSQC NMR spectroscopy) Human Gene Mutation Database (HGMD), 291 Human immunodeficiency virus (HIV), 37 Human R201C mutation, 303 Hydrochloride salt, 300 Hydrolysis, 247248 agents, 248249 of trehalose, 160161 Hydroponics culture test, 138, 141t 60 -Hydroxy derivative, 240, 243t [(1S)-[1(OH),2,4,5/3]-5-[[2-Hydroxy-1(hydroxymethyl) ethyl]amino]-1-Chydroxymethyl)-1,2,3,4cyclohexanetetrol], 245 N-[2-Hydroxy-1-(hydroxymethyl)ethyl] derivatives, 245, 246t

322

Index

(1)-Hydroxycalidamine, 214 N-(Hydroxycyclohexyl) valiolamines, 240 N-[(1R, 2R)-2-Hydroxycyclohexyl] isomer, 240, 243t N-[(1S, 2S)-2-Hydroxycyclohexyl] isomer, 240, 243t Hydroxyl group, 240 Hydroxylation, 150153 [(1S)-(1, 4, 6/5)-3-Hydroxymethyl-4, 5,6trihydroxycyclohex-2-enyl]. See Validoxylamine A N-(β-Hydroxyphenethyl) valiolamines, 240 Hydroxyvalidamine, 28, 238, 239f characterization of, 3739 chemical structures of, 29f synthesis, 214215, 214f, 215f Hyperglycemia, 3839, 269270 Hyperglycemic range, 265 Hyperinsulinemia, 237 Hypertension, 269 Hypha, 119, 120f hyphal extension of R. cerealis and R. solani, validamycin A on, 124127 hyphal morphology of R. cerealis and R. solani, validamycin A on, 124127 effect of validamycins on angle of branches, 124t Hyphae, 116117, 118f effect of validamycins on length of primary, first secondary, and rest of secondary, 123f Hypoglycemia symptoms, 267 IBS. See Integrated backscatter (IBS)

I L-Ido configuration, 145146 IGT. See Impaired glucose tolerance (IGT) Immunoreactive insulin (IRI), 254 Impaired glucose tolerance (IGT), 245, 253254, 268270, 272 IMT. See Intima-media thickness (IMT) Inflammation, 256257 Inhibitory activity, 133, 146, 240, 284 α-D-glucosidase, 115 against trehalase, 145148 against tyrosinase, 150153 N-alkyl-β-valienamines, 285t of validamycins and validoxylamines, 148t of validoxylamines, 147t Injected larvae, 155 Inorganic acids, 247248

Inorganic salts effects, 24 Insecticidal activity. See also Antifungal activities; Enzyme inhibitory activities of validoxylamine A, 155t, 156t validoxylamine A and related compounds, 153161, 153t, 154t, 156f, 157t, 158t, 159t, 161f Insulin, 237. See also Glucose add-on therapy to, 254255 insulin-sparing effect, 251252 secretagogue mitiglinide, 252 Insulinogenic indexes, 254 Integrated backscatter (IBS), 268 Interim analysis, 253254, 268269 Intima-media thickness (IMT), 268 Iodooxazolidinone, 180181, 180f 7-Iodooxazolidinone, 186187, 187f Ion exchange chromatography, liquid chromatography with, 6365 IRI. See Immunoreactive insulin (IRI) Isomaltase, 150 Isomeric mixture, 292, 293f 2,3-O-Isopropylidene acetate, 292, 293f 2,3-O-Isopropylidene derivatives, 193, 194f

K Ketone, 165167, 166f, 175176, 175f Kinetic analysis, 146

L Laminarinase, 129131 Larval stage, 153154 Larvalpupal intermediate, 155 LD50. See 50% lethal dose (LD50) LDL-C. See Low-density lipoprotein cholesterol (LDL-C) Lethal effect, 153155 Linear chromosome, 1619 LineweaverBurk plots, 146, 150 Liquid chromatography, 60 HPLC of validamycin mixture and radiochemical purity, 65f with ion exchange chromatography, 6365, 64f, 65f Liquid chromatographyatmospheric pressure chemical ionizationtandem mass spectrometry (LCAPCIMS/MS), 60, 7175, 73f fragmentation pattern with structures for productions of validamycin A, 74f

Index Lithium aluminum hydride, 209210, 210f, 249, 284 Lithium azide, 182183, 182f Locusta migratoria, 157, 159 Low-density lipoprotein cholesterol (LDL-C), 266 Lysosomal diseases, 279

M m-CPBA. See m-Chloroperbenzoic acid (mCPBA) MAGE. See Mean amplitude of glycemic excursions (MAGE) Maltase, 150, 238, 240, 243t, 245 Mamestra brassicae, 153155 Martin sulfurane, 297 Mass spectrometric analysis, 303305 MBGD. See Microbial Genome Database (MBGD) Meal tolerance test (MTT), 251252 Mean amplitude of glycemic excursions (MAGE), 265266 MEKC. See Micellar electrokinetic capillary chromatography (MEKC) Melanogenesis process, 150153 Mesylate, 297, 297f Metabolic diseases, 282 Metformin monotherapy, 265266 Methanolic potassium carbonate, 287290 p-Methoxybenzyl isothiocyanate, 180181 Methoxymethyl ether groups, 285287, 288f Methylene compounds, 294, 294f (1SR,2RS,3SR)-6-Methylenecyclohex-4-ene1,2,3-triol, 193 synthesis from, 183, 185f 5-Methylper-hydro-1,3,5-dithiazine. See Nmethylthioformaldine N-Methylthioformaldine, 165167 Micellar electrokinetic capillary chromatography (MEKC), 66 Microbes for validamycins. See also Biosynthesis of validamycins characteristics for microbes, 916 physiological properties of strains T-7545 and TH82, 15t genomes for two microbes, 1622 Microbial degradation of validamycin A by F. saccharophilum, 76, 77f by P. denitrificans, 75 Microbial Genome Database (MBGD), 20

323

Microbial α-D-glucosidase inhibitors, 238 Microorganisms, antimicrobial activity against, 142145 cylinder agar plate method, 145t fungi sensitive to validamycin A, 144t reversal of valienamine inhibition by sugars, 145t Miglitol, 256, 269270 additive effects, 254255 chemical structure, 258f voglibose comparison with drugs of, 257270 adverse events, 263 carotid plaque, 268 clinical recommendations, 260263 comparison of α-glucosidase inhibitors, 269270 cost-effectiveness ratio, 263264 drug summary, 258 glycemic control in T2DM, 264265 glycemic excursions, 265266 impaired glucose tolerance, 268269 inflammation, 266267 mechanism of action, 258259, 258f, 259f pharmacokinetic properties, 259260, 261t Milk peptonization tests, 10 “Miracle drugs”, 2 MOM ethers, 187189, 188f Monobromo compounds, 294296, 295f Monosaccharides, 237, 314 Morquio B disease, 280281, 291 MRM mode. See Multiple reaction monitoring mode (MRM mode) MS/MS. See Tandem mass spectrometry (MS/MS) MTT. See Meal tolerance test (MTT) Multiple reaction monitoring mode (MRM mode), 7172 Mutant enzyme, 281, 298 Mutant human genes, 301303 Mutant lysosomal misfolding protein, 281 Mutant protein, 282 Mutant β-galactosidase activity, 305 Mutation R201C, 301303 Myo-inositol, 294 bioconversion of, 294, 294f synthesis from, 206207 synthesis of valiolamine from, 207f Myzus persicae, 153157

324

Index

N Nacetyl-tetra-O-benzylvalienamine, 172173, 173f NBS. See N-Bromosuccinimide (NBS) NDP. See Nucleotidyldiphosphate (NDP) Neurological tests, 305 Neuropathology, 303 Neutral trehalase activity (Ntc1p), 142 Nicotinic aldehyde, 247248 NIDDM. See Noninsulin-dependent diabetes mellitus (NIDDM) Nitrofuranose, synthesis from, 211, 212f NMR spectrum. See Nuclear magnetic resonance spectrum (NMR spectrum) NOEV. See N-Octyl-4-epi-β-valienamine (NOEV) Non-ribosomal peptide synthases (NRPSs), 20 Noninsulin-dependent diabetes mellitus (NIDDM), 4, 237 Nonsubstituted cyclohexyl derivative, 240, 243t NOV. See N-Octyl-β-valienamine (NOV) NRPSs. See Non-ribosomal peptide synthases (NRPSs) Ntc1p. See Neutral trehalase activity (Ntc1p) Nuclear magnetic resonance spectrum (NMR spectrum), 2830, 31f, 245 13 C NMR spectral data, 31 1 H NMR spectral data, 31 of validamycins A and B, 31f Nucleophilic amine, 294296 substitution, 294296, 295f Nucleotidyldiphosphate (NDP), 45 Nymphs production, 155157

O n-Octanoyl chloride, 284 N-Octyl 2,3:4,6-di-O-isopropylideneβ-valienamine, 285287, 286f, 288f N-Octyl compound, 284 N-Octyl β-galacto-valienamine, 285287, 288f N-Octyl-(1)-conduramine F-4  HCl, 298, 299t N-Octyl-4-epi-β-valienamine (NOEV), 45, 5f, 288f, 292, 313314 chaperone therapy in genetically engineered GM1-gangliosidosis model mice, 303305 for chaperone therapy of GM1-gangliosidosis, 291305

chemical modification of, 296298, 296f, 297f, 299t effects on cultured human and mouse fibroblasts, 301303 HCl, 298, 299t on model mice, 305 physicochemical and biological characteristics of, 298301, 301t, 302f, 303t synthesis and screening of, 288f, 289f, 292296, 293f, 294f, 295f N-Octyl-5α0 -carba-β-lactosylamine, 287290, 289f N-Octyl-β-valienamine (NOV), 45, 5f, 283, 288f, 292, 313314 for chaperone therapy of Gaucher disease, 283291 biological activities of, 290291, 290t chemical modification of, 285290, 286f, 288f, 289f synthesis and screening of, 283284, 283f, 284f N-Octyl-β-valienamine (NOV), 45, 5f, 283, 288f, 292 for chaperone therapy of Gaucher disease, 283291 biological activities of, 290291, 290t chemical modification of, 285290, 286f, 288f, 289f synthesis and screening of, 283284, 283f, 284f n-Octylamine, 296 OFL. See Oral fat load (OFL) OGTT. See Oral glucose tolerance test (OGTT) Oligosaccharides, 251, 258259 Oligostatins, 238, 239f Oocyte development, 159 Oral antidiabetic agents, 270 Oral fat load (OFL), 266 Oral glucose tolerance test (OGTT), 254 Organic acid, 247248 Oxalic acid, 247248 Oxidation reaction, 247248 Oxidizing agents, 247249

P Palladium-catalyzed coupling, 223, 223f Pancreatic β-cells, 237, 251252 Parafilm method, 155157 Parenchyma cells, 128129 PCR. See Polymerase chain reaction (PCR)

Index PDMP. See Platelet-derived microparticles (PDMP) Pellicularia praticola, 117 Pellicularia sasakii, 9, 115, 119f, 121f effect on morphology of, 116124, 117t contents of glucose and glucosamine in cell wall, 124t effect of validamycins on elongation and branching of mycelium, 122t Penicillium notatum, 1 Penta-N,O,O,O,O-acetate, 212213, 212f Penta-N,O-acetate, 209210, 210f Pentaacetylvalidamine, 210211, 211f Pentaacetylvalienamine, 170171, 170f Pentose phosphate pathway (PPP), 20 Periplaneta Americana, 157 PFGE. See Pulsed-field gel electrophoresis (PFGE) PfitznerMoffatt oxidation, 177179, 178f Pfk. See 6-Phosphofructokinase (Pfk) PGase. See Polygalacturonase (PGase) pH, 247248 Pharmacology, pharmacokinetics, and pharmacodynamics glycemic/metabolic effects, 251252 mechanism of action, 251 pharmacokinetic properties, 251 voglibose in combination with mitiglinide for T2DM, 252 Phosphinimide, 165167 6-Phosphofructokinase (Pfk), 48 6-Phosphogluconate, 48 Phthalimido derivative, 171172, 172f Physicochemical properties of voglibose, 245247 Phytopathogenic fungi, 117, 118t Pioglitazone, 256257 PKSs. See Polyketide synthases (PKSs) PlackettBurman design, 9395, 94t Plant pathogens, agricultural antibiotics for, 12 Plasma concentrations, 251 Plasma glucose, 252, 254, 271272 Platelet-derived microparticles (PDMP), 266 Pneumatosis cystoides intestinalis, 257 Polygalacturonase (PGase), 133134 Polyketide synthases (PKSs), 20 Polymerase chain reaction (PCR), 51f Polysaccharides, 251, 258259, 314 Porcine maltase, 240 sucrase, 238

325

Postprandial glucose, 254 Postprandial hyperglycemia (PPG), 237, 269272 Postprandial insulin (PPI), 264 Postprandial plasma glucose, 252 PPG. See Postprandial hyperglycemia (PPG) PPI. See Postprandial insulin (PPI) PPP. See Pentose phosphate pathway (PPP) PPTS. See Pyridinium-p-toluene-sulfonate (PPTS) Protease inhibitors, 279280 Protein binding, 260 function, 282283 proteinchaperone complex, 281 ( 1 )-Proto-quercitol, 294, 295f, 297, 297f Pseudo-D-glucopyranose, 211, 212f Pseudo-β-galactose, 296297, 296f Pseudodisaccharides, 240, 243t Pseudoglucosidic linkage bond (-C-NH-C-), 245 N-Pseudoglycosyl linkage, 4748 Pseudomonas denitrificans, 37 degradation of validamycin A by, 75 Pseudomonas solanacearum, 136 antibacterial activity against, 136140, 139f control of cabbage black rot, 141f control of tomato bacterial wilt by foliar sprays, 140f effect of validamycin A against tomato bacterial wilt, 140f Pseudonitrosugar treatment, 168169 Pseudooligosaccharides, 238 Pseudotetrasaccharides, 133 Pulsed-field gel electrophoresis (PFGE), 91 Purification process, 296 Pyk. See Pyruvate kinase (Pyk) Pyridine, 285, 286f Pyridinium-p-toluene-sulfonate (PPTS), 294, 297 Pyruvate kinase (Pyk), 48 Quality-adjusted life years (QALY), 263264 L-Quebrachitol, synthesis from, 165167, 166f ()-Quinic acid, synthesis from, 181183, 182f, 184f, 202, 203f, 212213, 212f

R R201C model mouse, 303305 Radioactivity, 260

326

Index

RCEYM. See Ring-closing enyne metathesis (RCEYM) Reactive oxygen species (ROS), 100102 Relative standard deviation (RSD), 66 Repaglinide-treated patients, 264 Response surface methodology (RSM), 91 Retrospective post hoc analysis, 254 Reverse phase chromatography, HPLC with, 63 Reversed layer method, 6061 Rhizoctonia cerealis, 124127 validamycin A concentration on growth of, 125f validamycin A on hyphal extension and morphology of, 124127, 126t validamycin A on number of branches, 126t Rhizoctonia solani, 23, 56, 115 antifungal activity against, 115, 116t antifungal mechanism against, 115135 induction of validamycins for endochitinase and β-1,3-glucanase in rice plants, 134135 effect of validamycins on morphology and components, 116134 validamycin A concentration on growth of, 125f Ribonucleic acid (RNA), 123, 124t Rice plants destructive disease of, 9 induction of validamycins for endochitinase and β-1,3-glucanase in, 134135, 135t Rifampin, 1 Ring-closing enyne metathesis (RCEYM), 187189 RNA. See Ribonucleic acid (RNA) ROS. See Reactive oxygen species (ROS) RSD. See Relative standard deviation (RSD) RSM. See Response surface methodology (RSM)

S SAR. See Systemic acquired resistance (SAR) SARP-family regulatory gene, 50 SBF. See Soybean flour (SBF) Screening and breeding high-yield strains, 9092 amplification of val gene cluster, 92f Screening and breeding high-yield strains, 9092 amplification of val gene cluster, 92f L-Selectride, 176177

L-Serine, synthesis from, 187189, 188f Serum-free medium (SFM), 58 SFM. See Serum-free medium (SFM) ()-Shikimic acid, synthesis from, 190192, 191f, 208 SHJG0322 mutant, 5051, 51f, 52f sICAM-1. See Soluble intercellular adhesion molecule-1 (sICAM-1) Silkworm, 153 Silver trifluoromethanesulfonate (AgOTf), 228 Sodium borohydride (NaBH4), 171172 Sodium methoxide, 294296 Sodium periodate (NaIO4), 190192 Soilborne diseases, 135 Soluble intercellular adhesion molecule-1 (sICAM-1), 251252 Somatic cells, 280281 L-Sorbose, 195196, 195f Soybean flour (SBF), 2324 Spiro epoxy, 294, 294f Spiro oxiranes, 179, 179f Spodoptera litura (S litura), 145146, 153155 Stalk-tip soaking method, 155157 Starch, 2324, 237 hydrolysis, 10 starch-based food consumption, 9697 Stereochemistry, 240 STOP-NIDDM. See Study To Prevent NonInsulin Dependent Diabetes Mellitus (STOP-NIDDM) Streptomyces chromosomes, 1920 genomes, 20 S. hygroscopicus var. jinggangensis, 23 S. hygroscopicus var. limoneus, 23 Streptomyces hygroscopicus var. limoneus T-7545, 9, 10f, 11t Streptomyces hygroscopicus, 9, 215, 219 S. hygroscopicus var. jinggangensis 5008 genome, 1620, 19f central carbon and nitrogen metabolisms for validamycin, 21f Streptomyces chromosomes, 18t S. hygroscopicus var. jinggangensis Yen. TH82, 9, 10f, 11t S. hygroscopicus var. limoneus, 39 S. hygroscopicus var. limoneus KCCM 11405, 4344 S. hygroscopicus var. limoneus No. 7545, 22

Index subsp. limoneus KCTC 1717 genome, 2122, 22t Streptomyces scabies, 1920 Streptomycin, 2 Structureinhibitory activity, 285 Study To Prevent Non-Insulin Dependent Diabetes Mellitus (STOP-NIDDM), 269, 271272 Subacute neuronopathic phenotype, 283 N-Substituent unit, 245 N-Substituted ( 1 )-conduramine F-4 derivatives, 297, 297f N-Substituted valiolamine derivatives, 238 α-D-glucosidase inhibitory activity, 240245, 241t, 243t, 246t, 247f Sucrase, 240 Sugar hydrolases, 146148 Sulfonylurea agents, 256 Sulfur drugs, 1 Symbiotic fungi, 160 Symptomatic therapy, 291292 syn,syn-triol, 189190, 189f Systemic acquired resistance (SAR), 135, 138f

T T2DM. See Type 2 diabetes mellitus (T2DM) Tandem mass spectrometry (MS/MS), 4344 TAP. See Telomere-associated protein (TAP) Tartaric acid, synthesis from, 184187 D-Tartaric acid, 186187, 187f, 207208, 208f L-Tartaric acid, 184185, 186f synthesis from, 207208 TCA cycle. See Tricarboxylic acid cycle (TCA cycle) Telomere-associated protein (TAP), 1619 Temperature of submerged culture, 2223, 23t Terminal inverted repeats (TIRs), 1619 Termites, 160 Tert-butyldimethylsilyl derivative, 285287, 288f 2,3,4,6-Tetra-O-acetylα-α-glucopyranocshylolride, 224, 225f 1,3,4,5-Tetra-O-benzyl-6,7-dideoxy-L-xylohept-6-en-2-ulose, 172173, 173f Tetra-O-benzyl-D-glucono-1, 5-lactone, 171172, 249 2,3,4,6-Tetra-O-benzyl-D-glucopyranose, 172173, 173f 2,3,4,6-Tetra-O-benzyl-D-glucose, synthesis from, 173177, 174f, 175f

327

Tetrabenzyl glucose, 175176, 175f 2,3,4,6-Tetrabenzyl-D-glucono-1,5-lactone, 201, 201f Tetrahydrofuran (THF), 168169, 179, 285, 286f [(1S)-(1,2,4/3,5,6)-2,3,4,6-Tetrahydroxy-5(hydroxymethyl)cyclohexyl]-amine. See Validoxylamine B 1,1,3,3-Tetramethylurea (TMU), 228 Tetrasaccharide, 258259 TFA. See Trifluoroacetic acid (TFA) Tg. See Triglycerides (Tg) Tg mouse. See Transgenic mouse (Tg mouse) THF. See Tetrahydrofuran (THF) Thiazolidinediones, 237 Thin-layer chromatography (TLC), 24, 26f Thiol, 291292 Thionyl chloride (SOCl2), 190192 TIPS glucal, 170171, 170f TIRs. See Terminal inverted repeats (TIRs) TLC. See Thin-layer chromatography (TLC) TMS. See Trimethylsilyl (TMS) TMU. See 1,1,3,3-Tetramethylurea (TMU) Total correlation spectroscopy (TOCSY), 298 Trans-hydroxyl, 294296 Trans-isopropylidene group, 294, 295f, 297 Transgenic mouse (Tg mouse), 303 Trehalase, 157, 160, 217218 inhibition constant of validoxylamine A for, 148t inhibitor injection, 154t inhibitory activity against, 145148, 147t inhibitory activity of validamycins and validoxylamines, 148t LineweaverBurk plot of Spodoptera litura, 149f in rice plants, 134 in whole body homogenate of worker termites, 161f Trehalose, 136, 160 Trestatins, 238, 239f Tri-O-acetyl-N-(benzyloxycarbonyl) validamine, 203205 (1R)-(2)-1,2,3-Tri-O-acetyl-(1,3/2,4,6)-4bromo-6-bromomethyl-1,2,3cyclohexanetriol, 179 (1R,2S,3S,4R)-2,3,4-Tri-O-benzyl-5(benzyloxymethyl)-cyclohex-5-ene1,2,3,4-tetrol, 175176 2,3,6-Tribenzoate, 287290, 289f Tribenzyl ether, 182183, 182f

328

Index

(4S,5S,6S)-4,5,6-Tribenzyloxy-7(benzyloxymethyl)octa-1,7-dien-3-ol, 173174 Tribromide, 292, 293f Tricarboxylic acid cycle (TCA cycle), 20 Triethylamine, 249250 Trifluoroacetic acid (TFA), 173174 Trifluoroacetic anhydride, 249250 N-Trifluoroacetyl derivative, 287290, 289f Triglycerides (Tg), 255, 266 [(1S)-(1,4,6/5)-4,5,6-Trihydroxy-3hydroxymethyl-2-cyclohexenyl]. See Validoxylamine B [(1S)-(1, 2, 4/3, 5)-2, 3, 4-Trihydroxy-5hydroxymethylcyclohexyl]-amine. See Validoxylamine A Triisopropylidene compound, 165167, 166f Trimethylsilyl (TMS), 6163 Triphenylphosphine (PPh3), 196197 Type 1 diabetes, 237 Type 2 diabetes mellitus (T2DM), 253257, 268272 glycemic control in, 264265 patients, 251252 voglibose in combination with mitiglinide for, 252 Tyrosinase, inhibitory activity against, 150153 L-Tyrosine, 150153

U UDP-glucose pyrophosphorylase (Ugp), 7980, 80f, 8283, 82f cloning and expression of, 7984, 84f UDP-glucose-1-phosphate uridylyltransferases, 20 UDP-N-acetylglucosamine, 7678 Ultraviolet absorption spectra, 31 Uridine diphosphate-glucose (UDP-glucose), 105106, 105f UV detector, 70

V ValG. See Validamycin glycosyltransferase (ValG) Validamine (VD), 28, 238, 239f, 240, 241t, 245, 246t, 314 characterization of, 3739 chemical structures of, 29f synthesis, 203205, 204f, 205f, 209214

from ()-7-endo-oxabicyclo[2.2.1]-hept5-ene-2-carboxylic acid, 209210 from ()-quinic acid, 212213, 212f from D-glucose, 210211, 211f from D-xylose, 213214, 213f from nitrofuranose, 211, 212f Validamycin A, 2830, 30f, 41f, 115116, 125f, 126t, 165, 224, 225f, 238, 239f, 313314 detection of, 6075 colorimetric method, 61 CZE, 6671 GC, 6163, 62f, 63f HPLC with reverse phase chromatography, 63 LCAPCIMS/MS, 7175, 73f liquid chromatography with ion exchange chromatography, 6365 reversed layer method, 6061 on hyphal extension and morphology of R. cerealis and R. solani, 124127 microbial degradation of, 7576 treatment with, 128134, 129t, 130t Validamycin B, 30, 154155, 165, 225, 226f Validamycin C, 154155, 226227, 227f ( 1 )-Validamycin C, 226227 Validamycin D, 154155, 227, 228f Validamycin E, 228, 229f Validamycin F, 229, 230f Validamycin G, 154155, 229, 231f Validamycin glycosyltransferase (ValG), 47 bioconversion of validoxylamine A to 4v-epi-validamycin A, 78f to validamycin A, 79f cloning and expression of gene, 7679 expression and enzymatic activity of, 81f inactivation and complementation of, 85f knocking out, 8486, 85f knockout mutants, 105106 pJTU612, 86 protein, 4143 Validamycin H, 230231, 232f Validamycin(s), 3f, 4f, 56, 145146, 313 agricultural antibiotics for plant pathogens, 12 of antibiotics, 1 biosynthesis, 3959 biosynthetic gene clusters, 4344 characterization and validoxylamines, 2836 compounds, 160161 discovery of, 9

Index enzyme inhibitory activities, 145153 fermentation process for production, 90106, 93f hydroxyvalidamine characterization, 3739 magic agricultural antibiotics, 25 microbes for producing, 922 production and isolation of fermentation of validamycins, 2224 isolation of validamycins from broth, 2428 and related natural compounds, 115 antifungal activities, 115145 structures and natural compounds, 28, 29f synthesis, 192 validamine characterization, 3739 validamycin-ingested termites, 160161 validamycins effect on growth and morphology of fungi, 127128, 128t valienamine characterization, 3739 valiolamine characterization, 3739 Validoxylamine A, 27, 27f, 80, 81f, 146, 215218, 216f, 218f, 238, 239f, 314 by acid-catalyzed hydrolysis of validamycin A, 219f insecticidal activity, 153161, 153t, 154t, 155t, 156f, 156t, 157t, 158t, 159t, 161f by resin-catalyzed degradation of validamycin A, 219f synthesis, 215218, 216f Validoxylamine B, 27, 27f, 219220, 220f, 221f Validoxylamine G, 28, 221223, 222f, 223f, 224f Validoxylamine(s), 2836, 29f, 35t, 36t, 145146, 157 Valienamine (VE), 2122, 28, 238, 239f, 240, 241t, 245, 246t, 314 from ()-shikimic acid, 190192, 191f from (1SR,2RS,3SR)-6-methylenecyclohex4-ene-1,2,3-triol, 183 from acarbose, validamycin, and derivatives, 192 characterization, 3739 chemical structures, 29f from Garner’s aldehyde, 189190, 189f from L-serine, 187189, 188f synthesis, 165192, 203205, 204f, 205f from 2,3,4,6-tetra-O-benzyl-D-glucose, 173177 from cyclohexane skeleton, 179181, 179f, 180f from D-glucose, 167173, 168f

329

from D-xylose, 177179 from L-quebrachitol, 165167, 166f from ()-quinic acid, 181183, 182f from tartaric acid, 184187 ( 1 )-Valienamine, 165, 166f 1,10 -bis-Valienamine, 8688, 87f, 89f Valiolamine (VO), 28, 40t, 238, 239f, 240, 241t, 243t, 245, 246t, 248249 characterization, 3739 chemical structures, 29f synthesis, 200208 from D-glucose, 200202, 200f from myo-inositol, 206207, 207f from ()-quinic acid, 202, 203f from ()-shikimic acid, 208, 209f from L-tartaric acid, 207208 from valienamine and validamine, 203205, 204f from ()-vibo-quercitol, 205206, 206f Valiolone, 247248, 248f valN gene inactivation, 8688, 87f VD. See Validamine (VD) VE. See Valienamine (VE) ()-Vibo-quercitol, 294 synthesis from, 205206, 206f VldC, 45 VO. See Valiolamine (VO) Voglibose, 238, 239f, 313314 α-D-glucosidase inhibitory activity Nsubstituted valiolamine derivatives, 240245, 241t, 243t, 246t, 247f chemical structures of pseudoaminosugar glucosidase inhibitors, 238239, 239f clinical efficacy add-on therapy to insulin, 254255 combination therapy in patients on hemodialysis, 255 coronary atherosclerosis, 255256 impaired glucose tolerance, 253254 inflammation, 256257 oral antidiabetic agents, 252253 in combination with mitiglinide for T2DM, 252 comparison with drugs of acarbose and miglitol, 257270 adverse events, 263 carotid plaque, 268 clinical recommendations, 260263 comparison of α-glucosidase inhibitors, 269270 cost-effectiveness ratio, 263264 drug summary, 258

330

Index

Voglibose (Continued) glycemic control in T2DM, 264265 glycemic excursions, 265266 impaired glucose tolerance, 268269 inflammation, 266267 mechanism of action, 258259, 258f, 259f pharmacokinetic properties, 259260, 261t market and development, 270272, 271t NIDDM, 237 pharmacology, pharmacokinetics, and pharmacodynamics glycemic/metabolic effects, 251252 mechanism of action, 251 pharmacokinetic properties, 251 physicochemical properties and drug, 245247 safety and tolerability, 257 synthesis from compound 35, 247, 248f

synthesis from glucose, 249250, 250f synthesis from valiolamine, 247249, 248f, 249f

X X. campestris pv. campestris antibacterial activity against, 141142 control of cabbage black rot, 143t Xanthomonas campestris, 100101 Xylanase, 133134 D-Xylose, synthesis from, 177179, 213214, 213f Xylose mother liquid. See Corncob hydrolysate

Z Zemple´n deacylation, 294296, 295f