Organic Solvents: Properties, Toxicity, and Industrial Effects : Properties, Toxicity, and Industrial Effects [1 ed.] 9781611222296, 9781617618819

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Copyright © 2011. Nova Science Publishers, Incorporated. All rights reserved. Organic Solvents: Properties, Toxicity, and Industrial Effects : Properties, Toxicity, and Industrial Effects, edited by Ryan E. Carter, Nova Science

Copyright © 2011. Nova Science Publishers, Incorporated. All rights reserved. Organic Solvents: Properties, Toxicity, and Industrial Effects : Properties, Toxicity, and Industrial Effects, edited by Ryan E. Carter, Nova Science

CHEMICAL ENGINEERING METHODS AND TECHNOLOGY

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ORGANIC SOLVENTS: PROPERTIES, TOXICITY, AND INDUSTRIAL EFFECTS

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Organic Solvents: Properties, Toxicity, and Industrial Effects : Properties, Toxicity, and Industrial Effects, edited by Ryan E. Carter, Nova Science

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Organic Solvents: Properties, Toxicity, and Industrial Effects : Properties, Toxicity, and Industrial Effects, edited by Ryan E. Carter, Nova Science

CHEMICAL ENGINEERING METHODS AND TECHNOLOGY

ORGANIC SOLVENTS: PROPERTIES, TOXICITY, AND INDUSTRIAL EFFECTS

RYAN E. CARTER

Copyright © 2011. Nova Science Publishers, Incorporated. All rights reserved.

EDITOR

Nova Science Publishers, Inc. New York

Organic Solvents: Properties, Toxicity, and Industrial Effects : Properties, Toxicity, and Industrial Effects, edited by Ryan E. Carter, Nova Science

Copyright © 2011 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com

NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS.

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Additional color graphics may be available in the e-book version of this book.

LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA Organic solvents : properties, toxicity, and industrial effects / editor, Ryan E. Carter. p. cm. Includes bibliographical references and index. ISBN:  (eBook)

1. Organic solvents. I. Carter, Ryan E. TP247.5.O74 2010 660'.29482--dc22 2010034018

Published by Nova Science Publishers, Inc. † New York

Organic Solvents: Properties, Toxicity, and Industrial Effects : Properties, Toxicity, and Industrial Effects, edited by Ryan E. Carter, Nova Science

CONTENTS Preface Chapter 1

Co-Solvent Application for Biological Systems Satoshi Ohtake, Yoshiko Kita, Chiaki Nishimura and Tsutomu Arakawa

Chapter 2

Lipase-Catalyzed Synthesis of Edible Surfactants in Microaqueous Organic Solvents Yoshiyuki Watanabe and Shuji Adachi

Chapter 3

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Chapter 5

Chapter 6

Chapter 7

Analysis of the Organic Solvent Effect on the Structure of Dehydrated Proteins by Isothermal Calorimetry, Differential Scanning Calorimetry and FTIR Spectroscopy Vladimir A. Sirotkin Organic-Solvent Tolerant Gram-Positive Bacteria: Applications and Mechanisms of Tolerance Pedro Fernandes, Marco P.C. Marques, Filipe Carvalho and Carla C.C.R. de Carvalho Toxicity of Organic Solvents and Ionic Liquids to Lactic AcidProducing Microbes Michiaki Matsumoto Effect of Hydrogen Bond Accepting Organic Solvents on the Binding of Competitive Inhibitor and Storage Stability of Chymotrypsin Vladimir A. Sirotkin Regularities of Organic Solvents Penetration into Tetrafluoroethylene-Propylene Copolymer I. Yu Yevchuk, G. G. Midyan, R. G. Makitra, G. E. Zaikov, G. I. Khovanets’ and O. Ya. Palchykova

Index

Organic Solvents: Properties, Toxicity, and Industrial Effects : Properties, Toxicity, and Industrial Effects, edited by Ryan E. Carter, Nova Science

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31

57

89

105

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PREFACE A solvent is a liquid that becomes a solution by dissolving a solid, liquid, or gaseous solute. The most common solvent in everyday life is water. This book presents topical research data in the study of organic solvents, including co-solvent applications for biological systems; studying the structure of dehydrated proteins in the presence of organic solvents; the applications of Gram-positive bacteria in organic solvents; as well as the toxicity of organic solvents and ionic liquids to lactic acid-producing microbes. Chapter 1 - Biological macromolecules and viruses are used as therapeutic reagents and drugs. These important products are processed and stabilized using co-solvents, which are comprised of amino acids, salts, or organic solvents present at high concentrations. They exert their effects through weak interactions with the surface of biological macromolecules and viruses. Co-solvents can have a wide range of stabilizing, or destabilizing, effects on the solutes (i.e., proteins and viruses), depending not only on the type of co-solvent, but its concentration, as well as the type of solute examined. Furthermore, co-solvents can significantly affect the solubility of the system by either salting-in (solubility enhanced) or salting-out (precipitate) the macromolecules. Application of co-solvent systems to a wide variety of macromolecules as well as their mechanism of action will be reviewed. Chapter 2 - 6-O-Lauroyl saccharides were synthesized through condensation in acetonitrile, acetone and 2-methyl-2-propanol with various water contents using the immobilized lipase from Candida antarctica. Glucose, galactose, mannose and fructose were used as a hydrophilic substrate. The apparent equilibrium constant, KC, based on the concentrations of substrates and products in acetonitrile could be correlated to the dynamic hydration numbers of the saccharides. This indicates that the water activity played an important role during the condensation reaction in the microaqueous water-miscible solvent. The KC for the synthesis of lauroyl mannose also depended on the kind of solvent and could be correlated with the relative dielectric constant, r, of the organic solvent. Acyl mannoses with the acyl chain lengths of 8 to 16 were continuously produced using a plug-flow-type reactor packed with an immobilized lipase. Irrespective of the acyl chain length, a conversion of more than 0.5 was achieved at the superficial residence time, 0, equal to or longer than 20 min. A long-term operational stability of the enzyme was examined, and a conversion of ca. 40% was maintained for at least 16 days. The productivity was evaluated to be 350 g/Lreactor･day. The surfactant properties of the produced capryloyl, caproyl, lauroyl and myristoyl mannoses were also determined. Three lauroyl phenolic glycosides were synthesized through the condensation of phenolic glycoside such as arbutin, naringin and

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phloridzin with lauric acid by immobilized lipase in various organic solvents. The conversion depended on the polarity of the organic solvent used for the reaction. The suppressive effect of each lauroyl phenolic glycoside against the oxidation of linoleic acid was higher than that of the corresponding phenolic glycoside, whereas there was no difference between the radical scavenging activities of unmodified and lauroyl phenolic glycosides. It is suggested that enhancement of the suppressive effect against lipid oxidation is ascribed to the increase in the hydrophobicity of phenolic glycoside by the acylation. Chapter 3 - This review describes the basic principles of a novel method for studying the structure of the dehydrated proteins in the presence of organic solvents. This method, based on combined calorimetric and FTIR spectroscopic measurements, allows the simultaneous monitoring of the thermochemical parameters (interaction enthalpies, DSC thermograms) of the dried proteins and the corresponding changes in the protein structure in anhydrous organic solvents. This review aims to analyse the effect of organic solvents on dehydrated protein systems in order to understand what intra- and intermolecular processes produce the main effect on the structure and functioning of proteins in low water organic media. Two unrelated proteins with a high -helix content (human serum albumin, HSA) and with a high -sheet content (bovine pancreatic -chymotrypsin, CT) were used as models. Two groups of model organic solvents were used. The first group included hydrogen bond accepting solvents. The second group included hydrogen bond donating liquids. The results obtained showed that: 1) The enthalpy and integral structural changes accompanying the interaction of dried proteins with anhydrous organic solvents depend cooperatively on the solvent hydrophilicity. The solvent hydrophilicity was characterized by an excess molar Gibbs energy of water in organic solvent at infinite dilution and 25oC. Based on this solvent hydrophilicity parameter, the solvents were divided into two groups. The first group included hydrophilic solvents such as methanol, ethanol, and dimethylsulphoxide (DMSO). Considerable structural rearrangements were observed in this group of solvents. The interaction enthalpies of the dried proteins with hydrophilic liquids were strongly exothermic. The second group included the hydrophobic and medium hydrophilic liquids such as benzene, dioxane, butanol-1, and propanol-1. The enthalpy and structural changes in the second group of solvents were close to zero. 2) The FTIR spectroscopic results can be attributed to the formation of different unfolded states of CT and HSA obtained upon dehydration-, alcohol- and DMSO-induced denaturation. The denatured state obtained in DMSO has a maximal degree of unfolding compared with that observed in alcohols or in the presence of dry air. 3) The effect of the organic solvent on the protein structure is ―protein selective‖. On the other hand, the organic solvent-induced integral structural changes versus solvent hydrophilicity profiles do not depend on the predominant form of secondary structure in the protein. 4) Heat-induced exothermic peaks were observed on the DSC thermograms of the dried proteins in anhydrous organic solvents in the temperature range 60-105 oC. This means that dehydrated proteins in anhydrous solvents is the nonequilibrium state at room temperature. These results give strong support to the

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idea that the non-equilibrium status of the dehydrated proteins results from the protein–organic solvent interactions being ―frozen‖ at near room temperature. The thermodynamic and structural data were analysed to give a unified picture of the state of the dried proteins in anhydrous organic solvents. According to this model, the dehydration-induced protein-protein contacts and the potential of the organic solvent to form the hydrogen bonds are key factors in determining the structure of the dehydrated proteins in the liquids under study. Chapter 4 - Organic solvents have often a deleterious effect on microbial cells, yet the number of isolated bacteria able to thrive in organic environment has been steadily increasing since the first report of such a particular extremophile in the late 1980s. Solvent tolerant bacteria are particularly appealing for application in biocatalysis, since plenty of relevant educts and products are poorly water soluble. This feature critically limits process productivity in aqueous media, although an opposite picture results if bioconversions are performed in organic environment. Similarly, solvent tolerant bacteria can be of use in bioremediation. Given their potential and relevance, dedicated efforts have been made in order to get a significant insight on the mechanisms underlying solvent tolerance. This is mostly due to cell wall adaptation and rapid repair mechanisms, solvent efflux pumps or enzymatic pathways allowing the mineralization of the deleterious solvent. Although several Gram-positive bacteria (viz. Rhodococcus spp., Mycobacterium spp.) have been effectively used for biocatalysis and bioremediation in the presence of organic solvents, and the mechanisms for solvent endurance have been looked into, the information most widely disseminated on these matters relies on Gram-negative model systems. The present review aims to provide an updated perspective on the applications of Gram-positive bacteria in organic solvents environment, their mechanisms of tolerance and foreseen developments Chapter 5 - In situ extractive fermentation of lactic acid using organic solvents has already been extensively investigated. Now ionic liquids are emerging as alternative solvents for volatile organic compounds traditionally used in liquid-liquid extraction. We examined whether lactic acid producing-microbes can grow in the presence of a second phase of imidazolium-based ionic liquids or organic solvents. The lactic acid-producing bacteria used in this study are sensitive to organic solvents having 1 < log P < 4. Solvent toxicity to lactic acid-producing fungi, Rhizopus oryzae JCM 5568, clearly depended on the log P values compared with those of the lactic acid-producing bacteria. We found that Lactobacillus delbruekii subsp. lactis NRIC 1683 grew in the presence of a second phase of imidazoliumbased ionic liquids as well as in the absence of ionic liquids. Finally we discussed the greenness of an ionic liquid system. Chapter 6 - This review aims to analyse the studies of the competitive inhibitor binding and the storage stability of bovine pancreatic -chymotrypsin (CT) in organic solvents in order to elucidate what intermolecular processes produce the main effect on the state and functioning of enzymes at high and low water activities in organic media. The binding of competitive inhibitor proflavin and the storage stability of CT in water-organic mixtures were studied in the entire range of thermodynamic water activities (aw) at 25oC. The moderatestrength hydrogen bond accepting solvents (acetonitrile, dioxane, tetrahydrofuran, and acetone) were used as models due to their ability to vary significantly the size, polarity, denaturation capacity, and hydrophobicity.

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The state of water hydrogen bond network in organic solvents was characterized by thermodynamic and spectroscopic data. The absorption spectra of water in organic solvents were measured by FTIR spectroscopy. The state of water in organic solvents was defined in terms of variations in the integral intensity of water and the contour shape of the band of OH stretching vibrations. Excess chemical potentials, partial molar enthalpies, and entropies of water and organic solvents were simultaneously evaluated at 25oC. The results obtained showed that:

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1) The proflavin binding and storage stability curves can be unified in the water activity coordinates. At the highest water activities (aw>0.95), the water hydrogen bond network is bond –percolated. In this composition region, the storage stability values are close to 100%. 2) At the lowest water activities, the water molecules exist predominantly as single molecules complexed with organic solvent molecules. No proflavin binding was observed at low water activity values in the studied solvents. At aw>0.3, the proflavin binding is sharply increased reaching a maximal value at aw~0.5-0.6. This sharp increase in the enzyme activity occurs only above the threshold water activity level, when the self-associated (H-bonded) water molecules appear in the studied organic solvents. 3) In the intermediate composition region, the solution consists of two kinds of clusters, each rich in each component. There is a sharp transition from the waterrich region to the intermediate one. This transition is associated with an anomaly in the thermodynamic, structural, and enzyme activity properties. This transition may involve loss of the bond percolated nature of the hydrogen bond network of liquid water. The residual catalytic activity of CT changes from 100 to 0% in the transition region. A minimum on the competitive inhibitor binding and storage stability curves was observed at aw of 0.8-0.9. The thermodynamic, structural, and enzyme activity data were analysed to give a unified picture of the state of enzymes in low water organic solvents. According to this model, the dehydration-induced protein-protein contacts and the state of water hydrogen bond network play a key role in determining the enzyme activity – water activity profiles in organic liquids. Chapter 7 - The processes of swelling and diffusion of solvents into the structure of tetraflurethylene-propylene coolymer can not be described quantatively using only one characteristic of a solvent. In all cases a molar volume of liquid has the determining effect, hampering its penetration into the structure of polymer. However, the effects of other factors – both solvation and density of cohesion energy of solvents – are significant. Adequate quantitative generalization of abovementioned processes can be obtained on the basis of free energies linearity concept by using of linear multiparameter equations taking into account the effects of different factors.

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In: Organic Solvents Editor: Ryan E. Carter

ISBN 978-1-61761-881-9 © 2011 Nova Science Publishers, Inc.

Chapter 1

CO-SOLVENT APPLICATION FOR BIOLOGICAL SYSTEMS Satoshi Ohtake1, Yoshiko Kita2, Chiaki Nishimura3 and Tsutomu Arakawa4

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1. Aridis Pharmaceuticals, San Jose, CA 2. Department of Pharmacology, KEIO University School of Medicine, Tokyo, Japan 3. Faculty of Pharmaceutical Sciences, Teikyo Heisei University, Chiba, Japan 4. Alliance Protein Laboratories, Thousand Oaks, CA

ABSTRACT Biological macromolecules and viruses are used as therapeutic reagents and drugs. These important products are processed and stabilized using co-solvents, which are comprised of amino acids, salts, or organic solvents present at high concentrations. They exert their effects through weak interactions with the surface of biological macromolecules and viruses. Co-solvents can have a wide range of stabilizing, or destabilizing, effects on the solutes (i.e., proteins and viruses), depending not only on the type of co-solvent, but its concentration, as well as the type of solute examined. Furthermore, co-solvents can significantly affect the solubility of the system by either salting-in (solubility enhanced) or salting-out (precipitate) the macromolecules. Application of co-solvent systems to a wide variety of macromolecules as well as their mechanism of action will be reviewed.

 Address correspondences to Satoshi Ohtake, Aridis Pharmaceuticals, 5941 Optical Court, San Jose, CA 95138.  35 Shinanomachi,Shinjuku-ku, Tokyo 160-8582, Japan.  4-1 Uruido-minami, Ichihara, Chiba 290-0193, Japan.  3957 Corte Cancion, Thousand Oaks, CA 91360. Organic Solvents: Properties, Toxicity, and Industrial Effects : Properties, Toxicity, and Industrial Effects, edited by Ryan E. Carter, Nova Science

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1. INTRODUCTION Biological macromolecules and viruses are used as therapeutic reagents and drugs for basic research and drug development. Physico-chemical properties of biological macromolecules, such as their stability, solubility, and functional properties, are modulated by solvent composition. This review is concerned with solvent components that play a role only at high concentrations. High concentration signifies a high molar ratio of the additive with respect to the primary solvent (e.g., water), such that the solvent properties diverge from those of the pure primary solvent. For example, enzyme reaction at low water activity has been conducted at near-100% organic solvent [1-5]. Numerous compounds, including salts, amino acids, polyhydric alcohols, and organic solvents, have been used in a co-solvent system [6-20], however it would not be feasible to cover all of these compounds and their applications in this review. The general observations of the effects of the co-solvent system on biological macromolecules will first be summarized, followed by a description of specific applications of co-solvent systems on biological macromolecules, including proteins, DNA, and viruses.

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2. SOLVENT EFFECT OVERVIEW The classical and widely used application of a co-solvent system is phase separation, or precipitation, of proteins and other macromolecules by salts and organic solvents. In 1888, Hofmeister reported an innovative observation that inorganic salts have a universal order in decreasing protein solubility [21]. While the mechanism behind this principle is still under extensive investigation (after over 100 years), a significant leap in mechanistic understanding was made by Traube in 1910 [22], when he reported that the ―attraction pressure‖ of ions on water molecules significantly impacts the solubility of proteins, gases and other compounds. Hydration of ions leads to their repulsion from hydrated proteins, as both solutes tend to maximize their interactions with water molecules. The consequence of this repulsive effect is the thermodynamic destabilization of the entire system, resulting in phase separation or precipitation. There is an often-misquoted explanation that the ability of ions to adsorb water molecules depletes the hydration shell of the protein, exposing the protein surface for aggregation and precipitation. This is incorrect, as there are typically enough water molecules in the system to hydrate both proteins and salt ions, as long as the concentration is not too high (i.e., the two molecules are far apart). Furthermore, water desorption mechanism cannot explain the strong salting-out effects produced by the addition of organic solvents. The mechanism of salting-out is also through mutual repulsion between organic solvents and protein molecules, although the cause of repulsion is different from that operating in salt solutions [23].

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Figure 1. Illustration of cavity theory, demonstrating the various effects of salts on protein structure and stability.

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Figure 2. The order of salts according to their effects on macromolecules, either as a salting-in salt or salting-out salt.

The ―attraction pressure‖ theory was further extended by Sinanoglu and Abdulnur [24], in the ―cavity theory‖, to explain the effect of alcohol on DNA structure. They proposed that the surface tension is decreased by the addition of ethanol, which favors more solventexposed single stranded DNA, i.e., ethanol destabilized the DNA double helix. The ―cavity theory‖ was further formalized by Melander and Horvath [25] to explain the effects of salts on protein solubility and its mechanism of action in hydrophobic interaction chromatography. According to the ―cavity theory‖, the salts with high charge density have high ―attraction pressure‖ on water molecules, and hence increase the surface tension of water effectively. This creates an unfavorable free energy around the surface of proteins and macromolecules, thus their association is one way by which the unfavorable free energy can be released (Figure 1). As shown in Figure1, reversible association of native proteins (here into a trimer) will lead to decreased surface area per protein, with consequent reduction in free energy associated with ―attraction pressure‖, or in creating a cavity. An alternative way to release the unfavorable free energy is to prevent protein expansion (unfolding). As shown in Figure1, unfolding exposes the interior of the protein to solvent molecules and increases the surface area (expressed as an elongated ellipsoid in the right column). This would result in increased free energy associated with the ―attraction pressure‖. Thus, the salting-out salts stabilize the proteins and the salting-in salts destabilize them. The order of salts (see Figure 2), however, deviates at MgCl2. Mg2+ effectively increases the surface tension of water due to its high charge density, but it is classified as a salting-in salt [26-30]. Such deviation is also observed with its effects on the other properties of proteins

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and viruses [29-31], although the effects on virus stability are highly variable [32]. In all cases, the effects of MgCl2 deviate from the trend observed in the ―attraction pressure‖ principle. As described later, this deviation is due to the difference in the nature of proteinwater interface and air-water interface. More specifically, protein surface is not a homogeneous non-polar surface, as is the case with air-water interface. The protein surface can attract certain co-solvents over water molecules, even those molecules that increase the surface tension of water. When salting-out salts are used to precipitate proteins, there is no danger of protein denaturation due to their stabilizing effects. Water miscible organic solvents are strong protein precipitants [23, 29], and in general, they denature proteins at high concentrations [23, 29-38]. In fact, ethanol precipitation of plasma proteins is the most significant development in the realm of protein therapeutics, and has contributed greatly to the current progress in the production of recombinant biopharmaceuticals for a number of diseases and injuries that require blood component supplement [30, 39]. It is apparent that the effects of organic solvents on protein precipitation cannot be explained from the surface tension effects (cavity theory), as the addition of organic solvents decreases the surface tension [40-42]. Rather, the cavity theory explains the denaturing effects of organic solvents on the proteins, as described above for the destabilization of the DNA helical structure. How, then, does organic solvents decrease protein solubility on one hand and enhance unfolding on the other? This apparent paradox lies in the chemistry of the protein surface. The effects of salts can simply be related to the protein’s surface area: salting-out salts favor the compact structure and salting-in salts favor the expanded structure. Organic solvents interact favorably with the expanded structure, with greater hydrophobic-exposed surface area, and unfavorably with the native structure, with polar residues solvent-exposed. There are two critical observations that can be made to explain the complex nature of organic solvents: 1) preferential interaction of organic solvents with proteins and 2) solubility of polar and non-polar compounds in organic solvents.

Figure 3. Illustration demonstrating the competing effects of water and co-solvent molecules for the surface of a protein.

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Figure 4. The solubility of glycine in various organic solvents, including DMSO (dimethyl sulfoxide), ethanol, and dioxane. The concentration of each solvents, ranging from 10 to 80%, is indicated in the figure.

Figure 5. The transfer free energy of protein side chains, alanine (Ala), leucine (Leu), and tryptophan (Trp) in a DMSO co-solvent system, ranging in DMSO concentration from 20 to 80%.

A. Preferential interaction: Ligand binding to macromolecules can be readily determined by equilibrium dialysis. The co-solvents described here require high concentrations for their effects, suggesting that each individual interaction is weak, but can be large if sufficient number of bindings is present. In such case, the hydration of macromolecules contributes to the observed ligand binding as (g3/g2) = A1(g’3 - g3) = A3 - g3A1 where g3 is the concentration of co-solvent in the bulk and g’3 is the concentration of co-solvent at the protein surface, where both the properties of water and co-solvent are affected by the protein surface, as illustrated in Figure 3 [13, 18, 43, 44]. Their rotational and translational mobilities are

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different from those in bulk solution. Formally, the observed ligand binding is the sum of cosolvent binding, A3, and hydration, A1. When g3 (ligand concentration in binding experiment) is small, the second term (g3A1) is negligible. When ligand (co-solvent) concentration is high, the interaction of co-solvent with the protein is determined by both co-solvent binding and hydration, and hence is termed ―preferential interaction‖ to indicate which solvent component, co-solvent or water, is preferentially bound to the protein. It has been observed that (g3/g2) is negative in salting-out solutions, independent of the salt concentration, meaning that g’3 - g3 is negative and thus there is deficiency of salt concentration at the protein surface relative to the bulk concentration [45]. On the other hand, the salting-in salts have a tendency to bind to the proteins with g’3 - g3  0 [28, 30]. The negative interaction of salting-out salts correlates with the concept of ―attraction pressure‖ in that the salts and the protein repel each other. On the other hand, salting-in salts show no correlation with the ―attraction pressure‖. For salting-in salts, protein surface is not identical to the air-water interface and attracts the salts to its vicinity. Organic solvents exhibit unique interactions with the protein surface. When the proteins are in the native structure, they are strongly excluded from the protein surface with (g3/g2) = A1(g’3 - g3)< 0 [23, 38]. When the proteins are unfolded, they bind to the protein surface with (g3/g2) = A1(g’3 - g3) > 0 [38]. For example, 2-chloro-2, 4-methylpentanediol (MPD), ethanol and dimethyl sulfoxide (DMSO) are all repelled from the native protein surface, although they bind to the proteins at high enough concentrations to unfold the proteins [23, 38]. This repulsion causes the protein to phase separate and precipitate, as has been observed previously [38]. This property of MPD or ethanol is used for protein crystallization or fractionation of plasma proteins. They are used, however, under optimal conditions, e.g., at intermediate concentration and low temperature, to avoid denaturation. A more strongly binding 2-chloroethanol demonstrates positive (g3/g2), even at low concentrations, so that it cannot be used as a protein-precipitating agent [13, 18]. Instead, it is a good protein denaturing solvent, since it induces helical formation: it has been used to understand the helical structure of proteins. B. Solubility: How does the effect of organic solvent depend on the chemistry of protein surface? The solubility data most clearly demonstrate the effects of organic solvents to cause phase separation, or decrease the solubility of polar compounds, and to increase the solubility of non-polar and aromatic groups. Strongly crystallizing MPD phase-separates upon the addition of ions, suggesting that MPD and electrical charges repel each other [23]. The solubility of polar glycine decreases in organic solvents, as shown in Figure 4 [38, 46]. In Figure 4, the ratio (in log scale) of glycine solubility in organic solvent relative to that in water is plotted against organic solvent concentration (10-80 %). The solubility decreases similarly in DMSO, ethanol and dioxane, indicating that the interactions of glycine with these organic solvents are highly, and similarly, unfavorable. On the other hand, the solubility of non-polar and aromatic groups increases with organic solvent concentration. Figure 5 shows the results of interaction of the side chain of alanine, leucine and tryptophan with DMSO (2080%). The free energy is negative, indicating that the interaction is favorable. Thus, organic solvents are excluded (repelled) from the native protein with polar groups and charges that are solvent-exposed and bind to the non-polar and aromatic groups when they are solventexposed upon unfolding. This is why organic solvents can precipitate proteins in certain situations and cause denaturation in others.

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The effects of organic solvents are schematically illustrated in Figure 6. When hydrated native protein is transferred into an aqueous solution containing low concentrations of organic solvents (upper panel), repulsion of protein molecules increases due to the decreased dielectric constant. In addition, lower dielectric constant may alter the ion distribution at the protein surface, and thus, protein hydration. However, the most significant effect of organic solvent is its repulsion from the charged protein surface, leading to an overall decreased solubility (that overwhelms the repulsion between protein molecules). At high concentration, organic solvents bind to the hydrophobic region of the unfolded protein. Such unfolding also causes decreased charge density of the protein, which in turn reduces repulsion between protein molecules (this effect should enhance protein association) and between protein charge and organic solvent (this effect should reduce protein association). Binding (solvation) of the unfolded structure by organic solvents should favor the dissociated state, suggesting that the effects of organic solvents on the solubility of unfolded protein may be unpredictable.

APPLICATIONS OF CO-SOLVENTS

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1. Enhancing Protein Stability In their natural environments, most biological macromolecules are stabilized by a number of factors. Unlike in vitro systems, they are not in dilute solution and instead are immersed in a highly concentrated macromolecular solution. Such crowded condition itself can stabilize proteins and enzymes [47-50]. They are often bound by other proteins and small molecules, which also stabilize these macromolecules. Alternatively, they may be made intrinsically unstable and hence designed for rapid turnover. In order to use these macromolecules for research purpose, drug development, and industrial application, they must be stabilized in the native, functional structure. Various salts, amino acids, amines, polyhydric alcohols, and sugars have been used to enhance the stability of proteins [6-20]. For example, Weisenberg and Timasheff made a pioneering observation when isolating brain microtubules [51]; the microtubules were readily purified from brain tissue homogenate by a simple temperaturecontrolled assembly and disassembly process [52, 53]. However, the microtubule proteins had to be recovered in the functional structure, which is maintained in vivo by microtubuleassociated proteins (MAPs) [52, 53]. Prior to the reports by Weisenberg and Timasheff, the purified microtubules were always accompanied by incorporated MAPs, and it was assumed that the microtubule assembly required the presence of these proteins. This was proved to be incorrect when Weisenberg and Timasheff purified MAPs-free tubulins, which are subunit proteins of microtubules [51, 54, 55]. In other words, they purified tubulins in the absence of MAPs. What made this purification possible was the presence of a solvent additive, glycerol or sucrose, at a high concentration: ~30 % glycerol and ~1 M sucrose [54, 55]. Without these additives, tubulin lost the ability to polymerize into microtubules during purification and storage. More importantly, the purified tubulins were able to polymerize into microtubules in the presence of these solvent additives. Namely, these additives replaced the activity of MAPs, demonstrating that MAPs are not an essential factor for microtubule assembly.

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Figure 6. Illustration demonstrating the diverging effects of organic solvent at low and high concentrations on the solubility and structural stability of proteins.

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B. Protein Biopharmaceuticals Co-solvents also play a role in the production of recombinant proteins. Co-solvents (e.g., sugars and amino acids), as well as aggregation suppressing arginine, have been reported to increase the yield of protein production [56-61]. They may be used to enhance the in vivo expression of soluble, folded proteins or help assist the in vitro folding of denatured proteins. The recombinant proteins are the basis of biotechnology. As described in the research application section, the proteins that are developed for human therapeutic applications must meet a rigorous guideline. While it would be ideal to prepare the protein solution for injection close to physiological condition, it is often not possible and may require the presence of cosolvents for effective stabilization. Even with the help of co-solvents, however, proteins are often too unstable to store in the liquid form, in which case, proteins are stored frozen or upon lyophilization. In both cases, proteins are subjected to various stressful conditions, including ice and salt crystal formation, freeze-concentration of the protein to extremely high concentration, and removal of water from the protein’s hydration shell. The addition of cryoor lyo-protectants (as a co-solvent) is usually essential to preserve the native conformation of the proteins, however, due to the requirement of high quality product, organic solvents are seldom used for the production and formulation of biopharmaceutical proteins.

C. Chromatography Chromatography is a fundamental technology for scientific research and biotechnology development of proteins, nucleic acids, and even viruses. The performance of column chromatography can be improved by choosing the appropriate combination of salts and co-

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solvents, which modulate protein binding and elution [62-64]. Size exclusion chromatography (SEC), also termed gel filtration or gel permeation chromatography, is among the most frequently used techniques for analysis and purification of proteins [65-68]. The recovery and resolution of proteins are often compromised by the interaction of the proteins with the column matrix. Both electrostatic and hydrophobic interactions can occur between the protein and the column matrix, often complicating the purification process [69]. The electrostatic interactions can be suppressed by the addition of electrolytes, e.g., high phosphate concentration or moderate concentrations of NaCl (0.2-0.5 M) [70, 71]. Gagnon [72] demonstrated that the addition of both ammonium sulfate and phosphate retarded the elution of monoclonal antibodies (mAbs). High salt concentrations, however, may enhance protein adsorption through hydrophobic interaction, which in turn, can be suppressed by the addition of organic solvents. In fact, acetonitrile at high concentrations is often used for that purpose [73, 74]. It is thus evident that while salts are effective in suppressing the electrostatic interactions, it enhances hydrophobic interactions. Arginine has been shown to effectively suppress non-specific binding of proteins to silica-based and polysaccharide-based columns [75]. Electrostatic interactions determine the binding of proteins in ion exchange chromatography (IEC) and contribute partially to the binding in hydroxyapatite chromatography (HA). Salts, therefore, play a major role in modulating the binding and elution of the proteins from these columns [76]. In IEC, NaCl has been the choice of salt for elution. Ammonium sulfate (AS) at high concentration can either induce or retain binding of BSA (bovine serum albumin) to Q-Sepharose, in which it is expected to reduce the electrostatic interaction, and thus cause elution of the protein [77]; BSA was found to bind to Q-Sepharose in the presence of 4M AS and elute at lower concentrations. Mevarech et al [78] have also shown that halophilic proteins bind to IEC columns in the presence of 2.5M AS and elute with decreasing AS concentration. IEC purification conducted under normal conditions (i.e., binding at low salt concentration and eluting at high salt concentration) could not be applied to halophilic proteins, as they are inactivated by lowered ionic strength (i.e., low salt concentration). Thus, IEC columns were used as a weakly hydrophobic surface to which AS at high concentrations salted-out the proteins [79, 80]. Polyethylene glycol (PEG) has been demonstrated to enhance protein-protein interactions due to its large excluded volume: similar to the salting-out effects of salts and organic solvents, the large excluded volume of PEG causes repulsion from proteins, and this effect can be used to enhance their binding to IEC columns. Although PEG typically delays the elution of all species from the column, the oligomer elution is the most significantly affected, leading to effective separation of the oligomers from the monomer species [81-83].

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Figure 7. Elution profile of LDH (lactate dehydrogenase) from a blue-dye column using several elution buffers, including NaCl at 0.5M and 2M and arginine at 0.25M and 2M.

Hydrophobic interaction chromatography (HIC) is one of the fundamental analytical and separation tools for proteins [84, 85], and is now widely used as a platform technology for large scale manufacturing of therapeutic proteins [86]. Various salts have been used for binding, including AS, NaCl, phosphate, and citrate [87-89]. Amino acids (e.g. glycine), polyols (ethylene glycol and glycerol), and sugars (sucrose) have been shown to modulate protein binding as well as elution [90, 91]. Effective elution of bound proteins can be obtained in the presence of arginine, in a range of 0.5-1 M in concentration, and ethanol [75]. Ligand-affinity chromatography is a convenient way to purify proteins, as purification can be conducted in a single step. Ligands encompass dyes, substrates, inhibitors, and many other small molecules, which specifically bind target proteins. Co-solvents are often used for elution. Figure 7 shows the elution profile of lactate dehydrogenase (LDH) bound to a bluedye column [92]. 2M NaCl (first panel) was ineffective in eluting the protein, resulting in a skewed elution peak with protein recovery less than 60%. On the other hand, 0.25M arginine addition resulted in a sharp elution profile, with a higher recovery of ~65% (not shown). Further increasing the arginine concentration to 2M resulted in nearly quantitative recovery of the bound LDH (second panel). It appears that arginine is highly effective in eluting the bound LDH, more so than NaCl. Polyclonal antibodies are versatile reagents and have been used for the detection of therapeutic markers in a wide variety of diagnostic assay formats. The polyclonal antibodies used for such applications must be specific for their target antigens. Specific antibodies can readily be purified by antigen-affinity chromatography [93]. The bound antibodies are eluted under acidic conditions, but often result in low recovery [94]. This is due to the high affinity of the antibodies for antigens, arising from a multitude of interaction forces [95, 96]. While there are various elution conditions available for antigen chromatography, such as different

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elution buffer pH (acid or alkaline elution), high salt concentrations, and different types of buffer salt, the most significant development in solvent manipulation (i.e., to disrupt antibody-antigen interactions) is the use of MgCl2 at high concentrations. Potts and Vogt [62] have applied various chaotropic agents to dissociate sarcoma virus kinase (which was used to generate the antibodies) from antibody columns developed against this kinase. Of the seven solvents tested (urea, guanidine HCl, ethylene glycol, MgCl2, NaSCN, formic acid, and NH4OH), urea, MgCl2, NaSCN, and formic acid demonstrated substantial elution of the kinase. Among these four reagents, urea and formic acid destroyed the kinase activity. Although the kinase was effectively eluted with 1.9-2.5M MgCl2, higher concentrations of MgCl2 caused partial loss of activity. Durkee et al. [97] showed that the Russell’s viper venom factor X (RVV-X) can be purified by antibody-conjugated columns using 3.5M MgCl2 at pH 7.0 [97]. Narhi et al [98-100] used ligand-columns to purify the different monoclonal antibodies raised against the ligand, and demonstrated that low pH elution was generally ineffective and the use of denaturing co-solvents resulted in greater elution. Protein-A/G affinity chromatography is the most convenient purification process for mAb (monoclonal antibody) production [101, 102]. This affinity chromatography is a platform technology for commercial scale production of therapeutic antibodies. The binding of ProteinA with the Fc-region of an antibody is highly selective, such that the majority of contaminating proteins in the conditioned media is eluted from the column, resulting in increased capacity (i.e., selectivity) of the column for antibodies. The interaction between the Fc-region and Protein-A or Protein-G is not only selective, but also very strong that a harsh elution condition is often required. While the elution of antibodies bound to Protein-A or Protein-G resin using an acidic solution is a common practice, strong acid can cause antibody denaturation [103]. Co-solvent arginine and arginine derivatives incorporated at 0.3-2M were found to be effective in eluting the antibodies without demonstrating any deleterious effects on their structural integrity [104, 105].

Figure 8. Spectrum of SNase in 50% methanol. The native structure was obtained at pH7.0 while the unfolded structure was obtained at pH2.0. The intermediate structure was obtained by raining the pH2.0 sample to pH7.0 at -40°C.

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Figure 9. Folding of SNase in 50% methanol at pH7.0. Unfolded SNase prepared at pH2.0 was titrated to pH7.0 to initiate folding at different temperatures, as indicated in the figure.

Figure 10. Illustration of proton exchange reaction taking place on a protein. Readily accessible proton is indicated by the open circle and the open square represents a proton that undergoes less efficient hydrogen-deuterium (H/D) exchange. Filled symbols (i.e., black circle and square) represent protons that have been exchanged to deuterium.

Both the antigen and Protein-A/G columns bind target proteins with high affinity, making elution extremely difficult and often requiring the use of denaturing or destabilizing cosolvents. The strong binding occurs by two mechanisms, multi-mode interactions (electrostatic and hydrophobic) and specific interactions, resulting from the complementary structures of ligand and target proteins. Columns possessing only the former property have been developed to offer high affinity with mild elution property. Even with reduced affinity,

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the use of co-solvents is still often required to reduce both electrostatic and hydrophobic interactions.

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D. Sub-Zero Temperature Enzymology Protein folding of globular proteins, with some exceptions, and enzyme reactions are often too fast to follow at physiological temperature. As the rate of chemical reactions is decreased with lowered temperature, it is greatly reduced at sub-zero temperatures. To avoid freezing, such experiments require the presence of organic solvents to lower the freezing point of solution. Fink and Douzou extensively studied both protein folding and enzyme kinetics at sub-zero temperatures [106-111]. A number of organic solvents have been examined, including ethylene glycol, glycerol, dimethyl formamide, DMSO, and alcohols, however, certain organic solvents can destabilize proteins, so care must be taken when choosing the appropriate solvent system [23, 29-38]. For example, DMSO has been reported to denature (unfold) proteins above 40% at room temperature [23]. However, Staphylococcal nuclease (SNase) was demonstrated to retain the native structure at pH 7.0 even in 50% methanol: melting temperature (Tm) at neutral pH decreased from ~55°C in the absence of methanol to ~15°C in 50% methanol [111]. While the stability is greatly decreased (as evidenced by the lowered Tm), the native structure is still retained below 10°C in 50 % methanol at pH 7.0. Upon lowering the pH to 2.0, however, SNase denatured, even near 0°C. Figure8 shows the structure of SNase in 50% methanol at pH 2.0 (unfolded, U) and at pH 7.0 (native, N). Fluorescence peak is observed at 350 nm when unfolded and at 330 nm when folded. The protein is helical in the native state and disordered at pH 2.0 (U), as shown by CD. These spectra were obtained at 0°C. To initiate folding, the unfolded sample at pH 2.0 was adjusted to pH 7.0. The time course of folding (Figure 9) was followed by monitoring the fluorescence intensity at 330nm (see Figure 8). It is evident that folding is greatly slowed down at low temperatures, suggesting that the folding intermediate may be observed under these experimental conditions. Figure 8 shows the fluorescence and CD spectra of the intermediate structure at –40°C. The fluorescence peak is already shifted to 330 nm, indicating that the native-like structure is formed. The lower intensity suggests that SNase must undergo further structural rearrangement. CD spectrum is consistent with the observation that folding has occurred, though not fully native. It appears that more helices are formed in the intermediate state. Such overshooting of the helical formation has been observed from the rapid kinetic experiment using stopped-flow technique.

E. Application of the Organic Solvent for NMR Studies Hydrogen/deuterium (H/D) exchange methodology for protein structure analysis is useful not only for the elucidation of the secondary structure of proteins [112], but also for the identification of interfacial regions between two domains in a complex structure [113]. The technique measures the solvent-accessible exchangeable protons by hydrogen-deuterium exchange (H/D exchange). Figure 10 shows an example of a protein with two exchangeable protons (depicted by open circle and square). The open circle represents a more accessible

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Satoshi Ohtake, Yoshiko Kita, Chiaki Nishimura et al.

proton than the one indicated by the open square. A short exposure of the protein to D2O leads to a complete exchange by deuterium (D, filled symbol) for the accessible proton. If the measurement time is faster than the exchange rate for the less accessible proton, then it is possible to follow the time course of the exchange. Recently developed NMR technique (e.g., SOFAST) [114] made such a fast acquisition of 2D-NMR data feasible. Two-dimensional 1H15 N correlation spectra can now be obtained within 2-3 s using this pulse-sequence mode technique. While this measurement mode is fast enough to follow the time course of proton exchange, the time requirement for sample preparation, i.e., sample mounting and optimizing measurement parameters, may deter the use of this NMR technique. Alternatively, kinetics can be followed if the H/D exchange can be halted. The best approach involves the exchange of water with organic solvents, which contain no exchangeable protons. As described above, organic solvents are protein precipitants (and denaturants). Although no H/D exchange may occur, proteins must be sufficiently soluble in the chosen organic solvent. The 15N-labeled apomyoglobin was used to test its solubility in a wide variety of solvent systems. Several solvents, none of which contained exchangeable protons, were examined in the study, and included chloroform, acetone, acetonitrile, diethyl ether, dioxane, benzene, and DMSO. For comparison, alcohols that possessed exchangeable protons, e.g., ethanol, methanol and butanol, were also tested. The protein solution was quench-frozen in liquid nitrogen to stop the exchange reaction instantaneously, and then extensively dried to remove as much water as possible, as any residual water can cause artificial (post-reaction) exchange. The lyophilized protein was not soluble in benzene, chloroform, diethyl ether, and dioxane, and was slightly soluble in acetone, and more so in acetonitrile and several alcohols. Strikingly, the lyophilized apomyoglobin was readily soluble in DMSO [115], consistent with the ability of this organic solvent to dissolve a variety of compounds [35]. Next, the 1H-15N HSQC spectra were acquired for each solvent with apomyoglobin, as described above. In alcohols and actonitrile, the signals were not consistent with the number of amino acids in the protein: the number of signals was ~60 for methanol and ~30 for ethanol, whereas the putative number is 153. This suggests that most of the peaks overlapped with each other under the denaturing condition of organic solvents [116]. Another possibility is the existence of fluctuation in the protein backbone structure on the same time scale as that used in NMR measurements [117]. Subsequently, many signals disappear due to the exchange broadening. However, surprisingly, about 150 peaks were successfully observed in the presence of DMSO [115]. Although the protein is unfolded in DMSO, many peaks were observed clearly and separately in the high field NMR. The observed peaks reflect the average of different assembly of unfolded structures within the NMR time scale [118]. Acetonitrile gave no interpretable NMR spectra by itself, but generated interpretable spectra in the presence of DMSO, which may be again due to the ability of DMSO to dissolve proteins. The expanded denatured state for proteins has been shown in 60% methanol at pH 2 [119]. The high solubility and unfolded structure of dried protein in DMSO thus can be applied for the analysis of H/D exchange kinetic experiment [115, 120]. First, the H/D exchange sample was quench-frozen in liquid nitrogen, which immediately stopped the H/D exchange reaction. No H/D exchange should occur in the subsequent drying and DMSO dissolution steps. However, the H/D exchange did occur, though slowly, during the NMR data acquisition. It may be possible that residual water remained after lyophilization or that moisture was acquired from air. To mitigate the effects of artificial H/D exchange, the pH of

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the DMSO co-solvent system can be optimized [121]. Under the optimized condition, it is possible to analyze the structure and conformation of protein-protein or protein-ligand complexes, as well as the intermediate structures involved in protein folding/unfolding [122].

F. Powder Enzyme Reaction in Organic Solvent: Low Water Activity

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Many enzymes, except for those present in membranes, function in aqueous solution and use water-soluble substrates. For this reason, the vast knowledge of enzyme reaction mechanism has been accumulated in aqueous systems. Although poorly water-soluble substrates cannot be utilized in aqueous solutions, water-soluble enzymes can function in organic solvents. Enzyme reaction in organic solvents offers two advantages: unique substrate specificity utilizing non-polar substrates and higher thermal stability [4, 123-127]. There are two ways to allow water-soluble enzymes to function in organic solvents. One is to lyophilize the enzymes, followed by their suspension in organic solvents [4, 123-127]. In this case, the enzymes are not dissolved in the organic solvent, but rather are suspended as particulates. Second approach is to chemically conjugate the enzymes to a resin, which helps the enzymes disperse as a monomer, followed by dehydration [128, 129]. The resin is then suspended in an organic solvent. As the water activity is low, hydrolysis is not a favorable reaction under this condition. There are several unique properties of enzymes suspended in organic solvents: 1) Requirement of water: Small amount of water is essential for enzyme activity. Enzymes are typically active only in the presence of water, and their behavior in a co-solvent system comprised of organic solvents differs greatly depending on their polarity: Enzymes are more active in non-polar, water-immiscible organic solvents than in those that are water-miscible [3]. In fact, when the solvent becomes more polar, the enzyme activity decreases, and in organic solvents that are fully miscible with water, the enzyme is completely inactivated. This can be interpreted as follows: in non-polar organic solvents, small amount of water is trapped by the protein surface and is available for the enzyme to maintain its flexibility/mobility for function. Without the protein-bound water molecules, the enzyme cannot function. When polar solvents are used, the water molecules are solvated by the organic solvent and are no longer tightly bound by the protein. Addition of water to this system can restore activity, but may cause protein denaturation [3, 23, 29-38]. Any of these organic solvents are capable of denaturing the enzymes, as described above. Interestingly, enzymes are extremely stable in non-polar organic solvents [3]. This was interpreted in terms of kinetics of unfolding [3]. If unfolded dried protein is dispersed in a nonpolar organic solvent, it will never be refolded to the active structure. In equilibrium, the unfolded structure is more stable in organic solvents, regardless of the polarity. In a non-polar organic solvent, the protein molecule is so rigid that the conformation cannot be readily altered, in contrast to the protein structure in an aqueous solution. Proteins are fluctuating in aqueous solution between many different conformations, some of which are more unfolded than the others, and are kinetically converted to the unfolded structure. Such a structural fluctuation is less likely to occur in non-polar organic solvents, suggesting the importance of water in lowering the kinetic barrier between different conformations.

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Satoshi Ohtake, Yoshiko Kita, Chiaki Nishimura et al. 2) pH memory: Another interesting aspect of enzymes suspended in a non-polar organic solvent is pH memory [2, 3]. When the enzyme is formulated in a non-volatile buffer, dried, and then suspended in a non-polar organic solvent, one will find that the activity of the resulting enzyme is dependent on the original pH of the formulation; if the enzyme is formulated at an optimal pH and then dried, the enzyme in the organic solvent demonstrates the highest activity. On the other hand, if the enzyme is formulated at a sub-optimal pH followed by dehydration, it results in suboptimal activity upon suspension in an organic solvent. The enzyme appears to retain the charged state in the formulation during processing and resuspension. This is reasonable, as the amount of water is so miniscule that without any additional electrolytes, no alteration in the charged state can occur upon suspension. However, such pH memory disappears when volatile buffers are used [130], in which case a new charged state will be established during drying and after suspension in an organic solvent.

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G. Protein Precipitation An important application of co-solvent systems is protein precipitation. Since the discovery of the Hofmeister series [21], differential precipitation by salting-out ammonium sulfate has been the most simple, yet effective means to reduce the sample volume and fractionate the proteins. While this process is useful for protein purification, it has not been practical for plasma fractionation, which is one of the most important life-saving technologies available today [39]. A critical parameter for plasma-derived products is the determination of viral load, or viral safety [131]. While ammonium sulfate may be effective in plasma fractionation, it is not effective in virus inactivation. In fact, it may even stabilize viruses, as described later for the application of Na2SO4. Organic solvents that are effective in virus inactivation (in particular for enveloped viruses) have been the choice of agents for plasma fractionation [132]. Based on Mellanby’s observation of cold ethanol-induced fractionation of horse serum [133], Cohn developed a method for conducting differential precipitation of human plasma proteins by cold ethanol, which met the critical need of blood-derived products during World War II [39]. This process consists of successive addition of 10-40% ethanol at low temperature (i.e., –3 to –6˚C), while modulating the pH and ionic strength of the system, resulting in fractionation of albumin, antibodies, coagulation factors, and protease inhibitors simultaneously with certain level of virus inactivation. Although other water-miscible organic solvents may be used, ethanol is safe to humans and is not strongly denaturing to proteins. Care must be used with ethanol, however, as it can be denaturing at elevated temperatures. Organic solvents are also used to induce protein crystallization. Obtaining high quality crystals is critical to the successful determination of protein structure by x-ray crystallography. Salting-out salts, PEG, and organic solvents have been used to induce crystallization. Among them, MPD is a strong protein precipitant, although at elevated temperatures, it is a protein destabilizer [23, 38, 42]. As described earlier in the review, such effectiveness may derive from the strong repulsion of MPD from the charged protein surface, as evidenced by the phase separation of MPD upon the addition of ions.

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H. Virus Inactivation and Processing

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As mentioned above, co-solvent applications for viruses have been examined from two opposite spectrums; one in which virus inactivation is targeted and the other in which virus purification is desired. Virus inactivation may be desired in applications such as purification of recombinant proteins and antibodies, during which virus inactivation takes place concurrently as protein purification, and surface disinfection. Virus purification is employed in situations for which the identification of the level of contamination (and the species involved) is required, and in the application of live viruses, or virus components, as a vaccine.

Virus Inactivation Removal of contaminant products, such as viruses, from cell culture media containing recombinant proteins, represents an integral step in the production and purification of therapeutic proteins. Typical processing condition employs high temperature incubation and perhaps low pH, but these conditions can also be detrimental to the stability of the freshly prepared proteins. Arginine, employed at high concentrations (i.e., ~1M), have been demonstrated to lower the temperature, as well as raise the pH, at which virus inactivation takes place, thus reducing the risk of degrading the protein being purified concurrently [134, 135]. Herpes simplex virus type 1 (HSV-1) in the presence of 1.2M arginine has been reported to be inactivated at approximately 40°C, while in its absence, complete inactivation was accomplished only at higher temperatures (>50°C) [134]. Significant reduction in viral titer (i.e., >5.7 Log reduction) has been obtained in the presence of 1M arginine at pH4.3, while in its absence, similar reduction was obtained only under more acidic condition (pH 3.5) [135]. Thus through the addition of arginine at a relatively high concentration, the virus inactivation procedure can be conducted at a milder processing condition (i.e., less acidic pH and lower temperature). The addition of salts has also been reported to be effective in inactivating viruses. The JES strain of herpesvirus was inactivated by heat treatment in distilled water (15min at 50°C), resulting in 2 Log10 decrease in titer (PFU/mL). Inactivation was further enhanced to >6Log10 decrease in titer upon the addition of 1M MgCl2 (136). Addition of 1M Na2SO4, however, improved the stability of viruses to heat inactivation (0.1Log decrease in titer), thus the choice of salt must be made carefully. Although these salt effects appear to agree with their effects on the stability of proteins (i.e., MgCl2 being destabilizing and Na2SO4 stabilizing), the salt effects are much more variable on viruses than on proteins. Similarly, the inactivation of type I Inoue-Melnick virus, infectious bronchitis virus, measles virus (Edmonston strain), influenza, parainfluenza, vesicular stomatitis, and rubella, was enhanced upon the addition of 1M MgCl2, whereas it was decreased (i.e., stabilized) upon the addition of 1M Na2SO4 upon heat treatment at 50°C [137-140]. In general, enveloped viruses are inactivated by the addition of molar quantities of MgCl2 (salting-in salt), while a wide variety of non-enveloped viruses, such as poliovirus and coxsackievirus, are stabilized by MgCl2 [140, 141]. This suggests a difference in the interaction of the salt with the viral membrane components (i.e., lipids, proteins, carbohydrates, etc.) and with proteins alone, as is the case for non-enveloped viruses. Furthermore, enveloped viruses that are susceptible to MgCl2 are also susceptible to inactivation by KCl and NaCl, but at twice the effective concentration of MgCl2 [136, 137], suggesting the importance of salt solution osmolality (or ionic strength) on the stability of these viruses.

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Common cleaning reagents used to disinfect surfaces typically contain alcohol, and is the product of years of extensive research conducted in examining the efficacy of alcohol in inactivating a wide variety of viruses. Moorer reported a short inactivation time (20sec) required for disinfecting enveloped viruses, HIV, BVDV (bovine viral diarrhea virus), and pseudorabies virus, all with very high clearance factors of at least 6.5 (clearance factor = Log10 (total amount of virus added/total amount recovered from the treated sample)) with 80% ethanol containing 5% iso-propanol [142], while 80% isopropyl alcohol was demonstrated to be effective in reducing the titer of both type 1 and type 2 herpes simplex virus to 6.0 >6.0 >6.5 >6.3 ND 5.0 6.4 4.6 0.3 0.3 4.1 0.0

GnSCN 6M 5.7

Urea 8M >4.8

EtOH 20% 5.5

EtOH 70% >5.8

>4.8 >5.4 >4.0

>7.1 >5.6 4.3

>7.0 6.6 0.1

>7.3 >5.6 3.7

The values shown in the table under each reagent represents the Log10 decrease in virus titer. Adapted from reference 144.

Ethanol is also utilized in the purification of major plasma components, as in cold ethanol fractionation. Besides being used as the precipitating agent, ethanol is also employed due to its bactericidal and virucidal properties. Typically, the highest concentration of ethanol used in the fractionation process is 40% and much lower, around 10-25%, in other stages. HIV-1

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was completely inactivated from an initial titer of 8.5Log10 TCID50 to undetectable levels within 2h, and rapid inactivation of HIV-1 was achieved during the first 10min of treatment under 40% ethanol, pH6.0, -5.5°C [150].

Virus and Plasmid Processing/Purification Efficient purification of respiratory syncytial virus (RSV) was conducted using an affinity resin column (MatrexTM CellufineTM Sulfate (MCS) column) in place of the traditional density gradient centrifugation method [151-153]. In a single pass through the column, viable RSV was purified from crude cell lysate. Enveloped viruses, such as RSV, are bound by phospholipid membranes and are particularly difficult to purify due to their lower density and fragility compared to naked viruses. Effective eluent for RSV was found to be 0.8M NaCl at pH7.2. Several other salts, besides NaCl, were examined in order to determine the effect of elution buffer’s counterion specie, ionic strength, and osmolality on the efficiency of virus purification. MgSO4 was ineffective in eluting RSV at either equivalent ionic or osmotic strength, however, Na2SO4 was equally as effective as NaCl under either condition (See table below, Table 2). The former two salts have a qualitatively similar effect on proteins, e.g., stabilizing and salting-out [28], signifying the complex surface properties of viruses that interact with the column resin.

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Table 2. The effect of ionic strength and osmolality on the elution of respiratory syncytial virus from an affinity resin column, MatrexTM CllufineTM Sulfate (MCS) Salt Equivalent Ionic strengtha MgSO4 Na2SO4 MgCl2 Equivalent osmolalitya MgSO4 Na2SO4 MgCl2

Concentration (M)

% elution

0.24 0.32 0.32

24 100 68

1.44 1.0 0.58

35 100 100

a

based on effective concentration of NaCl (0.8M). The salts examined include, MgSO4, Na2SO4, MgCl2, and their concentrations are indicated in the table. Table adapted from reference 151.

Similarly to viruses, which can be used as a vehicle for targeted therapy, plasmids, which have been studied for their use in gene delivery, are also purified by co-solvents. A typical process of plasmid production begins with the culture of transformed E.coli followed by alkaline lysis of the harvested bacteria. Following selective precipitation and concentration of the genetic material of the cell lysate, plasmids can be purified by ion-exchange chromatography, using co-solvents to modulate the binding affinity (i.e., charge-charge interaction) of the various components. Elution buffer containing higher than 45% isopropanol was effective in separating plasmids from RNA and proteins, while at lower concentrations, separation through an anion exchange Q-sepharose was ineffective [154]. The authors suggested that the role of the co-solvent, isopropanol, was to modify the dielectric constant, which had a profound impact on the binding strength between both RNA and

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Satoshi Ohtake, Yoshiko Kita, Chiaki Nishimura et al.

plasmid DNA to the resins, but to different extents: the addition of isopropanol in column buffer will decrease the dielectric constant and hence enhance the overall electrostatic interactions, whether attractive or repulsive. The interaction strength between RNA and the resins increased less than that between DNA and the resins, resulting in efficient separation between those two components. Another alcohol, ethanol, at high concentrations (i.e., >80%) has been reported to lead to aggregation of DNA [155-157], thus care must be taken when using the alcohol co-solvent system for DNA purification: whether such aggregation is due to the repulsion between the surface charges of DNA and alcohol, as is observed for proteins, is an intriguing question. However, the presence of certain metal salts has been reported to modulate the ethanol content at which aggregation occurs [158], suggesting the strong interplay between the compositions present in the co-solvent system on the efficiency of purification. Another solvent that demonstrated efficacy in the purification of plasmid DNA is PEG (polyethylene glycol). Humphreys, et al. demonstrated that 10% PEG6000 can precipitate DNA present in lysates of E.coli carrying plasmids of a wide range of molecular weight (i.e., 6 – 123 x 106) (160). DNA precipitation was rapid (90% completion within 2h at 4°C), and in comparison to other purification methodologies, PEG precipitation was fairly gentle and caused no changes in the biological activity of the purified plasmids; E.coli K12 strain JC7623 was transformed at similar frequencies by plasmid DNA isolated with or without a PEG precipitation step. PEG precipitation method can also be used to separate the DNA fragments based on the size of the base pairs by modifying the amount of PEG present as a co-solvent. Lis reported that the minimally required concentration of PEG in general is less for larger and/or more anisometric structures [160]. Addition of 5% PEG caused precipitation of DNA fragments of Drosophila melanogaster larger than 1650 bp, and smaller fractions were collected by increasing the PEG concentration further. In another application, Sauer, et al. used 10% PEG8000 in the presence of 250mM NaCl to selectively precipitate plasmid DNA (5369 bp) maintained in E. coli DH5, which was previously processed by selective precipitation of high molecular weight RNA from the cleared lysate with 1.4M CaCl2 [161]. The main advantage of the PEG precipitation method is that it does not require the use of organic solvents nor does it require the use of RNase or spermidine, which can bind RNA and DNA indiscriminately. Another co-solvent system that can be used to purify closed circular DNA is the acidphenol extraction system. Phenol (present at 50%) has been demonstrated to selectively extract DNA species other than the covalently closed DNA at acidic pH and low ionic strength. Zasloff, et al. have reported on a number of systems that greater than 95% of the nicked circular species, and linear DNA molecules greater than about 1500bp, are cleared from the water phase (containing 50mM sodium acetate, pH4.0) [162]. After 3 extractions, more than 99% of the contaminants have been removed, while closed circular DNA remained in the water phase.

CONCLUSION Co-solvents modulate various properties of proteins, nucleic acids, and viruses. They are used to stabilize (or destabilize), precipitate, or solubilize proteins depending on the intended

Organic Solvents: Properties, Toxicity, and Industrial Effects : Properties, Toxicity, and Industrial Effects, edited by Ryan E. Carter, Nova Science

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application. Similarly, they are used to purify viruses and plasmids, and in certain cases, to inactivate them. Care must be taken in their use, as a slight change in their concentration, environmental condition (pH, temperature), or in the presence of other components (salts) can suddenly change their stabilizing effect to one in which macromolecules are destabilized. Furthermore, co-solvent systems that are stabilizing for one system (i.e., proteins) are not necessarily stabilizing for other systems (i.e., viruses), suggesting a highly selective and sensitive interaction between co-solvents and macromolecules. As indicated in the introduction, there are several other applications that have not been included in this review, partly because of our limited knowledge and experience. Nevertheless, we hope that the present review on the application of co-solvent systems and their underlining mechanism would help the readers understand the importance and utility of co-solvents in their work on biological macromolecules.

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[113] Mandell, JG; Baerga-Ortiz, A; Falick, AM; Komives, EA. Measurement of solvent accessibility at protein-protein interfaces. Methods Mol. Biol., 2005, 305, 65-80. [114] Schanda, P; Forge, V; Brutscher, B. Protein folding and unfolding studied at atomic resolution by fast two-dimensional NMR spectroscopy. Proc. Natl. Acad. Sci. USA., 2007, 104, 11257-11262. [115] Nishimura, C; Dyson, HJ; Wright, PE. Enhanced picture of protein-folding intermediates using organic solvents in H/D exchange and quench-flow experiments. Proc. Natl. Acad. Sci. USA., 2005, 102, 4765-4770. [116] Yao, J; Dyson, HJ; Wright, PE. Chemical shift dispersion and secondary structure prediction in unfolded and partly folded proteins. FEBS Lett., 1997, 419, 285-289. [117] Eliezer, D; Wright, PE. Is apomyoglobin a molten globule? Structural characterization by NMR. J. Mol. Biol., 1996, 263, 531-538. [118] Schwarzinger, S; Wright, PE; Dyson, HJ. Molecular hinges in protein folding: the ureadenatured state of apomyoglobin. Biochemistry, 2002, 41, 12681-12686. [119] Kamatari, YO; Ohji, S; Konno, T; Seki, Y; Soda, K; Kataoka, M; Akasaka, K. The compact and expanded denatured conformations of apomyoglobin in the methanolwater solvent. Protein Sci., 1999, 8, 873-882. [120] Nishimura, C; Dyson, HJ; Wright, PE. Energetic frustration of apomyoglobin folding: role of the B helix. J. Mol. Biol., 2010, 396, 1319-1328. [121] Zhang, YZ; Paterson, Y; Roder, H. Rapid amide proton exchange rates in peptides and proteins measured by solvent quenching and two-dimensional NMR. Protein Sci., 1995, 4, 804-814. [122] Dyson, HJ; Kostic, M; Liu, J; Martinez-Yamout, MA. Hydrogen-deuterium exchange strategy for delineation of contact sites in protein complexes. FEBS Lett., 2008, 582, 1495-1500. [123] Kazandjian, RZ; Klibanov, AM. Regioselective oxidation of phenol catalyzed by polyphenol oxidase in chloroform. J. Am. Chem. Soc., 1985, 107, 5448-5450 [124] Dordick, JS. Enzymatic catalysis in monophasic organic solvents. Enzyme Microb. Technol., 1986, 11, 194-211. [125] Zaks, A; Klibanov, AM. Enzyme catalysis in organic solvents at 100 C. Science, 1984, 224, 1249-1251. [126] Sakurai, T; Margolin, AL; Russell, AJ; Klibanov, AM. Control of enzyme enantioselectivity by the reaction medium. J. Am. Chem. Soc. USA., 1988, 110, 72367237. [127] Klibanov, AM. Asymmetric transformations catalyzed by enzymes in organic solvents. Acc. Chem. Res., 1990, 23, 114-120. [128] Blanco, RM; Guisan, JM; Halling, PJ. Agarose-chymotrypsin as a catalyst for petide and amino acid ester synthesis in organic media. Biotechnol. Lett., 1989, 11, 811-816. [129] Blanco, RM; Rakels, JLL; Guisan, JM; Halling, PJ. Effect of thermodynamic water activity on amino-acid ester synthesis catalyzed by agarose-chymotrypsin in 3pentanone. Biochim. Biophys. Acta, 1992, 1156, 67-70. [130] Zacharis, E; Halling, PJ; Rees, DG. Volatile buffers can override the ―pH memory‖ of subtilisin catalysis in organic media. Proc. Natl. Acad. Sci. USA., 1999, 96, 1201-1205. [131] Brorson, K; Norling, L. Current and future approaches to ensure the viral safety of biopharmaceuticals. Dev. Biol., 2004, 118, 17-29.

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[132] Burnouf, T. Chromatography in plasma fractionations: benefits and future trends. J. Chromatogr. B., 1995, 664, 3-15. [133] Mellanby, J. The precipitation of the proteins of horse serum. J. Physiol., 1907, 36 (45), 288-333. [134] Utsunomiya, H; Ichinose, M; Tsujimoto, K; Katsuyama, Y; Yamasaki, H; Koyama, AH; Ejima, D; Arakawa, T. Co-operative thermal inactivation of herpes simplex virus and influenza virus by arginine and NaCl. Int. J. Pharm., 2009, 366, 99-102. [135] Yamasaki, H; Tsujimoto, K; Koyama, AH; Ejima, D; Arakawa, T. Arginine facilitates inactivation of enveloped viruses. J Pharm Sci., 2008, 97(8), 3067-3073. [136] Wallis, C; Melnick, JL. Thermostabilization and thermosensitization of herpesvirus. J. Bacteriol., 1965, 90(6), 1632-1637. [137] Nishibe, Y; Inoue, YL; Melnick, JL. Thermostabilization of Inoue-Melnick virus by salt. J. Med. Virol., 1986, 20, 105-109. [138] Hopkins, SR. Thermal stability of infectious bronchitis virus in the presence of salt solutions. Avian Dis., 1967, 22, 261-267. [139] Rapp, F; Butel, JS; Wallis, C. Protection of measles virus by sulfate ions against thermal inactivation. J. Bacteriol., 1965, 90(1), 132-135. [140] Wallis, C; Melnick, JL; Rapp, F. Different effects of MgCl2 and MgSO4 on the thermostability of viruses. Virology, 1965, 26, 694-699. [141] Wallis, C; Melnick, JL. Cationic stabilization – a new property of enteroviruses. Virology, 1962, 16, 504-506. [142] Moorer, WR. Antiviral activity of alcohol for surface disinfection. Int. J. Dent. Hygiene, 2003, 1, 138-142. [143] Croughan, WS; Behbahani, AM. Comparative study of inactivation of herpes simplex virus types 1 and 2 by commonly used antiseptic agents. J. Clin. Microbiol., 1988, 26(2), 213-215. [144] Roberts, PL; Lloyd, D. Virus inactivation by protein denaturants used in affinity chromatography. Biologicals, 2007, 35, 343-347. [145] Macinga, DR; Sattar, SA; Jaykus, LA; Arbogast, JW. Improged inactivation of nonenveloped enteric viruses and their surrogates by a novel alcohol-based hand sanitizer. Appl. Environ. Microbiol., 2008, 74(16), 5047-5052. [146] Bidawid, S; Malik, N; Adegbunrin, O; Sattar, SA; Farber, JM. Norovirus crosscontamination during food handling and interruption of virus transfer by hand antisepsis: experiments with feline calicivirus as a surrogate. J. Food Prot., 2004, 67, 103-109. [147] Doultree, JC; Druce, JD; Birch, CJ; Bowden, DS; Marshall, JA. Inactivation of feline calicivirus, a Norwalk virus surrogate. J. Hosp. Infect., 1999, 41, 51-57. [148] Springthorpe, VS; Grenier, JL; Lloyd-Evans, N; Sattar, SA. Chemical disinfection of human rotaviruses: efficacy of commercially-available products in suspension tests. J. Hygiene, 1986, 97, 139-161. [149] Sattar, SA; Springthorpe, VS; Karim, Y; Loro, P. Chemical disinfection of non-porous inanimate surfaces experimentally contaminated with four human pathogenic viruses. Epidem. Inf., 1989, 102, 493-505. [150] Kim, IS; Eo, HG; Park, CW; Chang, CE; Lee, S. Removal and inactivation of human immunodeficiency virus (HIV-1) by cold ethanol fractionation and pasteurization

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during the manufacturing of albumin and immunoglobulins from human plasma. Biotechnol. Bioprocess. Eng., 2001, 6, 25-30. [151] Downing, LA; Bernstein, JM; Walter, A. Active respiratory syncytial virus purified by ion-exchange chromatography: characterization of binding and elution requirements. J. Virol. Methd., 1992, 38, 215-228. [152] Lambert, DM; Pons, MW; Mbuy, GN; Dorsch-Hasler, K. Nucleic acids of respiratory syncytial virus. Virology, 1980, 36, 837-846. [153] Levine, S; Dillman, TR; Montgomery, PC. The envelope proteins from purified respiratory syncytial virus protect mice from intranasal virus challenge. Soc. Exp. Biol. Med., 1989, 190, 349-356. [154] Tseng, WC; Ho, FL. Enhanced purification of plasmid DNA using Q-Sepharose by modulation of alcohol concentrations. J. Chromatography B, 2003, 791, 263-272. [155] Girod, JC; Johnson, WC, Jr.; Huntington, SK; Maestre, MF. Conformation of deoxyribonucleic acid in alcohol solutions. Biochemistry, 1973, 12(25), 5092-5096. [156] Herbeck, R; Yu, TJ; Peticolas, WL. Effect of cross-linking on the secondary structure of DNA I. Cross-linking by photodimerization. Biochemistry, 1976, 15(12), 2656-2660. [157] Lang, D. Regular superstructures of purified DNA in ethanolic solutions. J. Mol. Biol., 1973, 78(2), 247-254. [158] Rupprecht, A; Piskur, J; Schultz, J; Nordensiold, L; Song, Z; Lahajnar, G. Mechanochemical study of conformational transitions and melting of Li-, Na-, K-, and CsDNA fibers in ethanol-water solutions. Biopolymers, 1994, 34(7), 897-920. [159] Humphreys, GO; Willshaw, GA; Anderson, ES. A simple method for the preparation of large quantities of pure plasmis DNA. Biochim. Biophys. Acta., 1975, 383, 457-463. [160] Lis, JT. Fractionation of DNA fragments by polyethylene glycol induced precipitation. Meth. Enzymol., 1980, 65, 347-353. [161] Sauer, ML; Kollars, B; Geraets, R; Sutton, F. Sequential CaCl2, polyethylene glycol precipitation for RNase-free plasmid DNA isolation. Anal. Biochem., 2008, 380, 310314. [162] Zasloff, M; Ginder, GD; Felsenfeld, G. A new method for the purification and identification of covalently closed circular DNA molecules. Nuc. Acids Res., 1978, 5(4), 1139-1152.

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In: Organic Solvents Editor: Ryan E. Carter

ISBN 978-1-61761-881-9 © 2011 Nova Science Publishers, Inc.

Chapter 2

LIPASE-CATALYZED SYNTHESIS OF EDIBLE SURFACTANTS IN MICROAQUEOUS ORGANIC SOLVENTS Yoshiyuki Watanabe1, and Shuji Adachi2 1. Department of Biotechnology and Chemistry, School of Engineering, Kinki University, Higashi-Hiroshima, Japan 2. Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, Japan

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ABSTRACT 6-O-Lauroyl saccharides were synthesized through condensation in acetonitrile, acetone and 2-methyl-2-propanol with various water contents using the immobilized lipase from Candida antarctica. Glucose, galactose, mannose and fructose were used as a hydrophilic substrate. The apparent equilibrium constant, KC, based on the concentrations of substrates and products in acetonitrile could be correlated to the dynamic hydration numbers of the saccharides. This indicates that the water activity played an important role during the condensation reaction in the microaqueous water-miscible solvent. The KC for the synthesis of lauroyl mannose also depended on the kind of solvent and could be correlated with the relative dielectric constant, r, of the organic solvent. Acyl mannoses with the acyl chain lengths of 8 to 16 were continuously produced using a plug-flow-type reactor packed with an immobilized lipase. Irrespective of the acyl chain length, a conversion of more than 0.5 was achieved at the superficial residence time, 0, equal to or longer than 20 min. A long-term operational stability of the enzyme was examined, and a conversion of ca. 40% was maintained for at least 16 days. The productivity was evaluated to be 350 g/L-reactor･day. The surfactant properties of the produced capryloyl, caproyl, lauroyl and myristoyl mannoses were also determined. Three lauroyl phenolic glycosides were synthesized through the condensation of phenolic glycoside such as arbutin, naringin and phloridzin with lauric acid by immobilized lipase in various organic  Author to whom correspondence should be addressed telephone to: +81-82-434-7000; fax: +81-82-434-7890; email: [email protected]. 1 Umenobe, Takaya, Higashi-Hiroshima 739-2116, Japan. Organic Solvents: Properties, Toxicity, and Industrial Effects : Properties, Toxicity, and Industrial Effects, edited by Ryan E. Carter, Nova Science

32

Yoshiyuki Watanabe and Shuji Adachi solvents. The conversion depended on the polarity of the organic solvent used for the reaction. The suppressive effect of each lauroyl phenolic glycoside against the oxidation of linoleic acid was higher than that of the corresponding phenolic glycoside, whereas there was no difference between the radical scavenging activities of unmodified and lauroyl phenolic glycosides. It is suggested that enhancement of the suppressive effect against lipid oxidation is ascribed to the increase in the hydrophobicity of phenolic glycoside by the acylation.

Keywords: Acyl saccharide; Edible surfactants; Immobilized lipase; Microaqueous organic solvent; Packed-bed reactor

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INTRODUCTION Saccharides are hydrophilic components in food. If the hydrophobic and hydrophilic components were bound, the product would be edible surfactant and be usable as a food additive. For example, acyl saccharides have been of much interest for use in many industries not only for food but also for chemicals, cosmetics and pharmaceuticals [1, 2]. There are many reports about their antimicrobial properties [3, 4]. They have been produced on an industrial scale based on chemical procedures. Their syntheses through lipase-catalyzed transesterification [5-9] or condensation [5, 10-25] reaction would have some advantages; the simplicity of its reaction process, the moderate reaction conditions, and high regioselectivity of the enzyme. Because the lipase-catalyzed reaction in conventional aqueous system thermodynamically favors the hydrolysis, the reaction has been carried out in an organic medium with low water content [11, 12, 17, 18, 20-22], in a solvent-free system [5, 15, 19] or under reduced pressure and/or in the presence of a dessicant [12, 14] to shift the reaction equilibrium toward synthesis. Especially, lipase-catalyzed reactions in organic solvent have been examined by many researchers [26]. This study deals with the synthesis of acyl saccharides through the condensation of hexose and saturated fatty acid by immobilized lipase. The reaction equilibrium constant is an important parameter to predict the equilibrium conversion, but has scarcely been reported for the synthesis of acyl saccharides in microaqueous organic solvents. Although a batch reactor has mainly been used for the lipase-catalyzed synthesis of acyl saccharides, a packed-bed reactor would be preferred for a large-scale production. Furthermore, acyl saccharides are surfactants with good emulsifying properties [14, 27], but the surface-active properties of acyl mannoses have not been available. In Section 1, the apparent equilibrium constant, KC, based on the concentrations of substrates and products for the lipase-catalyzed synthesis of some lauroyl saccharides in various water-miscible organic solvents with different water contents were estimated, and the effects of the kind of hexose and solvent on the KC value were evaluated [28, 29]. In Section 2, a continuous production of 6-O-acyl mannoses through the immobilized-lipase-catalyzed condensation of mannose and saturated fatty acids in dehydrated 2-methyl-2-propanol using a packed-bed reactor was examined [30]. Surface tensions in aqueous solution of the products were measured, and their surfactant properties were determined. In Section 3, three lauroyl phenolic glycosides were synthesized through the condensation of arbutin, naringin or phloridzin with lauric acid by an immobilized lipase in various organic solvents, and the

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Lipase-Catalyzed Synthesis of Edible Surfactants in Organic Solvents

33

effect of a solvent on the conversion was examined [31]. The antioxidative activity of each lauroyl phenolic glycoside against lipid oxidation and the radical scavenging activity were also measured to evaluate the availability of acyl phenolic glycoside as an additive for cosmetic and food industry.

HO

CH2OH O OH

OH

HO

OH D-Glucose

HO

OH 6-0-Lauroyl D-glucose

CH2OH O OH

HO OH

OH

+ CH3(CH2)10 COOH Lauric acid

CH2OH O OH HO

OH

D-Mannose

HOCH2

O HO

CH2OCO(CH2)10CH3 O OH OH

OH 6-0-Lauroyl D-galactose

D-Galactose

HO

CH2OCO(CH2)10CH3 O OH OH

Lipase HO

CH2OCO(CH2)10CH3 O OH OH HO

+ H2O

6-0-Lauroyl D-mannose CH2OH OH

OH -D-Fructose

CH3(CH2)10COOCH2

O

CH2OH

HO

OH OH 6-0-Lauroyl -D-fructose

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Figure 1. Reaction scheme of lipase-catalyzed condensation of various hexoses with lauric acid.

1. EQUILIBRIUM CONSTANT FOR LIPASE-CATALYZED CONDENSATION OF SACCHARIDE AND LAURIC ACID IN WATERMISCIBLE ORGANIC SOLVENTS IN A BATCH REACTION Figure 1 shows the reaction scheme of the regioselective condensation of hexoses and lauric acid. In this section, the apparent equilibrium constant, KC, for the lipase-catalyzed synthesis of some lauroyl saccharides (hexoses) in various water-miscible organic solvents with different water contents were estimated, and the effects of the kinds of hexose and solvent on the KC value were evaluated. In advance, the organic solvent, such as acetonitrile, acetone, 2-methyl-2-propanol, and 2-methyl-2-butanol, was dehydrated over molecular sieves 5A. A specified amount of water was added to the dehydrated solvent to adjust the initial water content of the solvent to a desired level, which was measured by Karl Fischer titration. Hexose (0.25 mmol), which was D(+)-glucose, D(+)-galactose, D(+)-mannose, or D(-)-fructose, lauric acid (1.25 mmol), and the immobilized lipase (100 mg) from Candida antarctica were placed in a vial. Five milliliters of the solvent was then added to dissolve or disperse the substrates. The vial was tightly screw-capped and then immersed in a thermo-regulated water bath at 50oC. A portion of the reaction mixture was removed at appropriate intervals, and the transient changes in

Organic Solvents: Properties, Toxicity, and Industrial Effects : Properties, Toxicity, and Industrial Effects, edited by Ryan E. Carter, Nova Science

Yoshiyuki Watanabe and Shuji Adachi

34

conversion were observed for some condensations using HPLC with an ODS column (4.6 mmx 300 mm) and a refractometer. The eluent used was a mixture of acetonitrile and water (80/20 by vol.) for lauroyl hexoses synthesized in acetonitrile (the flow rate: 0.8 mL/min), of acetonitrile and water (70/30 by vol.) for lauroyl fructose synthesized in acetone and 2methyl-2-propanol (the flow rate: 1.0 mL/min), or of acetonitrile and water (65/35 by vol.) for lauroyl mannose synthesized in acetone, 2-methyl-2-propanol and 2-methyl-2-butanol (the flow rate: 0.8 mL/min). The calibration curves were prepared using the products isolated from the reaction mixture according to the reported method [21] with a slight modification.

Conversion [%]

25

20 15 10

5 0

0

20

40

60

80

100

120

Reaction time [h]

Figure 2. Transient changes in conversion of lauroyl glucose and galactose in acetonitrile with different initial water contents. The initial water contents were (○) 0.32% (v/v) and (□) 0.50% for synthesis of lauroyl glucose, and were (◆) 0.059% and (●) 0.32% for synthesis of lauroyl galactose. The curves were empirically drawn. 80 60 Equilibrium conversion [%]

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(a)

40 20

0 (b) 40

20 0 -2 10 10-1 100 101 Initial water content [% (v/v)]

Figure 3. Effect of initial water content on the equilibrium conversion of (○) lauroyl glucose, (◇) galactose, (□) mannose, and (△) fructose in (a) acetonitrile, and (b) lauroyl fructose in (▷) acetone, and (▽) 2-methyl-2-propanol. The curves were empirically drawn.

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Lipase-Catalyzed Synthesis of Edible Surfactants in Organic Solvents

35

Figure 2 shows the transient changes in the conversion of monolauroyl glucose or galactose in acetonitrile with various water contents. Di- and higher esters were not detected for galactose, glucose and mannose under the analytical conditions. HPLC chromatograms of the product from fructose and lauric acid, observed under various conditions, revealed that it consisted of at least three components. Judging from their retention times, they seemed to be positional or conformational isomers of monoesters, although the possibility of formation of di- or higher esters still remained. The conversion in the condensation was defined as the molar ratio of the amount of lauroyl hexose to the initial amount of hexose under the assumption that only monoester(s) was formed. The conversion of each ester reached equilibrium within 3 days. The equilibrium yield was higher at a lower water content for the synthesis of both lauroyl glucose and galactose. It also depended on the kind of hexose used as a substrate even if the initial water content of acetonitrile was the same (0.32% (v/v)). Figure 3(a) shows the effect of initial water content on the equilibrium conversion of lauroyl glucose, galactose, mannose and fructose. Except for lauroyl fructose, the equilibrium conversions were higher at the lower initial water contents. The synthesis of lauroyl fructose showed a peculiar dependence on the water content, and a maximum conversion appeared at about 0.3% (v/v) water content. A similar dependence had been observed during the synthesis of the lauroyl erythritol, and it was suggested that the activity coefficients of the substrates and products depended on the water content [20]. Fructose and erythritol have two primary hydroxyl groups, while other hexoses have only a primary hydroxyl group. Although Acros et al. [18] reported that fructose was quantitatively converted into its diester at a low temperature (5oC) in acetonitrile, a significant amount of the diester could not be observed under our experimental and analytical conditions. Figure 3(b) shows the equilibrium conversions of lauroyl fructose in acetone and 2-methyl-2-propanol with various initial water contents. The conversions were higher at lower water contents. At the higher water contents, the conversion was the highest in acetonitrile among the solvents tested, while the conversion in acetone became the highest at lower water contents. Figure 4 shows the solubilities of the hexoses at 50oC in the solvents with different water contents. Two or five hundred milligrams of hexose was placed into a vial together with 5 mL of solvent with a known water content, and then the vial was tightly screw-capped. The vial was held at 50oC with occasional stirring for a period until the corresponding condensation reached equilibrium. A portion of the solvent was carefully sampled so as not to include the undissolved hexose and was diluted by adding the same volume of eluent used for the HPLC analysis as quickly as possible. For determination of hexose solubilized in a solvent, a Cosmosil 5NH2 column (4.6 mmx 250 mm) was used. The eluent used was a mixture of acetonitrile, methanol, and water (70/15/15 by vol.) for fructose, glucose and galactose in acetonitrile, of acetonitrile and water (80/20 by vol.) for fructose in acetone and 2-methyl-2propanol, or of acetonitrile and water (60/40 by vol.) for mannose in all solvents used. The flow rate was 1.0 mL/min. The solubility depended on both the kind of hexose and solvent, and it could empirically be expressed as an exponential function of the water content w for all the cases: CS = exp(w)

Organic Solvents: Properties, Toxicity, and Industrial Effects : Properties, Toxicity, and Industrial Effects, edited by Ryan E. Carter, Nova Science

(1)

Yoshiyuki Watanabe and Shuji Adachi

36

where  and  are the constants, and the values are listed in Table 1. This indicated that a certain amount of hexose could be solubilized even in a completely dehydrated solvent. Mannose was the most soluble in acetonitrile, followed by fructose, galactose and glucose. 2Methyl-2-propanol possessed the highest ability to solubilize fructose among the solvents tested. 10-1

(a)

10-2

Solubility [mol/L]

10-3 10-4

(b)

10-1 10-2

(c)

10-1 10-2

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10-3 10-2 10-1 100 101 Water content [% (v/v)] Figure 4. Solubility at 50oC (a) of (○) glucose, (◇) galactose, (□) mannose and (△) fructose in acetonitrile, (b) of fructose in (▷) acetone and (▽) 2-methyl-2-propanol, and (c) of mannose in (■) acetone, (▲) 2-methyl-2-propanol, and (▼) 2-methyl-2-butanol. The curves were drawn using the parameters shown in Table 1.

Table 1. Parameters in Eq. 1 for solubility of hexoses in organic solvents at 50oC Hexose Glucose Galactose Mannose Mannose Mannose Mannose Fructose Fructose Fructose

Organic solvent Acetonitrile Acetonitrile Acetonitrile Acetone 2-Methyl-2-propanol 2-Methyl-2-butanol Acetonitrile Acetone 2-Methyl-2-propanol

a [mol/L] 9.27×10－5 2.11×10－4 5.46×10－3 7.73×10－3 4.06×10－2 3.23×10－2 9.78×10－4 3.17×10－2 1.67×10－1

b [% (v/v)－1] 0.398 0.415 0.251 0.307 0.270 0.354 0.353 0.233 0.238

Organic Solvents: Properties, Toxicity, and Industrial Effects : Properties, Toxicity, and Industrial Effects, edited by Ryan E. Carter, Nova Science

Lipase-Catalyzed Synthesis of Edible Surfactants in Organic Solvents

37

102

KC

101

100

10-1 10-1 CWe

100 [mol/L]

101

Figure 5. Dependence of equilibrium constants for lauroyl hexose synthesis on equilibrium water concentration. Symbols represent (○) lauroyl glucose, (◇) galactose, (□) mannose, and (△) fructose in acetonitrile, and lauroyl fructose in (▷) acetone, and (▽) 2-methyl-2-propanol. The curves were empirically drawn.

5

lnKC

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4 3 2 1 14

15

16

nDHN

17

18

19

Figure 6. Relationship between the apparent equilibrium constant KC for lauroyl hexose formation in acetonitrile and the dynamic hydration number nDHN of hexoses. Symbols represent mean±standard deviation.

The equilibrium constant is an important parameter for predicting the equilibrium conversion of the desired product under any conditions. The apparent equilibrium constant, KC, is defined by Eq. 2:

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Yoshiyuki Watanabe and Shuji Adachi

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KC 

CPe C We CSe CFe

(2)

where C is the concentration in units of mol/L, and the subscripts S, F, P and W represent saccharide (hexose), fatty acid (lauric acid), product (lauroyl saccharide) and water, respectively. The subscript e indicates equilibrium. The concentrations of product and water at equilibrium, CPe and CWe, were experimentally observed. The water content in units of % (v/v) was converted to the concentration in units of mol/L, assuming roughly the volume additivity among substrates, products and solvent. The molar volumes used were 0.114 L/mol for all hexoses [22], 0.231 L/mol for lauric acid, 0.0182 L/mol for water, 0.0548 L/mol for acetonitrile, 0.0740 L/mol for acetone, 0.0942 L/mol for 2-methyl-2-propanol, and 0.1090 L/mol for 2-methyl-2-butanol. The molar volumes except for the hexoses were calculated from the density and molecular mass of each compound. The molar volume of lauroyl hexose was assumed to be 0.326 L/mol, which was obtained by vS+vF－vW (v: molar volume). The concentration of lauric acid at equilibrium, CFe, was estimated by CF0－CPe, where CF0 is the initial concentration of lauric acid. In the present reaction system, hexose was not fully dissolved in the solvent, and only the hexose dissolved in the solvent would be effective as a substrate for the condensation. The smaller of either the solubility CS at CWe or CS0－CPe (CS0 : the overall initial concentration of hexose) was regarded as the concentration of hexose at equilibrium CSe. By substituting into Eq. 2 the concentrations of substrates and products at equilibrium estimated as above, the KC value was determined. Figure 5 shows the KC values for lauroyl hexoses at various equilibrium water concentrations. The KC value largely depended on the kind of hexose. Except for lauroyl fructose, there was a tendency that the KC was slightly higher at the lower CWe. The kind of solvent also significantly affected the KC value. The KC values were different by two orders of magnitude among the solvents. As shown above, the apparent equilibrium constants, KC, significantly depended on the kind of hexose and the solvent. The equilibrium constant, Ka, should, in principle, be defined based on activities, a, of the substrates and products, and is truly constant at specific temperature and pressures. The KC can be related to the intrinsic reaction equilibrium constant Ka, which is defined based on the activities, by the following equation:

KC 

γS γ F Ka γPγW

(3)

Unfortunately, it is impossible or very difficult to evaluate all the a values (or activity coefficients ) of the substrates and products in the present reaction system. When a saccharide hydrates, the water activity decreases. Therefore, it would be supposed that a hexose with stronger binding of water gives a larger KC value, although the activities of other components would also be affected by the presence of the hexose. The dynamic hydration number, nDHN, of hexose was chosen as a measure of the extent of hydration, and examined the relationship between the KC values observed in acetonitrile and the nDHN of hexoses [32, 33] used as substrates (Figure 6). Since the KC value depended on the CWe, the KC value averaged over all the CWe values for each product is plotted in the figure. As expected, there was a positive correlation between lnKC and nDHN. This indicates that the water activity plays

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Lipase-Catalyzed Synthesis of Edible Surfactants in Organic Solvents

39

an important role for the condensation in microaqueous organic solvents. The KC value for the formation of lauroyl fructose also depended on the kind of solvent. Although the correlation between the KC value and the solvent parameter such as logP (P: partition coefficient between 1-octanol and water phases) and the Dimroth-Reichardt parameter for polarity of solvents, ET, [34] was examined, no significant correlation was found.

Conversion [%]

80

60 40 20

0 0

20

40

60

80

100

Time [h]

Equilibrium conversion [%]

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Figure 7. Transient changes in conversion of lauroyl mannose through lipase-catalyzed condensation at 50oC in (●) acetonitrile, (■) acetone, (▲) 2-methyl-2-propanol, and (▼) 2-methyl-2-butanol with low water contents. The initial water contents of acetonitrile, acetone, 2-methyl-2-propanol, and 2-methyl-2butanol were 0.039, 0.043, 0.029 and 0.029% (v/v), respectively. The curves were empirically drawn.

80 60 40 20 0 10-2

10-1 100 Initial water content [% (v/v)]

101

Figure 8. Effect of the initial water content on the equilibrium conversion of lauroyl mannose in various water-miscible solvents. The symbols are the same as in Fig. 7. The curves were empirically drawn.

The condensation of mannose and lauric acid was carried out at 50oC in nine watermiscible organic solvents. Lauroyl mannose was formed in acetonitrile, acetone, 2-methyl-2propanol and 2-methyl-2-butanol, but not in isopropanol, propionitrile, dimethyl sulfoxide, Nmethyl formamide and N, N-dimethyl formamide. Therefore, subsequent experiments were performed using the former four solvents. Figure 7 shows the transient changes in the conversion of lauroyl mannose in the solvents with initial water contents of 0.029 to 0.043%

Organic Solvents: Properties, Toxicity, and Industrial Effects : Properties, Toxicity, and Industrial Effects, edited by Ryan E. Carter, Nova Science

Yoshiyuki Watanabe and Shuji Adachi

40

(v/v). The conversion of every ester reached equilibrium within 3 days. The conversion was the highest in acetonitrile and the lowest in 2-methyl-2-butanol, although the initial water content of each solvent was slightly different. Figure 8 shows the equilibrium conversions of lauroyl mannose in the four solvents with various initial water contents. In every solvent, the equilibrium conversion was higher at the lower initial water contents. This dependence of the conversion on the water content would be reasonable, because water is one of the products in the condensation. However, the conversion largely depended on the kind of solvent. Among the solvents, acetonitrile gave the highest conversion at any initial water content, and a conversion of ca. 70% was realized in acetonitrile dehydrated with molecular sieves, the water content of which was 0.014% (v/v). In order to elucidate whether the reaction equilibrium constant KC itself was different among the solvents or other factors affected the conversion, the KC values were estimated.

KC

101

100

100 CWe [mol/L]

10

Figure 9. Dependence of the apparent equilibrium constants KC for lauroyl mannose synthesis in various organic solvents on the equilibrium water concentration CWe. The symbols are the same as in Figure 7. The curves were empirically drawn. 2 Acetonitrile

1

lnKC

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10-1 10-1

Acetone

0 2-methyl2-butanol

－1

2-methyl-2-propanol

－2

0

10

20 r

30

40

Figure 10. Correlations of the apparent equilibrium constant KC for lauroyl mannose synthesis in various solvents with the relative dielectric constants, r, of the solvents. The curves were empirically drawn. Organic Solvents: Properties, Toxicity, and Industrial Effects : Properties, Toxicity, and Industrial Effects, edited by Ryan E. Carter, Nova Science

41

The solubility of mannose in each solvent was measured at 50oC to obtain CSe in the solvents with different water contents (Figure 4(c)). The solubility depended on the kind of solvent. Mannose was the most soluble in 2-methyl-2-propanol and 2-methyl-2-butanol, followed by acetone and acetonitrile. The solubility could empirically be expressed as an exponential function of the water content for each solvent. The parameters,  and , in Eq. 1 are listed in Table 1. By substituting the substrate and product concentrations at equilibrium into Eq. 2, the KC value was determined. Figure 9 shows the dependence of the KC on the equilibrium water concentration. The KC values were different by about two orders of magnitude among the solvents. The KC value was the highest in acetonitrile, intermediate in acetone, and small in 2-methyl-2-propanol and in 2-methyl- 2-butanol. As mentioned above, the KC value evaluated here is an apparent one, because it was defined based not on the activities but on the concentrations. If the water activity is only a parameter affecting the KC value, KC would be inversely proportional to W (Eq. 3). The reaction system consisted of five components even if the immobilized enzyme was assumed to be inert, and it was too complicated to estimate the W value. Therefore, we estimated the W value under the assumption of a binary system consisting of water and organic solvent according to the UNIFAC [35]. However, the W did not correlate with the KC value. This might indicate that the water activity was not the sole factor affecting the reaction equilibrium in an organic solvent. Another possibility for the failure was that the W was not adequately evaluated because the assumption of a binary system was too simple. As shown in Figure 9, the dependence of the KC value on the equilibrium water concentration CWe was relatively weak for every solvent. Therefore, the KC values were averaged for each solvent, and we then tried to find a solvent parameter that could correlate with the averaged KC value to obtain a criterion for selection of the solvent. The correlations of the averaged KC values with logP, the Dimroth-Reichardt parameter, ET, and the relative dielectric constant of the solvent, r, [36] were examined. There was a tendency for the KC to be smaller in a more hydrophobic solvent, but logP did not seem to be a satisfactory parameter to correlate with the KC value. The ET did not correlate with the KC value at all. As shown in Figure 10, the r value correlated best with the lnKC value among the parameters tested. 8

C /CWf

1.0

q－ q0 [mol/L-resin]

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Lipase-Catalyzed Synthesis of Edible Surfactants in Organic Solvents

6

0.5

0 0

4

1 t /(Vt/Q)

2

2

0 0

1

2

3

4

CWf [mol/L]

Figure 11. Adsorption isotherms of water onto immobilized-lipase particle observed at room temperature. The insets shows the breakthrough curves of water in the bed packed with immobilizedlipase particles at room temperature. The water concentrations in feed were (▲) 0.552 and (●) 1.61 mol/L. The curves were empirically drawn.

Organic Solvents: Properties, Toxicity, and Industrial Effects : Properties, Toxicity, and Industrial Effects, edited by Ryan E. Carter, Nova Science

42

Yoshiyuki Watanabe and Shuji Adachi

The KC value was evaluated based on the concentrations of substrates and products in the bulk phase. However, the condensation proceeds in the immobilized-enzyme particle. Therefore, the concentrations in the immobilized-enzyme phase would be desirable to estimate the KC value. In this study, the concentration of water in the phase was estimated because water played an important role in the condensation. The adsorption isotherm of water onto immobilized enzyme was measured. Immobilized-enzyme particles were packed in a cylindrical glass column with a 1.0 cm I.D. and 15 cm height. The bed was washed with acetonitrile dehydrated with molecular sieves, the water content of which was about 0.04 mol/L (C0). The acetonitrile of a given water concentration CWf was fed to the bed at a flow rate of 0.5 mL/min. The effluent was fractionated at appropriate intervals, and the water concentration in the effluent was determined. Thus, the breakthrough curve of water was obtained. The amount of water adsorbed on the immobilized enzyme, q, was estimated by the following equation: t

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(1   b )Vt ( q  q0 )  QCWf t E   bVt (CWf C 0 )  Q  E Cdt 0

(4)

where C is the concentration of water in the effluent, Q is the volumetric flow rate, Vt is the total volume of the bed, t is the elution time, tE is the time at the end point, and b is the bed voidage. q0 is the amount of water adsorbed at C0. Since we could not directly measure q0, the value of q－q0 was obtained. The integration of Eq. 4 was numerically carried out. The bed voidage, b, was estimated to be 0.406 by dividing the volume of solvent withdrawn from the void of the bed by the total bed volume, although such an estimation was somewhat rough. All measurements were carried out at room temperature (ca. 25oC). The apparent density of the wet immobilized enzyme was pycnometrically determined to be 1.044 g/mL. A rough estimation of the immobilized-enzyme particle porosity was made from the difference in weight between the wet and dry immobilized-enzyme particles, and the porosity was 0.588. The insets in Figure 11 shows examples of the breakthrough curves of water observed at different concentrations of water in the feed, CWf, using acetonitrile as a solvent. The curves were numerically integrated, and the q－q0 values were calculated according to Eq. 4. Figure 11 shows the adsorption isotherms of water onto the immobilized-enzyme. Although the condensation was conducted at 50oC, the isotherm was obtained at room temperature because of the experimental ease and to roughly estimate the extent of water adsorbability. The isotherm was almost linear. The slope of the line, which corresponds to the distribution coefficient of water onto the immobilized-enzyme phase, was 1.31. Since the porosity of the immobilized-enzyme was 0.588, the concentration of water in the pore was higher than that in the bulk phase by a factor of about 2. Thus, the possibility that the concentrations of substrates and products would be different from those in the bulk phase has been suggested. To more precisely discuss the KC value, we need information about the adsorption isotherms of all the components. The isotherms should be observed in a multi-component system because at least four components participate in the condensation.

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Lipase-Catalyzed Synthesis of Edible Surfactants in Organic Solvents

43

2. CONTINUOUS PRODUCTION OF ACYL ,MANNOSES BY IMMOBILIZED LIPASE USING A PACKED-BED REACTOR AND THEIR SURFACTANT PROPERTIES A batch reactor has mainly been used for the lipase-catalyzed synthesis of acyl saccharides, but a packed-bed reactor would be preferred for a large-scale production. As shown in Section 1, hexoses also dissolved in dehydrated water-miscible solvents at a concentration of 0.1 to 100 mmol/L, although the solubility largely depended on the kinds of hexose and of solvent. The surfactant properties of some acyl saccharides, which are enzymatically synthesized, have been reported [14, 27]. However, the properties of acyl mannoses have not been available. In this section, a continuous production of 6-O-acyl mannoses of acyl chain lengths of 8 to 16 through the immobilized-lipase-catalyzed condensation of mannose and fatty acids in dehydrated 2-methyl-2-propanol using a packedbed reactor was examined, and surface tensions in aqueous solution of the products were measured. A packed-bed reactor system is schematically illustrated in Figure 12. Immobilized-lipase particles from Candida antarctica were packed into a cylindrical glass column (10 mm x 150 mm). 2-Methyl-2-propanol was dehydrated over the molecular sieves 5A overnight to ca. 0.02% (v/v) of water content. Mannose and a fatty acid (caprylic, capric, lauric, myristic or palmitic acid) were dissolved with the dehydrated solvent at concentrations of 20 mmol/L and 100 mmol/L, respectively. The substrate solution was fed to the column at a specified flow rate by a delivery pump.

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⑤

④

① ②

③ ⑥

Figure 12. Scheme of a packed-bed reactor system used for continuous production of acyl mannoses; ① reservoir of substrate solution, ② magnetic stirrer, ③ pump, ④ reactor packed with immobilized lipase, ⑤ temperature-controlled chamber, ⑥ reservoir of product solution.

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44

80

60

40

Conversion [%] at  0 = 30 min

Conversion [%]

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The substrate reservoir, column and pump were installed in a thermo-regulated chamber at 50oC to prevent solidification of 2-methyl-2-propanol, the melting point of which is 25.4oC. After a steady-state being achieved (usually the substrate solution of 3 times the bed volume was fed), the effluent was sampled. These operations were carried out at various flow rates for every fatty acid to obtain the relationship between the conversion and superficial residence time, 0. A mixture of mannose (20 mmol/L) and myristic acid (100 mmol/L) was continuously fed to the column packed with immobilized lipase (10 mm x 50 mm) for 16 days at a flow rate of 0.33 mL/min, which corresponded to 0 = 12 min. At appropriate intervals, the effluent was sampled and the myristoyl mannose concentration in it was determined. An acyl mannose was quantified using an HPLC with an ODS column (4.6 mmx 300 mm) and a refractometer. The eluent used was a mixture of acetonitrile and water; 40/60 by vol. for capryloyl mannose, 50/50 for caproyl mannose, and 65/35 for lauroyl, myristoyl and palmitoyl mannoses. The flow rate was 0.8 mL/min. The calibration curves were prepared using the products isolated from the reaction mixtures. The effluent was rotaryevaporated to reduce its volume to about half. The concentrated effluent was applied to an YMC-pack ODS column (20 mm x 250 mm, Kyoto), and eluted with a mixture of acetonitrile and water (65/35 by vol.) at a flow rate of 7 mL/min. The effluent at the peak corresponding to a desired product was gathered, and the product was recovered by evaporation. This purification was repeated until a sufficient amount of the product for surface-tension measurement was obtained. Immobilized lipase from Candida antarctica exhibits its catalytic activity in some water-miscible solvent such as acetonitrile, acetone, 2methyl-2-propanol and 2-methyl-2-butanol as shown in Section 1. Since the solubility in 2methyl-2-propanol was the highest (Figure 4(c)), the solvent was used throughout this study. The substrate solution was fed to the column packed with immobilized lipase at various flow rates, and the conversion of mannose to a desired acyl mannose at a steady-state was observed.

20

80 60

40 8 12 16 Carbon number of acyl chain

0 0

10

20  0 [min]

30

Figure 13. Relationship between the superficial residence time, 0, and the conversion of mannose to (○) capryloyl, (□) caproyl, (◇) lauroyl, (△) myristoyl or (▽) palmitoyl mannose at 50oC. The dimensions of the bed were 10 mm I.D. and 150 mm height. The insets shows the dependence of the conversion at 0 = 30 min on the carbon number of acyl chain. The curves were empirically drawn.

Organic Solvents: Properties, Toxicity, and Industrial Effects : Properties, Toxicity, and Industrial Effects, edited by Ryan E. Carter, Nova Science

Lipase-Catalyzed Synthesis of Edible Surfactants in Organic Solvents

45

Conversion [%]

60 50 40 30 20 10 0

0

5 10 15 20 Operation time [days]

25

Figure 14. Continuous production of (▲) myristoyl, (◆) lauroyl and (■) caproyl mannoses using a packed-bed reactor of immobilized lipase at 0 = 12 min. The dimensions of the bed were 10 mm I.D. and 50 mm height. The arrows indicate the operating times when the substrate solution was changed. 80

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 [mN/m]

70

60 50 40 30 20 10－7 10－6 10－5 10－4 10－3 10－2 10－1 C [mol/L]

Figure 15. Surface tensions of aqueous solutions of (●) capryloyl, (■) caproyl, (◆) lauroyl and (▲) myristoyl mannoses at various concentrations and at 25oC.

Table 2. Surfactant properties of the prepared acyl mannoses at 25ºC Acyl chain

CMC a [mol/L]

Capryloyl Caproyl Lauroyl Myristoyl

1.31×10－2 2.25×10－3 2.13×10－4 1.81×10－5

gCMC b [mN/m] 27.7 27.3 25.3 27.7

G×106 c [mol/m2] 4.45 4.18 4.44 4.42

ad [nm2] 0.37 0.40 0.37 0.38

a

Critical micelle concentration. Surface tension at critical micelle concentration. c Surface excess. d Residual area per molecule. b

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46

Figure 13 shows the relationship between the superficial residence time, 0, and the conversion. For every fatty acid, the conversion of more than 50% was attained at 0  20 min. The plots for each fatty acid were connected empirically by a curve, as shown by a solid curve. The conversion at 0 = 30 min, which would be near the equilibrium conversion judging from the shape of the curve, was read, and is plotted versus the carbon number of acyl chain in the insets in Figure 13. There was a weak tendency that the equilibrium conversion was higher for the ester with the longer acyl chain. A long-term operational stability of the enzyme was examined for the synthesis of myristoyl mannose at 0 = 12 min. As shown in Figure 14, a conversion of ca. 40% was maintained for at least 16 days. The lauroyl and caproyl mannoses were also produced at similar conversions. The productivity during the operation was evaluated to be 350 g/L-reactor･day. The surface tensions of aqueous solutions of the produced acyl mannoses were observed at various concentrations and at 25oC by the Wilhelmy method using a surface tensiometer (Figure 15). The critical micelle concentration, CMC, was estimated from the intersection of the two lines for each acyl mannose. The surface excess, , was evaluated from the slope of the line drawn at the low concentrations according to the following equation:



d RT   d log C 0.434

(5)

where  is the surface tension, C is the concentration of acyl mannose, R is the gas constant, and T is the absolute temperature. The residual area per molecule, a, was calculated from the  value by the following equation:

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a

1 N A

(6)

where NA is Avogadro’s number. These surfactant properties of the acyl mannoses are listed in Table 2. The CMC of the acyl mannoses are plotted versus the carbon number of the acyl chains in Figure 16, together with the CMC at 37oC of monoacyl fructoses which were estimated from the literature [27]. Although the temperature was different between this study and the literature, the CMC of an acyl mannose was the almost same as that of the fructose ester with the same acyl chain. The change in CMC as a function of the chain length n is expressed by [37]

log CMC  

w nb kT

(7)

where w is the cohesive energy change per methylene group passing the bulk of the solution to the micelle, k is the Boltzmann’s constant, and b is a constant. The w values were estimated to be 2.0 x 10－21 and 1.6 x 10－21 J for 6-O-acyl mannoses and fructoses, respectively. These values were somewhat smaller than those for 1-O-alkyl glycosides (glucosides, galactosides, and fucosides; 2.2―2.7 x 10－21 J) [38]. As shown in Table 2, the a values were practically the same among the acyl mannoses tested, and were ca. 0.40 nm2. The a values of alkyl glycosides were also in a range of 0.37 to 0.49 nm2 [38]. Since acyl mannose molecules

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Lipase-Catalyzed Synthesis of Edible Surfactants in Organic Solvents

47

would be oriented so as to stick their acyl residues out in the air, the a value seemed to be exclusively determined by the saccharide moiety. The situation would be the same for alkyl glycosides. These would be the reason why the acyl mannoses and alkyl glycosides showed the similar a values.

3. SYNTHESES OF LAUROYL PHENOLIC GLYCOSIDES BY IMMOBILIZED LIPASE IN ORGANIC SOLVENT AND THEIR ANTIOXIDATIVE ACTIVITIES Recently, phenolic glycosides from crude plant materials are receiving attention in relation to their antioxidative activities which may correspond to a health protective action [39-41]. Arbutin found in plant, Uvae ursi, is used in cosmetics because of its whitening effect on the skin [42, 43]. Naringin is the primary bitter component in citrus fruits, though the bitterness in citrus fruit juices is one of the major problems of the citrus industry [44, 45]. Phloridzin is a polyphenolic compound included in apple skin and has the lowering effect on the postprandial blood glucose level in vivo [46]. These phenolic glycosides have some peculiar properties and would have also antioxidative activity due to their phenolic hydroxyl groups. The lipase-catalyzed syntheses of acyl ascorbate through the condensation of various fatty acids with L-ascorbic acid in an organic solvent has been reported [47, 48]. Acyl ascorbates have shown interesting emulsifier property and antioxidative activity against the oxidation of encapsulated lipid [49, 50]. It is expected that acyl phenolic glycoside is also synthesized through the acylation of hydrophilic phenolic glycoside and used as an amphiphilic antioxidant for foods and cosmetics.

10－2

CMC [mol/L]

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10－1

10－3 10－4

10－5 10－6 8

10 12 14 16 Carbon number of acyl chain

Figure 16. Relationship between the critical micelle concentration, CMC, and the carbon number of acyl chain. The symbols ● and ▲ indicate acyl mannose and fructose, respectively. The CMC values at 37oC for acyl fructoses were estimated from the literature [27].

The lipase-catalyzed condensation of arbutin, naringin or phloridzin and lauric acid was carried out in various organic solvents. Arbutin, naringin or phloridzin (0.0625 mmol) and lauric acid (0.469 mmol) were weighed into an amber glass vial with a screw-cap. Fifty milligrams of immobilized lipase from Candida antarctica and 5 mL of organic solvent, such

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Yoshiyuki Watanabe and Shuji Adachi

48

104

50 103 40 102

30 20

101

10 0

0

10

20

r

30

40

100

Solubility of phenolic glycoside [mmol/L]

60

Maximum conversion [%]

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as acetonitrile, 2-methyl-2-propanol, 2-methyl-2-butanol, and hexane, were also added to the vial. The headspace of the vial was filled with nitrogen gas, and the vial was tightly sealed over the blowing gas. The vial was then immersed in a water-bath at 60oC to commence the condensation reaction with vigorous shaking. These reaction conditions, such as temperature and the concentrations of two substrates and immobilized lipase, were determined in consideration of the results for the similar reaction systems in the previous reports [51]. At appropriate intervals, the reaction mixture was sampled and adequately diluted with an eluent, methanol/water/phosphoric acid (85:15:0.1, by vol.), for the HPLC analysis with an ODS column (4.6 mm x 150 mm) and a UV detector (280 nm). The mixture was applied to the column and eluted with the eluent at 1.0 mL/min. After the sampling, the headspace was filled with nitrogen gas again to prevent the oxidative degradation of the substrates and product. The solubility of each phenolic glycoside in various organic solvents was measured as follows: Two hundred milligrams of each phenolic glycoside was added to 5 mL of acetonitrile, 2-methyl-2-propanol, 2-methyl-2-butanol, or hexane in an amber glass vial. The vial was immersed in a water-bath at 60oC with shaking. After 24 h, the mixture was sampled and adequately diluted with an eluent for the HPLC analysis. The HPLC analysis was carried out under the above mentioned conditions. Lauroyl arbutin, naringin and phloridzin were purified by preparative HPLC with an ODS column (20 mm x 250 mm) and a UV detector (280 nm) using a mixture of methanol and water (85:15, by vol.) as the eluent. The volume of sample applied was 1 mL, and the flow rate of each eluent was from 5.0 to 7.0 mL/min. The effluent at the peak corresponding to the product was collected, rotary-evaporated and dried over phosphorus pentoxide in a desiccator. The purification was repeated until an amount of each product sufficient for oxidation experiments was obtained. The 1H-NMR (400 MHz, CD3OD, TMS, 297 K) and 13C-NMR (100 MHz, CD3OD, TMS, 297 K) charts consistently revealed that the primary hydroxyl group of C-6 position in glucose moiety included in the phenolic glycosides was esterified with lauric acid.

Figure 17. Relationship between relative dielectric constant, r, of organic solvent and the maximum conversion for the syntheses of (○) acyl arbutin, (□) acyl naringin and (△) acyl phloridzin or the solubilities of (●) arbutin, (■) naringin and (▲) phloridzin in organic solvent at 60oC. The solid and broken curves were empirically drawn.

Organic Solvents: Properties, Toxicity, and Industrial Effects : Properties, Toxicity, and Industrial Effects, edited by Ryan E. Carter, Nova Science

Fraction of unoxidized linoleic acid, Y

Lipase-Catalyzed Synthesis of Edible Surfactants in Organic Solvents 1.0

(a)

(b)

(c)

49

(d)

0.8 0.6 0.4 0.2

0

0

40 0

40

80

120 0 40 Time [h]

0

40

80

120

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Figure 18. Oxidation processes of linoleic acid with (○) no additive, (◇, ◆) arbutin, (□, ■) naringin and (△, ▲) phloridzin at 50oC and 12% relative humidity. The closed and open symbols represented unmodified and acyl polyphenol glycoside, respectively. The solid curves were calculated using the estimated kinetic parameters of the Weibull equation.

The transient changes in the conversions of lauroyl arbutin, naringin and phloridzin in acetonitrile, 2-methyl-2-propanol, 2-methyl-2-butanol, and hexane were measured. The conversion was calculated on the molar basis of the phenolic glycoside added, which was the limiting reactant. Each conversion was the highest in acetonitrile, and those in 2-methyl-2propanol and 2-methyl-2-butanol were followed. The conversion for the condensation using arbutin as a substrate was the highest in three substrates. The highest conversion was that of lauroyl arbutin in acetonitrile, and about 50% at 24h. The products in hexane were hardly detected, indicating that the solubility of each phenolic glycoside as a substrate for the enzymatic condensation in hexane of a hydrophobic solvent was very low due to its high hydrophilicity. Figure 17 shows the relationship between relative dielectric constant, r, of organic solvent used and the maximum conversion for the synthesis of lauroyl phenolic glycoside or the solubility of phenolic glycoside in the solvent. The maximum conversion depended on the relative dielectric constant of the solvent, and increased with increasing the constant for every lauroyl phenolic glycosides. The maximum conversions of lauroyl arbutin were much higher than those of lauroyl naringin and phloridzin in water-soluble solvents. In contrast to the conversion, the solubilities of every phenolic glycosides as a substrate for enzymatic reaction decreased with increasing the constant. For each solvent, the solubility of phloridzin was the highest, and those of naringin and arbutin followed. The intermolecular interaction between the phenolic glycoside and reaction solvent seemed to affect the conversion. The equilibrium constant for the formation of fatty acid butyl esters through the lipase-catalyzed condensation depended on the type of solvent polar group [52]. The phenolic glycoside having many hydroxyl groups would also be bound to the alcohol such as 2methyl-2-propanol and 2-methyl-2-butanol more strongly than the nitrile-type solvent such as acetonitrile. Therefore, the solubility of each phenolic glycoside in 2-methyl-2-propanol and 2-methyl-2-butanol may be high, and the mobility from solvent phase to the binding site in a lipase used as a catalyst would be low. Figure 18 shows the oxidation processes of linoleic acid with unmodified or lauroyl phenolic glycoside at 50oC and 12% relative humidity. The oxidation processes were measured as follows: Twenty milligrams of linoleic acid was dissolved in hexane at the

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Yoshiyuki Watanabe and Shuji Adachi

50

volume of 5 mL for the solution. Each unmodified or lauroyl phenolic glycoside dissolved in ethanol was added to the linoleic acid solution at the molar ratio of 0.1 to linoleic acid. Twenty five microliters of the mixture was placed in flat-bottomed glass cups (1.5 cm x 3.0 cm), and the hexane and ethanol were then evaporated under reduced pressure in a desiccator. The cups were placed in a plastic container in which a Petri dish filled with saturated lithium chloride aqueous solution to maintain the relative humidity at 12%. The container was then stored in the dark at 50oC. The cups were periodically taken out, and a mixture of methanol, benzene and methyl myristate (20/80/0.05 by vol.) was added to the cup. Linoleic acid was converted to its methyl ester by adding trimethylsilyldiazomethane solution dissolved in hexane and allowing it to stand at room temperature for 30 min [53]. After evaporation under reduced pressure, the remainder was dissolved in hexane, and the solution was used for the GC analysis. The amount of unoxidized linoleic acid was determined by a gas chromatograph with a hydrogen ionization detector and a capillary column (0.25 mm x 30 m) with polyethylene glycol. The injection or detection temperature was 200oC, and column temperature was 180oC. The fraction of unoxidized linoleic acid without unmodified and lauroyl phenolic glycoside rapidly decreased as shown in Figure 18(a), whereas Figure 18(b)(d) shows that the oxidation of linoleic acid with unmodified or lauroyl phenolic glycoside slowly proceeded. Furthermore, the suppressive effect of each lauroyl phenolic glycoside against the oxidation was higher than those of the corresponding phenolic glycoside. The oxidation kinetics of linoleic acid was empirically expressed by the Weibull equation, which is flexible and has a potential for describing many deterioration kinetics [54]: Y = exp[-(kt)n]

(8)

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where Y is the fraction of unoxidized linoleic acid at time t, k is the rate constant, the reverse of which is called the scale parameter, and n is the shape constant. Table 3. The parameters, k and n, of the Weibull equation for the oxidation of linoleic acid＊ with unmodified and lauroyl phenolic glycoside

Arbutin Naringin Phloridzin ＊

Unmodified phenolic glycoside k [h-1] n 0.0346 0.787 0.0893 2.92 0.0576 2.52

Lauroyl phenolic glycoside k [h-1] n 0.00766 3.12 0.00311 1.38 0.00110 1.34

The k and n values for linoleic acid with no additive were 0.105 h-1 and 3.29, respectively.

The kinetic parameters, k and n, were evaluated by fitting the experimental results by nonlinear regression. The curves in Figure 18 were drawn based on the equation using the estimated parameters. Table 3 shows the parameters for the oxidation of linoleic acid with unmodified and lauroyl phenolic glycoside. The k values show that the antioxidative activity of each lauroyl phenolic glycoside against lipid oxidation was higher than that of unmodified phenolic glycoside. There was no tendency between the n values for the oxidation with unmodified and lauroyl phenolic glycoside. The Weibull model has characteristics of a sigmoidal pattern that can be described when n > 1, that the model expresses the simple first-

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order kinetics at n = 1, and that Y value steeply decreases during the early stage when n < 1. Thus, it was indicated that each oxidation of linoleic acid except that with arbutin was represented as a sigmoidal pattern. There was no difference between the 1,1-diphenyl-2picrylhydrazyl free radical scavenging activities [55] of unmodified and lauroyl phenolic glycoside in ethanol solution. The radical scavenging activity of lauroyl phenolic glycoside, however, depended on the type of phenolic glycoside, and the acitivity was high in order of lauroyl arbutin, phloridzin and naringin. This order was same as that for their antioxidative effect against the oxidation of linoleic acid. The antioxidative activity of the glycoside would depend on the number and the placement of phenolic hydroxyl group exhibiting the activity in the molecule. Therefore, it is suggested that enhancement of the suppressive effect against lipid oxidation resulted due to increasing in the hydrophobicity of phenolic glycoside by the acylation. Enhanced hydrophobicity and high solubility in a lipid due to it would enable acyl phenolic glycoside to act to linoleic acid inside the bulky acid, resulting in enhancement of the antioxidative activity.

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CONCLUSION The equilibrium conversion of monolauroyl saccharide in acetonitrile significantly depended both on the kind of the saccharides and solvents tested. The apparent equilibrium constants, KC, based on the concentrations of substrates and products in acetonitrile could be correlated to the dynamic hydration numbers of the saccharides, indicating that the water activity played an important role during the condensation in the microaqueous water-miscible solvent. The equilibrium conversion for the synthesis of lauroyl mannose largely depended on the kind of solvent as well as the water content. The KC also depended on the kind of solvent and was found to correlate well with the relative dielectric constant, r, of the solvent. The correlation would be useful in the selection of the solvent though the reason remains unclear. Acyl mannoses with chain lengths of 8 to 16 were continuously produced using a plugflow reactor packed with the immobilized lipase. Irrespective of acyl chain, a conversion of more than 50% was achieved at the superficial residence time, 0, equal to or longer than 20 min. It was demonstrated that 6-O-myristoyl mannose could be produced at a conversion of ca. 40% for at least 16 days using the reactor operated at 0 = 12 min. Since the enzyme could catalyze the synthesis of other acyl hexoses such as glucose, galactose and fructose, the present reactor system would be applicable for their production. The surfactant properties of the produced capryloyl, caproyl, lauroyl and myristoyl mannoses were also determined at 25oC, and were similar to those of acyl fructoses. Using three phenolic glycosides, which were arbutin, naringin and phloridzin, as a hydrophilic substrate, lauroyl phenolic glycosides were found to be synthesized through the condensation with lauric acid by the immobilized lipase in organic solvents. The conversion, however, depended on the polarity of the organic solvent used, and the interaction between hydrophilic substrate and organic solvent should be considered for the synthesis of acyl phenolic glycoside. There was no difference in the radical scavenging activity between unmodified and lauroyl phenolic glycosides, although the suppressive effect of each lauroyl phenolic glycoside against the oxidation of linoleic acid was higher than that of the corresponding phenolic glycoside. It was indicated that enhancement of the suppressive effect

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against lipid oxidation resulted due to increasing in the hydrophobicity of phenolic glycoside by the acylation. Enhanced hydrophobicity and high solubility in a lipid would enable acyl phenolic glycoside to act to linoleic acid inside the bulky acid, resulting in enhancement of the antioxidative activity. Syntheses of various antioxidative and edible surfactants using phenolic glycosides, such as polyphenols, from crude plant materials seem to be promising for the production of new food additives.

ACKNOWLEDGMENTS This study was partly supported by a Grant-in-Aid from Satake Technical Foundation, Japan, and a research grant (GS16) from Kinki University, Japan. The authors are very grateful to Dr. Ryuichi Matsuno, Professor Emeritus of Kyoto University and currently the president of Ishikawa Prefectural University for his valuable discussions and advises throughout this study, and sincerely appreciate Dr. Kazuhiro Nakanishi, Professor of Okayama University, for his collaboration. The authors would like to thank all the members of the Laboratory of Biomolecular Engineering, Department of Biotechnology and Chemistry, School of Engineering, Kinki University, and those of Bioengineering, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University.

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[25] Jia, C., Zhao, J., Feng, B., Zhang, X. and Xia, W. (2010). A simple approach for the selective enzymatic synthesis of dilauroyl maltose in organic media. Journal of Molecular Catalysis B: Enzymatic, 62, 265-269. [26] Villeneuve, P. (2007). Lipases in lipophilization reactions. Biotechnology Advances, 25, 515-536. [27] Scheckermann, C., Schlotterbeck, A., Schmidt, M., Wray, V. and Lang, S. (1995). Enzymatic monoacylation of fructose by two procedures. Enzyme and Microbial Technology, 17, 157-162. [28] Watanabe, Y., Miyawaki, Y., Adachi, S., Nakanishi, K. and Matsuno R. (2000). Synthesis of lauroyl saccharides through lipase-catalyzed condensation in microaqueous water-miscible solvents. Journal of Molecular Catalysis B: Enzymatic, 10, 241-247. [29] Watanabe, Y., Miyawaki, Y., Adachi, S., Nakanishi, K. and Matsuno, R. (2001). Equilibrium constant for lipase-catalyzed condensation of mannose and lauric acid in water-miscible organic solvents. Enzyme and Microbial Technology, 29, 494-498. [30] Watanabe, Y., Miyawaki, Y., Adachi, S., Nakanishi, K. and Matsuno, R. (2001). Continuous production of acyl mannoses by immobilized lipase using a packed-bed reactor and their surfactant properties. Biochemical Engineering Journal, 8, 213-216. [31] Watanabe, Y., Nagai, M., Yamanaka, K., Jose, K. and Nomura, M. (2009). Synthesis of lauroyl phenolic glycoside by immobilized lipase in organic solvent and its antioxidative activity. Biochemical Engineering Journal, 43, 261-265. [32] Uedaira, H., Ikura, M. and Uedaira H. (1989). Natural-abundance oxygen-17 magnetic relaxation in aqueous solutions of carbohydrates. Bulletin of the Chemical Society of Japan, 62, 1-4. [33] Uedaira, H., Ishimura, M., Tsuda, S. and Uedaira, H. (1989). Hydration of oligosaccharides. Bulletin of the Chemical Society of Japan, 63, 3376-3379. [34] Reichardt, C. (1979). Empirical parameters of solvent polarity as linear free-energy relationships. Angewandte Chemie International Edition, 18, 98-110. [35] Fredenslund, A., Jones, R. L. and Prausnitz, J. M. (1975). Group-contribution estimation of activity coefficients in nonideal liquid mixtures. AIChE Journal, 21, 1086-1099. [36] Asahara, T., Tokura, N., Okawara, M., Kumanotani, J. and Seno, M. (1976). Solvent Handbook. Tokyo, Kodansha Scientific. [37] Shinoda, K., Yamaguchi, T. and Hori, R. (1961). The surface tension and the critical micelle concentration in aqueous solution of -D-alkyl glucosides and their mixture. Bulletin of the Chemical Society of Japan, 34, 237-241. [38] Kobayashi, T., Adachi, S. and Matsuno, R. (1999). Synthesis of alkyl fucosides through -glucosidase-catalyzed condensation of fucose and 1-alcohols. Biotechnology Letters, 21, 105-109. [39] Hagerman, A. E., Riedel, K. M., Jones, G. A., Sovik, K. N., Ritchard, N. T., Hartzfeld, P. W. and Riechel, T. L. (1998). High molecular weight plant polyphenolics (tannins) as biological antioxidants. Journal of Agricultural and Food Chemistry, 46, 1887-1892. [40] Ursini, F., Rapuzzi, I., Toniolo, R., Tubaro, F. and Bontempelli, J. (2001). Characterization of antioxidant effect of procyanidins. Methods in Enzymology, 335, 338-350.

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[41] Guyot, S., Bourvellec, C. L., Marnet, N. and Drilleau, J. F. (2002). Procyanidins are the most abundant polyphenols in desert apples at maturity. Lebensmittel-Wissenschaft und –Technologie, 35, 289-291. [42] Funayama, M., Arakawa, H., Yamamoto, R., Nishino, T., Shin, T. and Murano, S. (1995). Effects of - and -arbutin on activity of tyrosinases from mushroom and mouse melanoma. Bioscience Biotechnology and Biochemistry, 59, 143-144. [43] Kurosu, J., Sato, T., Yoshida, K., Tsugane, T., Shimura, S., Kirimura, K., Kino, K. and Usami, S. (2002). Enzymatic synthesis of -arbutin by -anomer-selective glucosylation of hydroquinone using lyophilized cells of Xanthomonas campestris WU9701. Journal of Bioscience and Bioengineering, 93, 328-330. [44] Horowitz, R. M. and Gentili, B. (1969). Taste and structure in phenolic glycosides. Journal of Agricultural and Food Chemistry, 17, 696-700. [45] Yusof, S., Ghazali, H. M. and King, G. S. (1990). Naringin content in local citrus fruits. Food Chemistry, 37, 113-121. [46] Takii, H., Matsumoto, K., Kometani, T., Okada, S. and Fushiki, T. (1997). Lowering effect of phenolic glycosides on the rise in postprandial glucose in mice. Bioscience Biotechnology and Biochemistry, 61, 1531-1535. [47] Humeau, C., Girardin, M., Rovel, B. and Miclo, A. (1998). Enzymatic synthesis of fatty acid ascorbyl esters. Journal of Molecular Catalysis B: Enzymatic, 5, 19-23. [48] Watanabe, Y., Sawahara, Y., Nosaka, H., Yamanaka, K. and Adachi, S. (2008). Enzymatic synthesis of conjugated linoleoyl ascorbate in acetone. Biochemical Engineering Journal, 40, 368-372. [49] Watanabe, Y., Fang, X., Minemoto, Y., Adachi, S. and Matsuno, R. (2002). Suppressive effect of saturated acyl L-ascorbate on the oxidation of linoleic acid encapsulated with maltodextrin or gum arabic by spray-drying. Journal of Agricultural and Food Chemistry, 50, 3984-3987. [50] Kuwabara, K., Watanabe, Y., Adachi, S., Nakanishi, K. and Matsuno, R. (2003). Emulsifier properties of saturated acyl L-ascorbates for preparation of O/W emulsions. Food Chemistry, 82, 191-194. [51] Watanabe, Y., Adachi, S., Nakanishi, K. and Matsuno, R. (1999). Condensation of Lascorbic acid and medium-chain fatty acids by immobilized lipase in acetonitrile with low water content. Food Science and Technology Research, 5, 188-192. [52] Kobayashi, T., Furutani, W., Adachi, S. and Matsuno, R. (2003). Equilibrium constant for the lipase-catalyzed synthesis of fatty acid butyl ester in various organic solvents. Journal of Molecular Catalysis B: Enzymatic, 24･25, 61-66. [53] Hashimoto, N., Aoyama, T. and Shioiri, T. (1981). A simple efficient preparation of methyl easters with trimethylsilyldiazomethane (TMSCHN2) and its application to gas chromatographic analysis of fatty acids. Chemical and Pharmaceutical Bulletin, 29, 1475-1478. [54] Cunha, L. M., Oliveira, F. A. R. and Oliveira, J. C. (1998). Optimal experimental design for estimating the kinetic parameters of processes described by Weibull probability distribution function. Journal of Food Engineering, 37, 175-191. [55] Fujinami, Y., Tai, A. and Yamamoto, I. (2001). Radical scavenging activity against 1, 1-diphenyl-2-picrylhydrazyl of ascorbic acid 2-glucoside (AA-2G) and 6-acyl-AA-2G. Chemical and Pharmaceutical Bulletin, 49, 642-644.

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In: Organic Solvents Editor: Ryan E. Carter

ISBN 978-1-61761-881-9 © 2011 Nova Science Publishers, Inc.

Chapter 3

ANALYSIS OF THE ORGANIC SOLVENT EFFECT ON THE STRUCTURE OF DEHYDRATED PROTEINS BY ISOTHERMAL CALORIMETRY, DIFFERENTIAL SCANNING CALORIMETRY AND FTIR SPECTROSCOPY Vladimir A. Sirotkin A.M. Butlerov Chemical Institute, Kazan Federal University, Kazan, Russia

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ABSTRACT This review describes the basic principles of a novel method for studying the structure of the dehydrated proteins in the presence of organic solvents. This method, based on combined calorimetric and FTIR spectroscopic measurements, allows the simultaneous monitoring of the thermochemical parameters (interaction enthalpies, DSC thermograms) of the dried proteins and the corresponding changes in the protein structure in anhydrous organic solvents. This review aims to analyse the effect of organic solvents on dehydrated protein systems in order to understand what intra- and intermolecular processes produce the main effect on the structure and functioning of proteins in low water organic media. Two unrelated proteins with a high -helix content (human serum albumin, HSA) and with a high -sheet content (bovine pancreatic -chymotrypsin, CT) were used as models. Two groups of model organic solvents were used. The first group included hydrogen bond accepting solvents. The second group included hydrogen bond donating liquids. The results obtained showed that: 5) The enthalpy and integral structural changes accompanying the interaction of dried proteins with anhydrous organic solvents depend cooperatively on the solvent 

Kremlevskaya str., 18, Kazan, 420008, Russia. vsirotkin2006.narod.ru/Sirotkin_engl_2009.html.

e-mail:

[email protected].

Personal

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Vladimir A. Sirotkin hydrophilicity. The solvent hydrophilicity was characterized by an excess molar Gibbs energy of water in organic solvent at infinite dilution and 25oC. Based on this solvent hydrophilicity parameter, the solvents were divided into two groups. The first group included hydrophilic solvents such as methanol, ethanol, and dimethylsulphoxide (DMSO). Considerable structural rearrangements were observed in this group of solvents. The interaction enthalpies of the dried proteins with hydrophilic liquids were strongly exothermic. The second group included the hydrophobic and medium hydrophilic liquids such as benzene, dioxane, butanol-1, and propanol-1. The enthalpy and structural changes in the second group of solvents were close to zero. 6) The FTIR spectroscopic results can be attributed to the formation of different unfolded states of CT and HSA obtained upon dehydration-, alcohol- and DMSOinduced denaturation. The denatured state obtained in DMSO has a maximal degree of unfolding compared with that observed in alcohols or in the presence of dry air. 7) The effect of the organic solvent on the protein structure is ―protein selective‖. On the other hand, the organic solvent-induced integral structural changes versus solvent hydrophilicity profiles do not depend on the predominant form of secondary structure in the protein. 8) Heat-induced exothermic peaks were observed on the DSC thermograms of the dried proteins in anhydrous organic solvents in the temperature range 60-105 oC. This means that dehydrated proteins in anhydrous solvents is the non-equilibrium state at room temperature. These results give strong support to the idea that the nonequilibrium status of the dehydrated proteins results from the protein–organic solvent interactions being ―frozen‖ at near room temperature.

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The thermodynamic and structural data were analysed to give a unified picture of the state of the dried proteins in anhydrous organic solvents. According to this model, the dehydration-induced protein-protein contacts and the potential of the organic solvent to form the hydrogen bonds are key factors in determining the structure of the dehydrated proteins in the liquids under study.

1. BACKGROUND AND SIGNIFICANCE This review aims to solve a topical problem - elucidating the physico-chemical regularities of the structure and functioning of proteins in organic solvents. Due to the ability to vary the size, polarity, and strength of hydrogen bonds, organic solvents are increasingly being used in biophysical chemistry, biotechnology and medicine to selectively modulate the properties of protein systems. As an example of an innovation-promising scientific area, enzymatic catalysis in nonaqueous media (including organic solvents, ionic liquids, and supercritical fluids) has been intensively developed [1-7]. There are many advantages in employing nonaqueous media for biocatalysis, including the high solubility of hydrophobic reagents, the synthesis of useful chemicals, the suppression of undesirable side reactions caused by water and enhanced thermostability. Enzymatic catalysis in organic solvents is a competitive and cost-saving technology for producing substances with a high optical purity. High selectivity (perhaps the most attractive feature of enzymes in organic liquids) can be markedly affected, and sometimes even inverted, by the solvent. On the other hand, the reactions catalyzed by enzymes in organic liquids can take several hours [8-13]. The high long-term stability of biocatalysts is necessary

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to make enzyme catalysis in organic solvents industrially successful. Some studies have been published on the operational stability of enzymes in low water organic liquids. However, the mechanism of enzyme inactivation in low water organic solvents is still unclear. It is well known that water bound to proteins (hydration or biological water) plays a key role in determining the structure, dynamics and functions of proteins [14-16]. The structure and properties of proteins in organic liquids depends markedly on the hydration level, but in a complicated way [1-6]. This ―organic solvent‖ approach can be successfully extended to other, non-catalytic, biochemical processes, such as protein folding/unfolding [17]. However, the detailed mechanism of protein unfolding in the presence of organic solvents remains elusive. Therefore, it is clear that studying the regularities of the structure and functions of proteins in organic media makes it possible not only to optimize various biotechnological processes but also to substantially extend our fundamental knowledge about the forces maintaining the catalytically active conformation of enzymes in organic media and the role that water plays in protein functioning.

2. METHODOLOGY

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2.1. FTIR Spectroscopy FTIR-spectrometry was carried out on a Vector 22 (Bruker) FTIR-spectrophotometer at 4 cm-1 resolution as described previously [19, 20]. Vibration spectra were obtained using glassy-like protein films cast from 2% (w/v) water solution onto the CaF2 window at room humidity. After mounting the windows in the sample cell, the film was dehydrated by flushing with air dried over P2O5 powder. The relative vapor pressure over P2O5 at 25 oC does not exceed 0.01 [21]. The protein film was flushed until no further spectral changes were detected in the 3450 cm-1 water absorbance region and the amide A contour represented a smooth line without any visible shoulders. The cell was then filled with the chosen organic solvent. Spectra were recorded as a function of time until equilibrium was achieved. Then, spectra of the solvent without the protein sample were recorded and subtracted from the protein + solvent spectra. Protein spectra in a water environment were obtained from the protein films by flushing with air with a relative humidity of 98%. The spectrum of liquid water was then subtracted from the spectra of wet films. Changes in the protein structure were analyzed using the established correlation between secondary structural elements in proteins and the peak positions in the amide I spectra [22,23].

2.2. Isothermal Calorimetry The enthalpy changes upon the immersion of the dried and hydrated enzyme preparations into pure liquid water or anhydrous organic solvents were measured at 25 oC with a Setaram BT-2.15 calorimeter as described previously [24]. Typically, a sample of 5-10 mg of CT or human serum albumin came into contact with 4.0 ml of a solvent in the calorimetric cell. The

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calorimeter was calibrated using the Joule effect and tested by dissolving sodium chloride in water according to the recommendations. The dried protein preparations (zero hydration level) were obtained by drying under vacuum using a microthermoanalyser ‖Setaram‖ MGDTD-17S at 25oC and 0.1 Pa until a constant sample weight was reached. The water content of the dried proteins was estimated as 0.2  0.1 % (g water/g protein) using the Karl Fischer titration method according to previous recommendations [24].

2.3. Differential Scanning Calorimetry DSC measurements were carried out using a DSC 111 Setaram instrument as described previously [24]. The recording of the DSC curves was performed in a tightly closed titanium cell with a Teflon liner. The total volume of this cell was 120 l. Typically, 5–10 mg of a dried protein sample and 100 L of an organic solvent were placed in this titanium cell. The reference cell contained the same organic solvent in the absence of protein sample. The sample and reference cells were allowed to stabilize at 20 oC prior to the initiation of the scanning experiment. DSC thermograms were determined in the temperature range 25–150oC with the heating rate of 2 oC/min. Second runs of the dried proteins in organic solvents were also performed. No reversible phenomena were found. Naphthalene (Tfus=80.3 oС) was used as a reference compound for enthalpy calibration. Benzoic acid (Tfus=122.4 oС) was used to perform temperature calibration.

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2.4. Solubility Control The solubility of the dried human serum albumin and -chymotrypsin was controlled by optical density measurements using a Specord M-40 spectrophotometer at 260 - 300 nm (sensitivity limit of assay – 0.01 mg/ml). No protein was observed in the liquid phase. No noticeable variation in the absorbance of the liquid phase was observed after exposing the protein sample for at least 6 hours to the studied anhydrous organic solvents. In our experiments although DMSO did not dissolve the proteins it transformed the samples to a transparent dense gel. It is known that some non-aqueous solvents, for example, DMSO and formamide, can dissolve proteins [25, 26]. However, the mechanism underlying the protein dissolving potential of anhydrous organic solvents remains elusive. The solubility of proteins in organic solvents may depend strongly on a variety of factors such as pH, temperature, solvent humidity and electrolyte additives [25-27]. It is likely that deep dehydration of the proteins and the absence of salts may greatly decrease the solubility of macromolecules in anhydrous organic solvents.

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2.5. Water Content of Organic Solvents The water concentrations in organic solvents were determined using the Karl Fischer titration as described previously [24]: 0.02 mol/L in 1,4-dioxane, 0.01 mol/L in acetone, 0.005 mol/L in THF, 0.01 mol/L in acetonitrile, 0.07 mol/L in pyridine, 0.1 mol/L in DMFA, 0.2 mol/L in DMSO, 0.1 mol/L in methanol, 0.02 mol/L in ethanol, 0.005 mol/L in butanol-1, 0.02 mol/L in propanol-1, and 0.002 mol/L in hexanol-1. The water activities (aw) in organic solvents did not exceed 0.01. The water activities were calculated as aw = w*xw (where xw is the mole fraction of water in the solution and w is the activity coefficient of water (in mole fraction; the standard state is pure water)). The water contents of tetrachloromethane and benzene were 0.5; , relative activity 0.5; -, relative activity < 0.5. Relative activity is defined as ratio of lactic acid concentration of sample to that of control. Organic Solvents: Properties, Toxicity, and Industrial Effects : Properties, Toxicity, and Industrial Effects, edited by Ryan E. Carter, Nova Science

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When lactate was not produced, the Rhizopus oryzae JCM 5568 used in this study also failed to grow. Figure 1 shows microscopic photographs of Rhizopus oryzae JCM 5568 in the absence of solvent (A) and in the presence of decane (B) and heptane (C). We observed decomposition of the cell wall of Rhizopus oryzae JCM 5568 and oil droplets within the cells in the presence of heptane (C). Although the difference in the biocompatibilities of lactic acid-producing bacteria and fungi cannot be explained clearly, we expect that the solvent-tolerant mechanisms considered in the solvent-tolerant bacteria, such as the hydrophobicity of cell surfaces and membranes and the ability of the solvent-efflux pump, can be applied [13].

2. TOXICITY OF IMIDAZOLIUM-BASED IONIC LIQUIDS ON LACTIC ACID-PRODUCING BACTERIA

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The activities of the bacteria retained by cells exposed to three imidazolium-based ionic liquids, whose structures are shown in Figure 2, are shown in Figure 3. All bacteria produced lactic acid in the presence of ionic liquids. Among nine species of lactic acid-producing bacteria, L. delbruekii subsp. lactis NRIC 1683 exhibited relatively high lactic acid production. The lactic acid-producing activities of the bacteria generally decreased with increasing alkyl chain length in the imidazolium cation moiety. Figure 4 shows the effect of ionic liquids on the number of viable cells. All bacteria grew in the presence of a second phase of ionic liquids, although they failed to grow in the presence of a second phase of toluene.

Figure 1. Microscopic photographs of JCM 5568 in absence of solvent (A) and presence of decane (B) and heptane (C). Organic Solvents: Properties, Toxicity, and Industrial Effects : Properties, Toxicity, and Industrial Effects, edited by Ryan E. Carter, Nova Science

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We found that L. delbruekii subsp. lactis NRIC 1683 grew in the presence of imidazolium-based ionic liquids as well as in the absence of ionic liquids and that the increase in the alkyl chain length of imidazolium resulted in low microbial activity.

Figure 2. Imidazolium-based ionic liquids: R=C4H9; [Bmim]: R=C6H13; [Hmim]: R=C8H17; [Omim].

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Bar [14] studied the effect of solvent toxicity on microbial cells in a two-liquid phase system to distinguish between molecular toxicity, which was caused by solvent molecules dissolved in the aqueous phase, and phase toxicity, which was caused by the presence of a distinct second phase. This result suggests that phase toxicity might be caused by direct cellsolvent contact and the extraction of nutrients from the aqueous solution due to adherence of the cells to the interface. Molecular toxicity might result from enzyme inhibition, protein denaturation, and membrane modification due to the accumulation of solvent in the membrane. In this study, presence of solvents as a second phase may enhance the toxic effect of the ionic liquids. The increase in the alkyl chain length of imidazolium is expected to result in a decreased molecular toxicity effect. The mechanism of molecular toxicity in the presence of ionic liquids may be different from that of solvents, because organic solvents with log P > 3 are non-polar and the ionic liquids, which formed a second phase with water, were liquid salts with polarity similar to that of acetonitrile or methanol [15].

Figure 3. Microbial activities of lactic acid-producing bacteria in presence of a second phase of imidazolium-based ionic liquids (5%[v/v]). Relative activity is defined as ratio of lactic acid concentration of sample to that in absence of ionic liquids. Organic Solvents: Properties, Toxicity, and Industrial Effects : Properties, Toxicity, and Industrial Effects, edited by Ryan E. Carter, Nova Science

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Figure 4. Number of viable cells of lactic acid-producing bacteria in absence and presence of a second phase of imidazolium-based ionic liquids and organic solvents (5%[v/v]).

3. GREENNESS OF IONIC LIQUIDS AS AN ALTERNATIVE SOLVENT In the previous section, we described the nonfatal effect of the imidazolium-based ionic liquids on the lactic acid production process using bacteria. Although the evaluation of greenness for the lactic acid production process using microbes and immiscible solvent has not been reported yet, Reinhardt et al. [16] evaluated the greenness of the solvent systems for the Dies-Alder reaction of cyclopentadiene and methyl acrylate by employing a Simplified Life Cycle Assessment. Figures 5 and 6 show the energy factor (EF) and environmental and human health factor (EHF) of the various solvent systems, respectively. The results presented in Figure 5 indicate that the energy demand for the supply of the reaction media of the ionic liquid system is higher than that for the supply of conventional solvents. One major disadvantage of ionic liquid is caused by its relatively low thermal and pressure stability compared to the other solvents used. Therefore, ionic liquids become an attractive alternative to conventional solvents, if their separation efficiency and recyclability become high and their producing cost is greatly reduced. In Figure 6, methanol is classified as a toxic substance and therefore significantly impacts human health. Although the risk is assumed to be high for [Hmim][BF4], acute toxicity for humans is lower than for methanol systems because of its negligible vapor pressure. In their paper [16], only one example of a number of ionic liquids ([Hmim][BF4]) was presented. On the other hand, potential exists for the use of ionic liquids

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in the extractive fermentation of lactate, which was conducted under normal temperature and pressure. The advantage of ionic liquids regarding human and environmental factors can be directly utilized.

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Figure 5. Dependence of EF on solvent for Diels-Alder reaction of methyl acrylate and cyclopentadiene (solvent systems, 1: methanol, 2: methanol/water(50/50), 3: acetone, 4: cyclohexane, 5: [Hmim][BF4], 6: solvent-free).

Figure 6. Dependence of EF on solvent for Diels-Alder reaction of methyl acrylate and cyclopentadiene (solvent systems, 1: methanol, 2: methanol/water(50/50), 3: acetone, 4: cyclohexane, 5: [Hmim][BF4], 6: solvent-free). Organic Solvents: Properties, Toxicity, and Industrial Effects : Properties, Toxicity, and Industrial Effects, edited by Ryan E. Carter, Nova Science

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CONCLUSION The tolerance of lactic acid-producing bacteria and fungi to organic solvents and imidazolium-based ionic liquids was studied. A solvent with a high log P exhibits the highest metabolic activities and solvents with lower log P were toxic to lactic acid-producing microbes. Lactic acid producing-fungi showed higher tolerance to aliphatic alcohols compared to hydrocarbons. The log P value was found to be a reliable index for the toxicity of organic solvents to lactic acid-producing microbes. The greenness of extractive fermentation using ionic liquids depends on their production costs. Therefore, as an alternative of the ionic liquid-broth two phase system, a supported ionic liquid membrane process has great potential because a supported liquid membrane process, which combines the extraction and stripping processes, has several advantages over liquid-liquid extraction systems, such as low energy consumption, high selectivity, and a considerably reduction in the amount of solvent used [17].

REFERENCES

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[1]

Matsumoto, M.; Isken, S.; de Bont, J. A. M., Organic solvents in microbial production processes. p. 856-865. In Wypych, G. (ed.), Handbook of solvents. Chemtec, Toronto (2000) [2] Pham, T.P.T; Cho, C.-W.; Yun, Y.-S., Environmental fate and toxicity of ionic liquids: A review. Water Res., 2010, 44, 352-372. [3] Ranke, J.; Stolte, S.; Strmann, R.; Arning, J.; Jastoff, B., Design of sustainable chemical products –The example of ionic liquids. Chem. Rev., 2007, 107, 2183-2206. [4] Wasewar, K. L.; Heesink, A. B. M.; Versteeg, G. F.; Pangarkar, V. G., Equilibria and kinetics for reactive extraction of lactic acid using Alamine 336 in decanol. J. Chem. Technol. Biotechnol., 2002 77, 1068-1075. [5] Hartl, J.; Marr, R.: Extraction processes for bioproduct separations. Separ. Sci. Technol., 1993, 28, 805-812. [6] Siebold, M.; Frieling, P. V.; Joppien, R.; Rindfleisch, D.; Schügerl, K., Comparison of the production of lactic acid by three different Lactobacilli and its recovery by extraction and electrodialysis. Process Biochem., 1995, 30, 81-95. [7] Tong, Y.; Hirata, M.; Takanashi, H.; Hano, T.; Matsumoto, M.; Miura, S., Solvent screening for production of lactic acid by extractive fermentation. Separ. Sci. Technol., 1998, 33, 1439-1453. [8] Playne, M. J.; Smith, B. R., Toxicity of organic extraction reagents to aerobic bacteria. Biotechnol. Bioeng., 1983, 25, 1251-1265. [9] Roffer, S. R.; Blanch, H. W.; Wilke, C. R., In situ recovery of fermentation products, Trends Biotechnol., 1984, 2, 129-136. [10] Demirci, A.; Pometto, A. L., III; Harkins, K. R., Rapid screening of solvents and carrier compounds for lactic acid recovery by emulsion liquid extraction and toxicity on Lactobacillus casei (ATCC 11443). Bioseparation, 1999, 7, 297-308. [11] Laane, C.; Boeren, S.; Vos, K.; Veeger, C., Rules of optimization of biocatalysis in organic solvents. Biotechnol. Bioeng., 1987, 30, 81-87.

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[12] León, R.;, Fernandes, P.; Pinheiro, H. M.; ;Cabral, J. M. S., Whole-cell biocatalysis in organic media. Enzyme Microb. Technol., 1998, 23, 483-500. [13] Matsumoto, M.; de Bont, J. A. M.; ;Isken, S., Isolation and characterization of the solvent-tolerant Bacillus cereus strain R1. J. Biosci. Bioeng., 2002, 94, 45-51. [14] Bar, R., Effect of interphase mixing on a water organic solvent two-phase microbial system: Ethanol fermentation. J. Chem. Technol. Biotechnol., 1988, 43, 49-62. [15] Aki, S. N. V. K.; Brennecke, J. F.; Samanta A., How polar are room-temperature ionic liquids? Chem. Commun., 2001, 413-414. [16] Reinhardt, D.; Ilgen, F.; Kralisch, D.; Kӧ nig, B.; Kreisel, G., Evaluating the greenness of alternative reaction media. Green Chem., 2008, 10, 1170-1181. [17] Matsumoto, M.; Hasegawa, W.; Kondo, K; Shimamura, T.; Tsuji, M., Application of supported ionic liquid membranes using a flat sheet and hollow fibers to lactic acid recovery, Desal. Water Treat., 2010, 14, 37-46

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In: Organic Solvents Editor: Ryan E. Carter

ISBN 978-1-61761-881-9 © 2011 Nova Science Publishers, Inc.

Chapter 6

EFFECT OF HYDROGEN BOND ACCEPTING ORGANIC SOLVENTS ON THE BINDING OF COMPETITIVE INHIBITOR AND STORAGE STABILITY OF -CHYMOTRYPSIN 1

Vladimir A. Sirotkin Kazan Federal University, A.M. Butlerov Chemical Institute, Kremlevskaya str., 18, Kazan, 420008 Russia, e-mail: [email protected], Personal site: http://vsirotkin2006.narod.ru/Sirotkin_engl_2009.html

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ABSTRACT This review aims to analyse the studies of the competitive inhibitor binding and the storage stability of bovine pancreatic -chymotrypsin (CT) in organic solvents in order to elucidate what intermolecular processes produce the main effect on the state and functioning of enzymes at high and low water activities in organic media. The binding of competitive inhibitor proflavin and the storage stability of CT in water-organic mixtures were studied in the entire range of thermodynamic water activities (aw) at 25oC. The moderate-strength hydrogen bond accepting solvents (acetonitrile, dioxane, tetrahydrofuran, and acetone) were used as models due to their ability to vary significantly the size, polarity, denaturation capacity, and hydrophobicity. The state of water hydrogen bond network in organic solvents was characterized by thermodynamic and spectroscopic data. The absorption spectra of water in organic solvents were measured by FTIR spectroscopy. The state of water in organic solvents was defined in terms of variations in the integral intensity of water and the contour shape of the band of OH stretching vibrations. Excess chemical potentials, partial molar enthalpies, and entropies of water and organic solvents were simultaneously evaluated at 25oC. The results obtained showed that: 1

A version of this chapter was also published in Advances in Medicine and Biology, Volume 5, edited by Leon V. Berhardt published by Nova Science Publishers, Inc. It was submitted for appropriate modifications in an effort to encourage wider dissemination of research.

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Vladimir A. Sirotkin 4) The proflavin binding and storage stability curves can be unified in the water activity coordinates. At the highest water activities (aw>0.95), the water hydrogen bond network is bond –percolated. In this composition region, the storage stability values are close to 100%. 5) At the lowest water activities, the water molecules exist predominantly as single molecules complexed with organic solvent molecules. No proflavin binding was observed at low water activity values in the studied solvents. At aw>0.3, the proflavin binding is sharply increased reaching a maximal value at aw~0.5-0.6. This sharp increase in the enzyme activity occurs only above the threshold water activity level, when the self-associated (H-bonded) water molecules appear in the studied organic solvents. 6) In the intermediate composition region, the solution consists of two kinds of clusters, each rich in each component. There is a sharp transition from the water-rich region to the intermediate one. This transition is associated with an anomaly in the thermodynamic, structural, and enzyme activity properties. This transition may involve loss of the bond percolated nature of the hydrogen bond network of liquid water. The residual catalytic activity of CT changes from 100 to 0% in the transition region. A minimum on the competitive inhibitor binding and storage stability curves was observed at aw of 0.8-0.9. The thermodynamic, structural, and enzyme activity data were analysed to give a unified picture of the state of enzymes in low water organic solvents. According to this model, the dehydration-induced protein-protein contacts and the state of water hydrogen bond network play a key role in determining the enzyme activity – water activity profiles in organic liquids.

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1. BACKGROUND AND SIGNIFICANCE This review aims to solve a topical problem - elucidating the physico-chemical regularities of the functioning of enzymes in organic media with low water content. This problem is of considerable interest for nonaqueous enzymology, an innovation-promising scientific area [1-8]. The use of organic solvents as a reaction medium makes it possible to successfully conduct enzymatic reactions with hydrophobic compounds poorly soluble in water. Nonaqueous organic media provide the possibility of conducting industrially important synthetic reactions that do not occur in aqueous media (for example, peptide synthesis and esterification). The enzymatic catalysis in organic solvents is competitive and cost-saving technology for producing substances with a high optical purity. On the other hand, reactions catalyzed by enzymes in organic liquids can take some hours. High long-term stability of biocatalysts is necessary to make enzyme catalysis in organic solvents industrially successful. Some works have been published on the operational stability of enzymes in low water organic liquids. Enzymes lost most of its activity exponentially upon storage in organic solvents [9-12]. However, the mechanism of enzyme inactivation in low water organic solvents is still unclear. Therefore, it is clear that studying the regularities of biocatalysis in organic media makes it possible not only to optimize various biotechnological processes but also substantially extent fundamental knowledge on the stability of enzyme macromolecules and on the forces maintaining the catalytically active conformation of enzymes under conditions of low water content. At present, there is solid evidence that reactions catalyzed by α-chymotrypsin in water and organic media proceed via a single mechanism [13]. The classical competitive inhibitors

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of reactions catalyzed by α-chymotrypsin are aromatic compounds, including proflavin (3,6diaminoacridine, Fig. 1) [14-18].

H2N

N

NH2

Figure 1. Structure of proflavin.

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One advantage of proflavin is its ability to form a 1:1 complex with the active site of the α-chymotrypsin molecule. Therefore, studying the regularities of the binding of the competitive inhibitor may be very informative for understanding the nature of the intermolecular forces that determine the state of the enzyme active site in the presence of organic media. For example, it was previously shown that hydrophobic effect is responsible for the affinity of competitive inhibitor to enzyme in the mixtures with high water content [14]. Binding of proflavin to chymotrypsin was studied in water-dimethylsulfoxide (DMSO) mixtures (water content more than 40 vol. % or water activity more than 0.65) [15]. Fink showed that enzyme affinity to proflavine decreases with the increase of organic solvent content. This effect was contributed to both decrease of medium polarity and competitive inhibition of enzyme by DMSO. However, no attempt has been undertaken to investigate the interaction of proflavin with chymotrypsin in organic solvents with low water content. The aim of the present work was to study the effect of organic solvents on the binding of the competitive inhibitor proflavin and the storage stability of -chymotrypsin in order to elucidate what intermolecular processes produce the main effect on the state and functioning of enzymes at high and low water activities in organic media.

2. BINDING OF THE COMPETITIVE INHIBITOR PROFLAVIN AND THE STORAGE STABILITY OF -CHYMOTRYPSIN IN ORGANIC SOLVENTS 2.1. Choice of Organic Solvents The choice of acetonitrile, dioxane, acetone, and tetrahydrofuran as model solvents was determined by the following reasons: a) Due to the ability to vary the size, polarity, denaturation capacity, and hydrophobicity (Table 1, Fig.2), these organic solvents are used increasingly in biophysical chemistry, biotechnology, and nonaqueous enzymology to selectively modulate the properties of enzymes [1-12]. b) They are water-miscible organic liquids. Therefore, it is possible to study the effect of these low molecular organic substances on the enzyme activity over the whole range of thermodynamic water activity.

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c) Acetonitrile, dioxane, acetone, and tetrahydrofuran are hydrogen bond accepting solvents. They are capable of forming hydrogen bonds with various hydrogen bond donors. However, in contrast to water, they have no evident hydrogen bond donating ability. O O

O

tetrahydrofuran

1,4-dioxane

H3C N

C

CH3

CH3

acetonitrile

O

acetone Figure 2. Structures of 1,4-dioxane, tetrahydrofuran, acetonitrile, and acetone.

Table 1. Physico-chemical parameters of organic solvents at 25oC. Log P is the measure of solvent hydrophobicity (where P is the partition n-octanol-water coefficient); ε is the Copyright © 2011. Nova Science Publishers, Incorporated. All rights reserved.

dielectric constant; Vm is the molar volume of organic solvent;

H 2O / S H int (spec.)

is

the enthalpy of specific interaction (hydrogen bonding of water with organic solvent).

Solvent

log P [20]

ε [21]

Denaturation capacity [22]

Vm, cm3/mole

Acetonitrile 1,4-Dioxane THF Acetone

-0.33 -1.1 0.46 -0.24

37 2.2 7.6 20.5

64.3 92.1 100 78.2

52.6 85.2 81.1 73.4

H 2O / S H int (spec.) , kJ/mole [23] -18.0 -19.3 -19.7 -20.5

2.2. Thermodynamic Activity of Water in Organic Solvents It is well known that water – organic mixtures are typical examples of nonideal systems where the thermodynamic characteristics of the state of water differ for the same molar ratio of components due to strong specific interactions between components (first of all, hydrogen bonding). To apply a correction for nonideality of a system, the concentrations of the substances are recalculated into thermodynamic activity scale. The thermodynamic activity of water (or organic solvent) is related to the chemical potential of the solute as (Eq. 1):

Organic Solvents: Properties, Toxicity, and Industrial Effects : Properties, Toxicity, and Industrial Effects, edited by Ryan E. Carter, Nova Science

Effect of Hydrogen Bonding Accepting Organic Solvents… μi = μio + RT lnai

119 (1)

where μio is the standard chemical potential, and ai is the activity of the ith component in the solution. Accordingly, the Gibbs energy change ( G ) for the transfer of one mole of water from pure liquid state to aqueous organic medium with a mole fraction of water x w equals (Eq. 2):

G  RT ln a w .

(2)

Therefore, the use of the thermodynamic activity scale allows one to normalize the concentration dependencies of characteristics of the effects under study with the respect to the Gibbs energy of water transfer from pure liquid to an water – organic medium. Water activity (aw) in organic solvent was calculated using the Eq. 3:

aw   w x w

(3)

where xw is the mole fraction of water in the solution and w is the activity coefficient of water (in mole fraction; the standard state is pure water). Water activity coefficients (w) were calculated using literature data [24-26] on the vapor-liquid equilibrium according to the Eq. 4.

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y P  w  w tot o x wPw

(4)

where yw is the measured mole fraction of water in vapor phase, Pt is the total pressure, Po is the saturated vapor pressure of pure water at the same temperature and xw is the mole fraction of water in the liquid phase.

Figure 3. Thermodynamic water activity plotted versus water mole fraction in organic solvent at 25oC: 1 – THF, 2 – acetonitrile, 3 – acetone, 4 - dioxane. The dashed line corresponds to the ideal binary mixture. Organic Solvents: Properties, Toxicity, and Industrial Effects : Properties, Toxicity, and Industrial Effects, edited by Ryan E. Carter, Nova Science

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Fig. 3 shows the thermodynamic activity of water plotted versus the water mole fraction in organic solvents at 25oC.

2.3. Binding of Proflavin in Water

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The measurements conducted on a Perkin-Elmer Lambda 35 double-beam scanning spectrophotometer at 25oC. The concentration of Tris-HCl buffer was 0.05 M (the pH value of the aqueous solution was 8.0). The initial state of -chymotrypsin in the experiments with proflavin was a solid enzyme preparation with a humidity of 8.6 % (g water/g protein). Proflavin shows a pronounced tendency toward micelle formation at concentrations more than 120 µM [17]. The formation of dye aggregates leads to deviations from the LambertBeer relationship. In order to avoid complications arising from proflavin aggregation, all the spectrophotometric measurements were performed at the proflavin concentrations not greater than 10 µM. Therefore, it is more convenient to study the -chymotrypsin-competitive inhibitor interactions in an excess of enzyme. Proflavin concentrations were determined by absorption spectroscopy at 444 nm using an extinction coefficient of 34,000 cm M-1. Typical absorbance spectra of proflavin in water at pH 8.0 are presented in Fig. 4A. The interaction of proflavin with α-chymotrypsin results in a shift of the spectrum of the dye (λmax=444 nm in water) to a longer wavelength. Fig. 4B shows the difference spectra of proflavin in the presence of α-chymotrypsin recorded with respect to the initial solution of proflavin in water. The optical density of the difference spectrum at the maximum (λmax=465 nm in water) is proportional to the concentration of proflavin bound in the complex. As the concentration of α-chymotrypsin increases, the amplitude of the maximum in the difference spectra increases tending to its limiting value (Fig. 5A). The binding of proflavin (P) to α-chymotrypsin (E) can be represented as:

E+P

KEP

EP

(5)

where KEP is the association constant. The enzyme has negligible absorbance in the region in which the difference between the absorbances of free and bound proflavin attains its maximum value. Therefore, it can be shown that: ΔА = (εЕР - εр)[EP]

(6)

where ∆A is the measured difference in the absorption at a given wavelength; εр and εЕР are the extinction coefficients of free and bound proflavin, respectively, at the same wavelength. The difference in the absorption attains its maximum value (Eq. 7) ΔАmax = (εЕР - εр)[P]0

(7)

when all the proflavin present is bound in complex and the concentration of complex is equal to the initial concentration of proflavin. At [E]0>>[EP]0 (corresponds to our experimental conditions), the expression for ΔА reads:

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Effect of Hydrogen Bonding Accepting Organic Solvents…

A 

Amax K EP [ E ]0 1  K EP [ E ]0

121

(8)

where [E]0 is the total concentration of the enzyme. The processing of the experimental data within the framework of this model makes it possible to obtain the value of KEP. In the 1/ ΔА versus 1/[E]0 coordinates, Eq. (8) becomes linear (Fig. 5B):

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1 1 1   A A max A max  K EP  [E]0

(9)

Figure 4A. Typical absorption spectra of proflavin in water (0.05 Tris buffer, pH 8.0) at various αchymotrypsin concentrations and 25oC. Proflavin concentration is 10 μM.

Figure 4B. Typical difference spectra of proflavin in water (0.05 Tris buffer, pH 8.0) at various αchymotrypsin concentrations and 25oC. Proflavin concentration is 10 μM. Organic Solvents: Properties, Toxicity, and Industrial Effects : Properties, Toxicity, and Industrial Effects, edited by Ryan E. Carter, Nova Science

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Figure 5A. The ΔA465 value as a function of the enzyme concentration. Initial proflavin concentration is 10 μM.

Figure 5B. Plot of Eq. (8): the intercept, 1/ΔAmax = 6.5 (0.4); the slope, 1/(ΔAmaxKEP) = 2.6×10–4 (5.5×10–6); the number of experimental points, N = 7; the standard deviation, s0 =0.69; and the correlation coefficient, R = 0.999.

The value of KEP can be obtained by dividing the intercept by the slope. As a result, we obtained KEP = 24,500±2000 M–1 (pH 8.0, 25oC). Table 2 shows that the KEP value obtained

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in this work is in good agreement with the previously published results. This is indicative of the reliability of the UV spectrophotometric method used. At a given concentration of α-chymotrypsin, Eq. (8) makes it possible to estimate the fraction of bound proflavin from the ratio ∆A/∆Amax. For example, at the α-chymotrypsin concentration of 200 μM (pH 8.0, 25oC), the fraction of bound proflavin in pure water solution was found to be 83.1%. Table 2. Association constants for the -chymotrypsin-proflavin complex. KEP, M-1 24,500 ±2,000 25,700 22,000 24,000

Reference pH 8.0 [This work] pH 8.0 [16] pH 7.6 [17] pH 8.0 [18]

2.4. Spectra of Proflavin in Water-Organic Mixtures

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Fig. 6-9 shows spectra of proflavin in water-organic mixture. As can be seen from Fig. 69, organic solvents produce significant effect on the shape of the proflavin spectra. For example, at high water activities, the shape of the spectra and the position of the maximum are similar to those observed in pure water at pH 8.0. At aw < 0.5, however, the shape of the proflavin spectrum is markedly different: a new shortwave absorption band appears. The intensity of this band increases with the organic solvent concentration, while the intensity of the longwave band concurrently decreases. This behavior was interpreted as reflecting the coexistence of two forms of proflavin, protonated and deprotonated. As the water content in the water-organic mixtures decreases, the equilibrium shifts towards the deprotonated form. This conclusion is supported by the results of the following experiment.

Figure 6. Absorbance spectra of proflavin in acetonitrile at various water activity values. Organic Solvents: Properties, Toxicity, and Industrial Effects : Properties, Toxicity, and Industrial Effects, edited by Ryan E. Carter, Nova Science

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Figure 7. Absorbance spectra of proflavin in dioxane at various water activity values.

Figure 8. Absorbance spectra of proflavin in THF at various water activity values.

Figure 9. Absorbance spectra of proflavin in acetone at various water activity values.

Fig. 10 displays the spectra of proflavin in water at various pH values. As seen, as in the case of water-organic mixtures, variations in the pH values are accompanied by the Organic Solvents: Properties, Toxicity, and Industrial Effects : Properties, Toxicity, and Industrial Effects, edited by Ryan E. Carter, Nova Science

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redistribution of the abundances of the protonated (predominant at low pH values) and deprotonated (predominant at high pH values) forms of proflavin.

Figure 10. Typical absorption spectra of proflavin in water at various pH values.

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2.5. Binding of Proflavin in Organic Solvents Fig. 11A shows typical difference spectrum of proflavin measured in the presence of αchymotrypsin with respect to the initial solution of proflavin in an aqueous-acetonitrile mixture with low water activity (aw=0.12). The presence of the enzyme produces no effect on the proflavin spectra. Similar results were obtained for dioxane, THF, and acetone (Fig. 12A14A). This means that in these mixtures no binding of proflavin to the enzyme occurs. Fig. 11B-14B show the difference spectra of proflavin recorded in acetonitrile, dioxane, THF, and acetone in the water activity range from 0.4 to 0.9. Within this range of water activities, α-chymotrypsin was insoluble. Therefore, the dip in the difference spectra was attributed to a decrease in the concentration of proflavin due to its binding by the enzyme.

Figure 11. Typical difference spectra of proflavin in water-acetonitrile mixtures with various thermodynamic activities of water in the presence of -chymotrypsin: (A) aw =0.12, (B) aw =0.49. The initial concentrations of proflavin and enzyme were 10 µM and 200 µM, respectively. The Tris-HCl buffer concentration was 0.05 M.

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The binding of proflavin was qualitatively characterized by the absorbance at the isosbestic point (at 414 nm). This was motivated by the following circumstances. It is unknown what form of proflavin, protonated or deprotonated, is bound by the enzyme in aqueous-organic mixtures. On the other hand, at the isobestic point the absorption coefficients of both forms coincide. Correspondingly, by measuring changes in the absorbance at the isosbestic point one can determine changes in the concentration of the competitive inhibitor in the solution irrespective of the bound form. The ratio of the absorbance at the the isosbestic point of the difference spectrum to the absorbance at the isosbestic point of the spectrum of the initial solution is a measure of the fraction of bound proflavin at a given concentration of the enzyme. The degree of binding of proflavin to α-chymotrypsin at a constant concentration of the latter (200 µM) is displayed in Fig. 15 and 16.

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Figure 12. Typical difference spectra of proflavin in water-dioxane mixtures with various thermodynamic activities of water in the presence of -chymotrypsin: (A) aw =0.24, (B) aw =0.5. The initial concentrations of proflavin and enzyme were 10 µM and 200 µM, respectively. The Tris-HCl buffer concentration was 0.05 M.

Figure 13. Typical difference spectra of proflavin in water-tetrahydrofuran mixtures with various thermodynamic activities of water in the presence of -chymotrypsin: (A) aw =0.2, (B) aw =0.5. The initial concentrations of proflavin and enzyme were 10 µM and 200 µM, respectively. The Tris-HCl buffer concentration was 0.05 M.

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Figure 14. Typical difference spectra of proflavin in water-acetone mixtures with various thermodynamic activities of water in the presence of -chymotrypsin: (A) aw =0.33, (B) aw =0.6. The initial concentrations of proflavin and enzyme were 10 µM and 200 µM, respectively. The Tris-HCl buffer concentration was 0.05 M.

Figure 15. Binding of proflavin by -chymotrypsin in organic solvents as a function of water mole fraction.

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Figure 16. Binding of proflavin by -chymotrypsin in organic solvents as a function of water volume content.

Figure 17. Binding of the competitive inhibitor proflavin by -chymotrypsin in organic solvents as a function of water activity. All values are the averages of three measurements.

No correlation was observed when proflavin binding was plotted versus water mole fraction or water volume content in organic solvents (Fig. 15 and 16). However, the proflavin binding curves can be unified in the water activity coordinates (Fig. 17). No proflavin binding was observed at low water activity values in acetonitrile, dioxane, acetone, and THF. At aw~ 0.35, the proflavin binding is sharply increased reaching a maximal value at aw~0.5-0.6. A minimum in the competitive inhibitor binding was observed at aw of 0.8-0.9.

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2.6. Enzyme Storage Stability Enzyme stability was determined by measuring the enzyme activity after storage in water - organic mixtures as described previously [27]. The model process was the hydrolysis of Nacetyl-L-tyrosine ethyl ester (ATEE, Fig. 18) catalyzed by -chymotrypsin. The measurements were performed on a Hiranuma Comtite-101 potentiometric titrator (Japan) in the pH-static mode at pH 8.0 and 25oC (Fig. 19). CH3 O O

H2C

O

NH

H2C

OH

H3C

Figure 18. Structure of N-acetyl-L-tyrosine ethyl ester.

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The concentration of the substrate was 4.0 mM. In the course of the experiment, the pH value was maintained at a constant level by adding a titrant (a potassium hydroxide solution of known concentration), which neutralized the acid (N-acetyl-L-tyrosine) released during the hydrolysis. The kinetic curve obtained was the time dependence of the amount of the reagent spent for titrating the acid released. Each kinetic curve was reproduced not less than three times.

Figure 19. Schematic representation of the experimental set-up of potentiometric measurements. The components of the experimental set-up: 1 - Hiranuma Comtite-101 potentiometric titrator, 2 – computer, 3 – pump, 4 – electrodes, 5 – electrochemical cell, 6 – thermostat.

The reaction mixture was prepared as follows. A lyophilized -chymotrypsin preparation with a humidity of 8.60.2 % (g water/g enzyme) was immersed in an aqueous-organic mixture of required composition and was incubated at 25oC for 1 h. This time period exceeded the time corresponding to the completion of the calorimetric heat effect accompanying the interaction of dehydrated proteins with pure organic solvents and waterorganic mixtures [28,29]. Water content of the initial chymotrypsin samples was measured by drying using a microthermoanalyzer ‖Setaram‖ MGDTD-17S at 25oC K and 0.1 Pa until the

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constant sample weight was reached. The content of the -chymotrypsin in the mixture was 1 mg/ml. Adding 100-µL aliquots of the aqueous-organic solution of -chymotrypsin to the aqueous solution of the substrate, we initiated the enzymatic reaction. A special experiment was performed to test the reliability of our method. For this purpose, we calculated the kinetic parameters of the enzymatic reaction in water at pH 8.0 and 25oC by using the Michaelis-Menten equation in the integral form: KM=1.1(0.1) mM, Vmax/[E]o=209 (15) s-1. Under similar conditions (25oC, pH 8.2), the authors of [30] obtained KM=1.2 mM, Vmax/[E]o=155 s-1. These results show that our method is quite reliable. The content of organic solvents in the final reaction mixture did not exceed 0.5 vol% in all cases. A special experiment was performed to test this effect on the enzyme deactivation. For this purpose, we studied the enzymatic reaction in the water-organic mixtures (0.5 vol% of organic solvent). The KM and Vmax/[E]o values determined in the presence of 0.5% of organic solvents are given in Table 3. Table 3. The KM,I, Vmax/[E]o, and KI values determined in the presence of 0.5% of hydrogen bond accepting solvent at 25oC. Solvent Acetone Acetonitrile 1,4-Dioxane Tetrahydrofuran

KM,I (mM) 1.16 1.22 1.30 1.40

Vmax/[E]o (s-1) 205 200 195 201

KI (mM) 1.2 [this work] 1.23 [31] 0.79 [this work] 0.83 [31] 0.33 [this work] 0.32 [31] 0.23

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The competitive effect of organic solvents was quantitatively estimated. When organic solvent acts as a competitive inhibitor, the Michaelis constant in the presence of competitive inhibitor can be defined as follows:

K M,I  (1 

[I] ])K M KI

(10)

where [I] is the competitive inhibitor conсentration (M), KI is the dissociation constant of the enzyme-competitive inhibitor complex (M), KM,I is the Michaelis constant in the presence of competitive inhibitor, KM is the Michaelis constant in the absence of any inhibitor (M). It can be concluded from Table 3 that the KI values for acetonitrile, acetone, and dioxane are in agreement with the results of Bender et al [31]. They determined the dissociation constants of the chymotrypsin-competitive inhibitor complex for hydrogen bond accepting solvents. For example, the KI value for 1,4-dioxane was found to be 0.32 (pH 7.8, 25oC). For comparison, the KI value for benzene (aromatic organic solvent, typical competitive inhibitor of chymotrypsin) is 0.0047 [14]. This means that the inhibition effect of the hydrogen bond accepting solvents is weak in the mixtures studied.

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Figure 20. Typical kinetic curves for the hydrolysis of N-acetyl-L-tyrosine ethyl ester catalyzed by chymotrypsin preliminary incubated in water-acetonitrile mixtures with various thermodynamic activities of water: (1) 1.0, (2) 0.11, (3) 0.49, (4) 0.60, (5) 0.75, (6) 0.81, (7) 0.87, (8) 0.92.

Fig. 20-23 display typical kinetic curves for the hydrolysis of ATEE catalyzed by chymotrypsin preliminary incubated in water-organic mixtures. The catalytic activity was characterized by the ratio of the extent of hydrolysis attained within 200 s with chymotrypsin incubated in a water-organic mixture to the same quantity measured using chymotrypsin incubated in pure water (Fig. 20, curve 1).

Figure 21. Typical kinetic curves for the hydrolysis of N-acetyl-L-tyrosine ethyl ester catalyzed by chymotrypsin preliminary incubated in water-dioxane mixtures with various thermodynamic activities of water: (1) 1.0, (2) 0.876, (3) 0.80, (4) 0.01.

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Figure 22. Typical kinetic curves for the hydrolysis of N-acetyl-L-tyrosine ethyl ester catalyzed by chymotrypsin preliminary incubated in water-THF mixtures with various thermodynamic activities of water: (1) 1.0, (2) 0.01, (3) 0.7, (4) 0.87.

Figure 23. Typical kinetic curves for the hydrolysis of N-acetyl-L-tyrosine ethyl ester catalyzed by chymotrypsin preliminary incubated in water-acetone mixtures with various thermodynamic activities of water: (1) 1.0, (2) 0.01, (3) 0.872.

As can be concluded from Fig. 24-27, organic solvents affect the catalytic activity of the biocatalyst in a complicated way. At water activities from 0 to 0.4, the residual catalytic activity remains virtually constant, equal to ~60-90% compared with that observed after incubation in pure water. As the water activity increases from 0.4 to 0.8, the residual catalytic activity decreases sharply. At aw > 0.8-0.9, the residual catalytic activity increases, approaching the level corresponding to pure water. The Km and Vmax values for the hydrolysis of N-acetyl-L-tyrosine ethyl ester catalyzed by -chymotrypsin preliminary incubated in water-organic mixtures were calculated by using the Michaelis-Menten equation in the integral form. As can be seen from Fig. 28B-31B, the Km values do not depend noticeably on the water activity in organic solvents studied. On the other hand, the shape of the Vmax – water activity curves are consistent with the storage stability dependences (Fig. 28A-31A). The Vmax values include two contributions: k2 and ET (the total amount of enzyme). Overall, this means that the changes in the V max values occur probably due to the decrease in the amount of the catalytically active form of -chymotrypsin during the incubation in water – organic mixtures.

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Figure 24. (1) Solid-state solvent-free hydrolysis of N-succinyl-L-phenylalanine-p-nitroanilide. Modified data from [32]. (2) Hydrolysis of N-acetyl-L-tyrosine ethyl ester by -chymotrypsin preliminary incubated in water-acetonitrile mixtures. (3) Binding of proflavin by -chymotrypsin in water-acetonitrile mixtures.

Figure 25. (1) Solid-state solvent-free hydrolysis of N-succinyl-L-phenylalanine-p-nitroanilide. Modified data from [32]. (2) Hydrolysis of N-acetyl-L-tyrosine ethyl ester by -chymotrypsin preliminary incubated in water-dioxane mixtures. (3) Binding of proflavin by -chymotrypsin in waterdioxane mixtures.

Figure 26. (1) Solid-state solvent-free hydrolysis of N-succinyl-L-phenylalanine-p-nitroanilide. Modified data from [32]. (2) Hydrolysis of N-acetyl-L-tyrosine ethyl ester by -chymotrypsin preliminary incubated in water-THF mixtures. (3) Binding of proflavin by -chymotrypsin in waterTHF mixtures.

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Storage stability of α-chymotrypsin depends markedly on the hydration history. There is a strong difference between the storage stability of the initially hydrated and dehydrated CT. As follows from Fig. 32, at water activities from 0 to 0.4 the residual catalytic activity of the initially hydrated enzyme is close to zero. On the other hand, in the same water activity range, the residual catalytic activity of the initially dehydrated enzyme equals to 80-90% compared with that for pure water. At high water activities, the storage stability of CT does not depend on the hydration history. At aw>0.85, the residual catalytic activity of the initially hydrated and dehydrated enzyme increases, approaching the level corresponding to pure water.

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Figure 27. (1) Solid-state solvent-free hydrolysis of N-succinyl-L-phenylalanine-p-nitroanilide. Modified data from [32]. (2) Hydrolysis of N-acetyl-L-tyrosine ethyl ester by -chymotrypsin preliminary incubated in water-acetone mixtures. (3) Binding of proflavin by -chymotrypsin in wateracetone mixtures.

Figure 28. α-Chymotrypsin preliminary incubated in water-acetonitrile mixtures. Dependences of Vmax and Km values on the water activity in acetonitrile.

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Figure 29. α-Chymotrypsin preliminary incubated in water-dioxane mixtures. Dependences of Vmax and Km values on the water activity in dioxane.

Figure 30. α-Chymotrypsin preliminary incubated in water-THF mixtures. Dependences of Vmax and Km values on the water activity in tetrahydrofuran.

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5

Vmax*10 , mol/L*s

136 4

A

3 2 1 0 0.0

0.2

100

KM, mM

10

0.4

0.6

0.8

1.0

0.8

1.0

Water activity B

1

0.1 0.01 0.0

0.2

0.4

0.6

Water activity

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Figure 31. α-Chymotrypsin preliminary incubated in water-acetone mixtures. Dependences of Vmax and Km values on the water activity in acetone.

Figure 32. Hydrolysis of N-acetyl-L-tyrosine ethyl ester by -chymotrypsin preliminary incubated in water-acetonitrile mixtures. (1) – the initially hydrated enzyme. The initial state for the hydrated chymotrypsin was an enzyme solution in pure water at 25oC. (2) – the initially dehydrated enzyme. The initial state for the dehydrated chymotrypsin was a solid enzyme preparation with water content of 8.6% (g water/g protein).

2.7. The State of Hydrogen Bond Network of Water in Hydrogen Bond Accepting Organic Solvents as Studied By FTIR Spectroscopy One of the straightforward and informative methods used to study intermolecular interactions in aqueous solutions of nonelectrolytes is vibrational spectroscopy. In this work

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the state of hydrogen bond network of water in hydrogen bond accepting organic solvents was studied by FTIR spectroscopy. FTIR spectroscopic experiment. Absorbance spectra were measured on a Vector 22 Bruker Fourier IR spectrophotometer at 25°C in the range of 4000-1000 cm-1 at a resolution of 4 cm-1 in the CaF2 cells with layer thicknesses of 10 and 100 mm [33]. For the water activity values close to unity, thin layers of liquid between two plane parallel plates of CaF2 were employed. Since in this case the layer thickness was unknown, the integral coefficients of water extinction were not determined at high values of water activity. The integral water extinction coefficient (B) was calculated by the Eq. 11:

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B 2.303 / CdDd



(11)

where D = log I0/I; D is optical density at a frequency; I0 is the intensity of incident light; I is the intensity of transmitted light; C is the concentration, mole/L; d is the thickness of the liquid layer, cm. For pure water, it was taken that Bo =96,000 L×mole-1×cm-2 [34]. IR spectra of water in hydrogen bond accepting organic solvents. Typical spectra of water in dioxane and acetonitrile in the region of the OH stretching vibration band are presented in Fig. 33 and 34. As follows from Fig. 33 and 34, the contour shape changes in both systems with increasing fraction of the organic component. The contour is shifted to the highfrequency region and decomposes into a number of discrete components. The high-frequency shift is due to the weakening of hydrogen bonds. The narrowing of the components and the appearance of new bands is explained by the weakening of the interaction between the water molecules and formation of the water–organic solvent complexes. Above a certain value of activity (aw >0.4), the contribution of the water–organic solvent complexes is low, and the spectrum shape depends on the absorption of different forms of water associated by hydrogen bonds. The wide diffuse bands characteristic of this region of compositions reflect the fluctuation character of the three-dimensional network of hydrogen bonds in water with a wide distribution according to the size of associate and the number and strength of hydrogen bonds per molecule.

Figure 33. Typical absorbance spectra of water in the region of OH stretching vibrations for solutions in dioxane. The spectra are normalized based on area. The IR spectra were measured at the following activity values of water: 0.02, 0.05, 0.29, 0.34, 0.62, 0.65, 0.75,0.80, 0.87, 1.0. The extreme right spectrum corresponds to pure water.

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Figure 34. Typical absorbance spectra of water in the region of OH stretching vibrations for solutions in acetonitrile. The spectra are normalized based on area. The IR spectra were measured at the following activity values of water: 0.02, 0.08, 0.35, 0.43, 0.57, 0.65, 0.77, 0.80, 0.87, 0.89, 0.90, 1.0. The extreme right spectrum corresponds to pure water.

Integral coefficients of water extinction. Fig. 35 shows the dependences of the relative

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integral extinction coefficient of the water OH groups,

B B0

, on the water activity in dioxane

and acetonitrile obtained in the present work. The same figure represents the relative integral extinction coefficients of the OD stretching vibration band of the HOD molecule obtained for dioxane and acetonitrile in [36,37]. These data were recalculated into water activity coordinates. As follows from Fig. 35, our data are in good agreement with the published data for dioxane and acetonitrile over the whole range of water activity values. Therefore further discussion of results is conducted with respect to the unified set of experimental data. Below aw ~ 0.4 in dioxane, the spectra have an isosbestic point at 3495 cm1, which is due to the decomposition of the water associates and formation of 1:2 water–dioxane complexes with frequencies of 3515 and 3585 cm1 corresponding to the symmetric and antisymmetric OH bond vibrations. In dioxane with the minimal water content (aw 0.01), apart from the bands of the 1:2 complexes one can also observed a weak component of free OH bonds at 3685 cm1, and the single band of the 1:1 complex is believed to overlap the 3515 cm1 component.

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1.1 1.0

A

B/B 0

0.9 0.8 0.7 0.6 0.5 0.4 1.0

B/B 0

0.9

B

0.8 0.7 0.6 0.5 0.0

0.2

0.4

0.6

0.8

1.0

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Water actvity Figure 35. Dependencies of the relative integral extinction coefficient of the stretching vibration band of OD groups in HOD and OH groups in HOH versus water activity in dioxane (A) and acetonitrile (B); and  - modified data from [33]; — modified data from [36], o — modified data from [36].

In acetonitrile, the spectra of water behave in a more complicated way. The network of hydrogen bonds of water is destructed within a narrow range of activity from 1 to approximately 0.9. For aw = 0.9 to 0.4, the first isosbestic point appears at 3470 cm1. The second isosbestic point (3512 cm1), is observed below aw~0.4. The first process is probably due to decomposition of the linear associates of water and formation of 1:2 complexes with components at 3543 and 3617 cm1. The second process corresponds to formation of 1:1 complexes with a single component at 3653 cm1. The 3685 cm1 band of free OH groups in acetonitrile is almost indiscernible because of the overlap with this component. Water sorption by α-chymotrypsin was controlled in the region of OH stretching vibration band at 3450 cm-1, which is the most intensive band of sorbed water [35]. Fig. 3 from review [35] shows the absorbance spectra of water sorbed by -chymotrypsin in the presence of dioxane at low and high water activity. The contour of spectra of sorbed water does not differ essentially from that for pure liquid water (Fig. 33). No bands at 3515, 3585, 3534 or 3617 cm1 were observed. This means that, at low water activities, the environment of water sorbed by CT differs essentially from that observed in low water dioxane and acetonitirle.

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The shapes of the curves of

35). At aw0.95 in all

the solvents studied. This indicates that there is virtually no difference when a water molecule is in the pure liquid state or in the solution in this water activity range. The hydrogen bond network of water is bond-percolated in this region. At aw~0.95-1.0, the storage stability values are close to 100% (Figures 24-27). At the highest water concentrations, the storage stability curves can be unified in the water activity coordinates in all the solvents studied (Figures 2427). In the water-rich region, organic solvent molecules enhance the hydrogen bond network of water in their immediate vicinities, i.e., the so-called the ―iceberg formation‖,

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―structure enhancement of water‖ or the ―hydrophobic attraction‖ [36,37, 43-46]. However, the hydrogen bond probability in the bulk water away from solute is reduced. At the highest water activities, the aw decreases. The

E Gorg

E Gorg

values are positive. However, they decrease as

values become less positive on addition of an extra organic solvent

molecule into the solution. This indicates the organic solvent-organic solvent interaction is attractive in terms of Gibbs energy. At the highest water activities, the solvent-solvent interaction is repulsive in terms of enthalpy but attractive in terms of entropy. The first organic solvent molecule dissolves in water with a large negative enthalpy and larger negative entropy at aw~0.95-1.0 (Figures 44 and 45). As the concentration of organic solvent increases, both the enthalpy gain and the entropy loss become smaller. B) The organic solvent-rich region. In the organic solvent-rich region, the

E Z org

values

are close to 0. This means that organic solvent molecules retain the same molecular arrangement as in the pure liquid state. The

Z wE

quantities are virtually constant in all the solvents. The

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negative. The

H wE

(

B E )w B0

values are

values are positive. This means that the hydrogen bonds between the

water and organic solvent molecules are weaker than those in pure water. No significant contribution of the self-associated forms of water was observed in this water activity region (Figures 33-35) [33]. Water molecules interact with organic solvents as single molecules. Hence, there is no hydrogen bond network of liquid H2O. No proflavin binding was observed in this region (Figure 17). The residual catalytic activity remains virtually constant, equal to 60-90% compared with that observed in pure water (Figures 2427). C) The intermediate region. In the intermediate composition region, the solution consists of two kinds of clusters, each reminiscent to the water-rich and organic solvent-rich region [42, 43]. There is a sharp transition from the water-rich region to to the intermediate one. This transition is associated with an anomaly in the thermodynamic, structural, and enzyme activity properties (Figures 24-27, 36-45). It occurs in a narrow water activity range. There is a ―knee‖ on the

E Zorg -aw curves at aw~0.95. At aw 0.4, the storage stability of the enzyme and its ability to bind proflavin vary in a similar way, both passing through a minimum at aw ~ 0.8-0.9. The position of this minimum on the storage stability and proflavin binding curves is consistent with the extreme on the

Z iE -aw

curves (Fig. 36-45). This water activity level likely corresponds to loss of the

hydrogen bond percolation transition. At moderate water activities, the degree of proflavin binding (Fig. 17) passes through its maximum. Notice also that, as follows from Fig. 17, the shapes of the isotherms of binding of proflavin by α-chymotrypsin and the positions of the maximum degree of binding in THF, acetone, dioxane, and acetonitrile are similar. These nonelectrolytes possess identical hydrogen bond accepting abilities with respect to water, as indicated by the similarity

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between the enthalpies of specific interaction

H 2O / S H int (spec.)

(–18.0 kJ/mole for

acetonitrile, –19.3 kJ/mole for dioxane, –19.7 kJ/mole for THF, and –20.5 kJ/mole for acetone, Table 1). For comparison, the enthalpies of specific interaction of water with benzene and DMSO equal –1.5 and –33.1 kJ/mole, respectively. This means that the formation of the enzyme - competitive inhibitor complex in the moderate-strength hydrogen bond accepting solvents, such as THF, acetone, dioxane, and acetonitrile, exhibits similar regularities at low water activities. On the other hand, the enzymatic activity in the absence of the organic solvent shows no minimum at high water activities (Fig. 24-27). This suggests that, at a high degree of hydration of the enzyme, when most of the interprotein contacts are already broken, the interaction with the organic solvent determines significantly the characteristics of αchymotrypsin and water. The storage stability (Fig. 32), structural and sorption properties of chymotrypsin in the presence of organic solvents [35, 47] depend markedly on the hydration history. These results are consistent with the idea that the dehydrated proteins are in a non-equilibrium state (or kinetically ―frozen‖ state). However, no maximum was observed on the storage stability curve for the initially hydrated enzyme (Fig. 32). It is expected that thу initially hydrated enzyme does not have significant kinetic limitations. This means that the maximum on the proflavin binding curves in the intermediate region exists due to kinetic reasons reflecting the interplay between the following processes: (i) Self-associated water molecules play an activating role breaking the dehydrationinduced interprotein contacts. The hydration of the enzyme is already high enough, so that its conformation is close to the native one. (ii) Some interprotein contacts remain unbroken in the initially dehydrated enzyme, a factor that plays a positive role by hindering the denaturation of the enzyme by the organic solvent. Relatively large organic solvent molecules do not penetrate into the solid enzyme due to steric (kinetic) reasons.

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Vladimir A. Sirotkin

REFERENCES [1] [2] [3] [4] [5] [6]

[7]

[8] [9]

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[10]

[11]

[12] [13] [14]

[15] [16]

[17]

Klibanov, A.M., Improving enzymes by using them in organic solvents, Nature, 409 (2001) 241-246. Carrea, G., Riva, S., Properties and synthetic applications of enzymes in organic solvents, Angew. Chem. Int. Ed., 39 (2000) 2226-2254. Gupta, M.N., Methods In Non-Aqueous Enzymology, (Ed: M.N. Gupta), Birhäuser Verlag, Basel-Boston-Berlin, 2000. Halling, P.J., What can we learn by studying enzymes in nonaqueous media? Philos. Trans. R. Soc. Lond. B Biol. Sci., 359 (2004) 1287– 1297. Micaelo, N.M., Soares, C.M., Modeling hydration mechanisms of enzymes in nonpolar and polar organic solvents, FEBS Journal, 274 (2007) 2424–2436 Persson, M, Bornscheuer, U.T., Increased stability of an esterase from Bacillus stearothermophilus in ionic liquids as compared to organic solvents., J Mol. Cat B, 22 (2003) 21-27. Clark, D.S., Characteristics of nearly dry enzymes in organic solvents: implications for biocatalysis in the absence of water, Philos. Trans. R. Soc. Lond. B Biol. Sci., 359 (2004) 1299–1307. Serdakowski, A.L., Dordick, J.S., Enzyme activation for organic solvents made easy, Trends Biotechnol., 26 (2007) 54-48. Gonzalez Martinez, S., Alvira, E., Vergara-Cordero, L., Ferrer, A., MontañesClemente, I., Barletta, G., High initial activity but low storage stability observed for several preparations of subtilisin Carlsberg suspended in organic solvents. Biotechnol Prog. 18 (2002) 1462–1466. Castillo, B., Pacheco, Y., Al-Azzam, W., Griebenow, K., Devi, M., Ferrer, A., Barletta, G., On the activity loss of hydrolases in organic solvents: I. Rapid loss of activity of a variety of enzymes and formulations in a range of organic solvents. J. Mol. Catalysis B: Enz. 35 (2005) 147-153. Castillo, B., Bansal, V., Ganesan, A., Halling, P, Secundo, F., Ferrer, A., Griebenow, K., Barletta, G., On the activity loss of hydrolases in organic solvents: II. A mechanistic study of subtilisin Carlsberg, BMC Biotechnology, 6 (2006) 51-63. Fernandez, J.F.A., Halling, P.J., Operational stability of high initial activity protease catalysts in organic solvents. Biotechnol. Prog, 18 (2002) 1455-1457. Klibanov, A.M., Enzymatic catalysis in anhydrous organic solvents, Trends Biochem. Sci., 14 (1989) 141-144. Martinek, K., Levashov, A. V., Berezin, I. V., On the modes of interaction between competitive inhibitors and the -chymotrypsin active centre. FEBS Lett., 7 (1970) 2022. Fink, A. L., Effect of dimethyl sulfoxide on the interaction of proflavine with alphachymotrypsin, Biochemistry, 13 (1974) 277-280. Bernhard, S. A., Lee, B.F., Tashjian, Z. H., On the interaction of the active site of alpha-chymotrypsin with chromophores: proflavin binding and enzyme conformation during catalysis. J. Mol. Biol., 18 (1966) 405-420. Glazer, A. N., Spectral studies of the interaction of α-chymotrypsin and trypsin with proflavine, Biochemistry, 54 (1965) 171-176.

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[18] Bruylants, G., Wintjens, R., Looze, Y., Redfield., C., Bartik, K., Protonation linked equilibria and apparent affinity constants: the thermodynamic profile of the achymotrypsin–proflavin interaction, Eur. Biophys. J., 37 (2007) 11–18. [19] Perrin, D.D., Armarego, W.L.F., Perrin, D.R., Purification of Laboratory Chemicals, Oxford: Pergamon Press, 1980. [20] Chin, J. T., Wheeler, S. L. Klibanov, A.M., On Protein Solubility in Organic Solvents, Biotech.Bioeng., 44 (1994) 140-145. [21] Reichardt, C., Solvents and solvent effects in organic chemistry. 2nd edition. VCH, Weinheim, 1988. [22] Khmelnitsky, Yu., Mozhaev, V.V., Belova, A.B., Sergeeva, M.V., Martinek, K., Denaturation capacity: a new quantitative criterion for selection of organic solvents as reaction media in biocatalysis, Eur. J. Biochem, 198 (1991) 31-41. [23] Borisover, M.D., Stolov, A.A., Cherkasov, A.R., Izosimova, S.V., Solomonov, B.N., Calorimetric and infrared spectroscopic study of intermolecular interactions of water in organic solvents, Russ. J. Phys. Chem, 68 (1994) 48-53. [24] Kogan, V.B., Fridman, B.N., Kafarov, V.V., Ravnovesie mezhdu zhidkost’ju i parom: Spravochnoe posobie (Equilibrium between Liquid and Vapor. Handbook), Moscow, Nauka, 1966. [25] Lyudmirskaya, G.S., Barsukova, T.A., Bogomolnij, A.M., Ravnovesie Zhidkost-Par. (Liquid-Vapour Equilibria. Handbook), Khimia, Moscow, 1987. [26] Gmehling, G., Onken, U., Vapor-Liquid Equilibrium Data Collection, DECHEMA, Frankfurt, 1977. [27] Sirotkin, V.A., Mukhametzyanov, T.A., Komissarov, I.A., Effect of tetrahydrofuran on the binding of the competitive inhibitor proflavin and the storage stability of bovine pancreatic α-chymotrypsin, Eng. Life Sci. 9 (2009) 82–88. [28] Borisover, M.D., Sirotkin, V.A., Zakharychev, D.V., Solomonov, B.N., Calorimetric methods in evaluating hydration and solvation of solid proteins immersed in organic solvents, in Enzyme in Nonaqueous Solvents, (Eds: E.N. Vulfson, P.J. Halling), Humana Press, Totowa, 2001, 183-202. [29] Sirotkin, V.A., Zinatullin, A.N., Solomonov, B.N., Faizullin, D.A., Fedotov, V.D., Calorimetric and Fourier transform infrared spectroscopic study of solid proteins immersed in low water organic solvents, Biochim. Biophys. Acta, 1547 (2001) 359-369. [30] M. R. Eftink, R. E. Johnson, and R. L. Biltonen, The application of flow microcalorimetry to the study of enzyme kinetics, Anal. Biochem. 111 (1981) 305-320. [31] G.E. Clement, M.L. Bender, The effect of aprotic dipolar organic solvents on the kinetics of -chymotrypsin-catalysed hydrolyses. Biochem. 2 (1963) 836-843. [32] Khurgin, Y.I., Maksareva, E.Y., Study of the solid-state enzyme reactions. 3. Irreversible inactivation of -chymotrypsin by benzylsulfonyl fluoride. Bioorg. Khim. 17 (1991) 76-80. [33] Sirotkin, V.A., Solomonov, B.N., Faizullin, D.A., Fedotov, V.D. IR spectroscopic study of the state of water in dioxane and acetonitrile: relationship with thermodynamic activity of water, J. Struct. Chem. 41 (2000) 997–1003. [34] Zolotarev, V.M., Demin, A.V., Optical constants of water in wide band of wavelengths, Optik. i Spektroskop., 43 (1977) 271-279.

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[35] Sirotkin, V.A., Analysis of the organic solvent effect on the hydration-dehydration and structure of proteins by FTIR spectroscopy, in Methods in Protein Structure and Stability Analysis: Vibrational spectroscopy, (Eds: V.N. Uversky, E.N. Permyakov), Nova Science Publishers, Inc., Hauppauge, NY. 2007, 195-230. [36] Gorbunov, B.Z., Kozlov, V.S., Naberukhin, Y.I., Study of structure of aqueoussolutions of nonelectrolytes by methods of vibrational spectroscopy, J. Struc. Chem., 16 (1975) 748-754. [37] Gorbunov, B.Z., Naberukhin, Y.I., Study of structure of aqueous-solutions of nonelectrolytes by methods of vibrational spectroscopy, J. Struc. Chem., 16 (1975) 755762. [38] Pimentel, G. C., McClellan, A.L., The Hydrogen Bond, Freeman, San Francisco, 1960. [39] Atkins, P.W., Physical Chemistry, Oxford University Press, 8th ed., 2006. [40] Belousov, V.P., Morachevski, A.G., Heats of Mixing of Liquids, Khimiya, Leningrad, 1970. [41] Belousov V.P., Panov M.Y., Thermodynamic properties of aqueous solutions of organic substances, Boca Raton, Fla.: CRC Press, 1994. [42] Nikolova, P., Duff, S.J.B., Westh, P., Haynes, C.A., Koga, Y., A thermodynamic study of aqueous acetonitrile: excess chemical potentials, partial molar enthalpies, entropies and volumes, and fluctuations, Can. J. Chem., 78 (2000) 1553 – 1560. [43] Koga, Y., Siu, W.W.Y., Wong, T.Y.H., Excess partial molar free energies and entropies in aqueous tert-butanol solutions at 25 oC J. Phys. Chem., 94 (1990) 7700 - 7706. [44] Franks F., Hydrophobic interactions - a historical perspective, Faraday Symp. Chem. Soc., 17 (1982) 7-10. [45] Bender, T.M., Pecora, R., A dynamic light-scattering study of the tert-butyl alcohol water-system, J. Phys. Chem, 90 (1986) 1700-1706. [46] Nakanishi, K., Ikari, K., Okazaki, S., Computer experiments on aqueous-solutions. 3. Monte-carlo calculation on the hydration of tertiary butyl alcohol in an infinitely dilute aqueous-solution with a new water butanol pair potential, J. Chem. Phys. 80 (1984) 1656-1670. [47] Sirotkin, V.A., Effect of dioxane on the structure and hydration-dehydration of chymotrypsin as measured by FTIR spectroscopy. Biochim. Biophys. Acta., 1750 (2005) 17-29. [48] Gregory, R.B., in Protein-Solvent Interactions, (Ed: R.B. Gregory), Marcel Dekker, New York, 1995, 191-264. [49] Rupley, J.A., Careri, G., Protein hydration and function, Adv. Protein Chem. 41 (1991) 37-172.

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In: Organic Solvents Editor: Ryan E. Carter

ISBN 978-1-61761-881-9 © 2011 Nova Science Publishers, Inc.

Chapter 7

REGULARITIES OF ORGANIC SOLVENTS PENETRATION INTO TETRAFLUOROETHYLENEPROPYLENE COPOLYMER 1

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I. Yu Yevchuk2, G. G. Midyan, R. G. Makitra*, G. E. Zaikov 3**, G. I. Khovanets’ and O. Ya. Palchykova* Department of Physical Chemistry of Combustible Minerals L. M. Lytvynenko Institute of Physico-organic Chemistry and Coal Chemistry NAS of Ukraine 79060 Lviv Naukova St., 3a, Ukrane *Institute of Geology and Geochemistry of Fossil Fuels NAS of Ukraine 79060 Lviv Naukova St., 3a, Ukrane **N. M. Emanuel Institute of Biochemical Physics RAS 19991 Моscow, Kosygin St., 4, Russia

ABSTRACT The processes of swelling and diffusion of solvents into the structure of tetraflurethylene-propylene coolymer can not be described quantatively using only one characteristic of a solvent. In all cases a molar volume of liquid has the determining effect, hampering its penetration into the structure of polymer. However, the effects of other factors – both solvation and density of cohesion energy of solvents – are significant. Adequate quantitative generalization of abovementioned processes can be obtained on the basis of free energies linearity concept by using of linear multiparameter equations taking into account the effects of different factors.

1

A version of this chapter was also published in Emergin Topics in Organic Chemistry, edited by Rattaporn Thonggom, Thevarak Rochanapruk and Gennady E. Zaikov published by Nova Science Publishers, Inc. It was submitted for appropriate modifications in an effort to encourage wider dissemination of research. 2 e-mail: [email protected]. 3 e-mail: [email protected].

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Keywords: regularities, membranes, liquids.

solvends,

duffusion,

copolymers,

swelling,

transport,

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Growing interest to investigation of liquids transport through fluoropolymer membranes processes is due to their high strength and stability to external effects. Therefore studing the behaviour of such membranes in different liquids, including aggressive ones, becomes especially actual. Parameters of polymers swelling in organic solvents and diffusion of the last ones into polymer structure can not be described unambiguously by means of simple dependences on physico-chemical properties of liquids. As in the most widespread model of these processes, based on the regular solvents theory, the solvation effects are not taken into account, the complicated bell-shaped relations between the equilibrium degree of swelling Sм and the Hildebrand solubility parameter  are revealed even in the case of such simple carbon-chain polymers as poly(ethylene) or poly(propylene) [1, 2] accompanied by the numerous deviations. Introduction of correction coefficients does not lead to essential improvement of generalizing relations [3]. The attempts to describe the swelling processes by means of another characteristics of solvents, such as their molar volume Vм [4] or the Reichardt’s parameter of specific electrophilic solvation ЕТ [5], give positive results only for the series of similar in properties solvents, mainly homologues. We have shown since the processes of swelling of polymers and diffusion of the solvents into their structure are accompanied by solvation processes they should depend on summary effect of some factors in complicated way and acceptable relation between characteristics of these processes and properties of liquids may be obtained only by means of multiparameter equations [6, 7]. It was found as effective the use of the Koppel-Palm ―solvation‖ equation [8], which takes into account the influence of medium on kinetics of chemical reactions , supplemented with some parameters which characterize the structure of the solvent:    lg Sм = a0 + a1 n2  1 + a2 + a3B + a4ET + a52 + a6Vм .

n 2

  

(1)

In equation (1) Sм is the degree of swelling (expressed in moles of solvent, adsorbed by 100 g of polymer); n is refractive index and  is permittivity of liquids, which characterize their polarizability and polarity, determining their ability to nonspecific solvation; B is the Koppel-Palm basicity and Ет is the Reichardt’s electrophilicity, that characterize the ability to specific (acid-base) interactions; square root of solubility parameter 2 is proportional to cohesion energy of liquids and reflects inputs of energy on disturbance of their structure; Vм is the molar volume of liquids. Here it is necessary to accentuate the advisability of representation of the values of swelling in moles of adsorbed liquid, because if calculations are accomplished with the values of S expressed in weight or volume parts, usually cited in literature, the results of their generalizations are essentially worse. The use of the equation (1) gives the possibility of the satisfactory generalization of the data on swelling in organic solvents of series of polymers – poly(ethylene) [6], poly(cisisoprene) [9], poly(butadiene) [10], as well as of correlation of them with the properties of the solvents. So, in accordance with elementary logic, it was found that in all three cases the coefficients at Vм have negative value, that is, the larger is molar volume of the solvent, the

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more difficult is its penetration into polymer structure. In the case of poly(ethylene) the factors f(n) and f() (ability of solvents to nonspecific solvation of polymer) promote its swelling. The signs ―+― at solvation members point out on this fact. At the same time parameter Ет (ability of solvents to electrophilic solvatation) counteracts this process, what is no wonder, taking into account hydrophobicity and nonpolarity of poly(ethylene), and high values of Ет are typical for alcohols, protoncontaining amines etc. Similar generalizations were revealed for swelling relatively low-polar poly(cis-isoprene) and poly(butadiene). In [11-15] the experimental data on swelling of membranes made from tetrafluoroethylene-propylene copolymer Aflas FA 150P containing carbon black (30 parts per 100 parts of polymer), d =1.55g/сm3 in 22 solvents at 30, 45 and 60ºС are given. Although in original works high termal and chemical stabilities of this polymer are mentioned, nevertheless it can absorb up to 100% and even 200% of solvent from polymer weight. At the same time in the cited works reliable relationship between the properties of solvents and the swelling degree of polymer was not established. That is why the attempt of generalization of these data by means of equation (1) appears to be advisible. In Table 1 corresponding data on swelling abovementioned copolymer are represented. Since in [11-15] these data were given in g of solvent per 100 g of polymer, they were recalculated in moles of solvent absorbed by 100 g of polymer according to our previous works [9, 10]. Solvents characteristics were taken from reviews [16, 17], calculation procedure was realized according [18]. Generalization of available data obtained at 30ºС for all 22 solvents leads to expression with unsatisfactory low values of multiple correlation coefficient R = 0.891. However, the exclusion (according [18]) of the most deviating data for only three solvents – cyclohexane, dimethylacetamide and dichloroethane – makes it possible to obtain expression (2) with acceptable value of R: lg (Sм-102) = 0.527 + (11.506±2.047)f(n2) + (1.020±1.188)f() + (1.484±0.431)·10-3B +(0.062±0.042) ET – (7.305±0.935) 10-32 – (0.014±0.002) Vм (2) N = 19, R = 0.964, s = ± 0.127. Low values of pair correlation coefficients of Sм values with separate parameters of equation r (the order of magnitude is 0.2-0.6) do not allow to define their significance. It may be determined (according to [18]) by alternating exception of individual members of the regression equation with calculating of the value of R of the equations with lesser amount or members in every case. If R of such equations falls down unsufficiently the exceptioned member is considered as insignificant. In this way insignificance of the factors of polarity f() and electrophilic solvation ET was found and so the degree of swelling of polymer can be characterized adequately by fourparameter equation (3): lg (Sм-102) = 2.736 + (9.155±1.515)f(n2) + (1.366±0.411) 10-3B – (5.997±0.624) 10-32 – (0.016±0.001) Vм (3) R = 0.957, s = ± 0.139.

Organic Solvents: Properties, Toxicity, and Industrial Effects : Properties, Toxicity, and Industrial Effects, edited by Ryan E. Carter, Nova Science

Table 1. Logarithms of swelling degree of copolymer Aflas FA 150P at 30, 45 and 60ºС according to [11-15] and values, calculated by means of equations (3), (5) and (7)

N

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Solvent Hexane Heptane Octane Isooctane Nonane Decane 1,2-Dichlorethane Chloroform Trichlorethylene Tetrachlormethane 1,1,1Trichlorethane Tetrachlorethylene Tetrachlorethane Dichlormethane Acetone Methylethylketone Acetonitrile Tetrahydrofuran 1,4-Dioxane Dimethyl acetamide Cyclohexanone Cyclohexane

lg (Sм102) 30ºС

45ºС

exp.

calc.

1.1526 0.9638 0.8698 1.2370 0.7709 0.6042 1.2993* 1.9666 1.8878 1.8017 1.9258

1.4252 1.1805 0.9175 1.1116 0.6827 0.4177 1.5750 1.7866 1.7867 1.8717 1.8312

lg (Sм 102) 0.2726 0.2167 0.0477 -0.1254 -0.0882 -0.1865 0.2757 -0.1800 -0.1011 0.0701 -0.0946

1.5821 1.1316 1.8132 1.5563 1.5038 0.4800 1.9671 1.5561 -

1.6623 1.3528 1.6273 1.4630 1.5426 0.5453 1.9379 1.5855 -

1.2543 1.0777*

1.2964 1.6764

60ºС

exp.

calc.

1.2497 1.0645 0.9703 1.3164 0.8739 0.8482 1.3981 2.0342 1.9669 1.8842 1.9794

1.4261 1.2458 1.0435 1.2234 0.8658 0.6599 1.6455 1.8046 1.8052 1.8475 1.8816

lg (Sм 102) 0.1765 0.1813 0.0731 -0.0930 -0.0081 -0.1883 0.2474 -0.2296 -0.1617 -0.0367 -0.0978

exp.

calc.

1.3339 1.1511 1.0558 1.4028 0.9518 0.8609 1.5120 2.0644 2.0057 1.9473 2.0072

1.4899 1.3118 1.1124 1.2888 0.9371 0.7340 1.7191 1.8727 1.8724 1.9095 1.9440

lg (Sм 102) 0.1560 0.1607 0.0566 -0.1139 -0.0147 -0.1269 0.2071 -0.1918 -0.1333 -0.0378 -0.0632

0.0803 0.2212 -0.1859 -0.0933 0.0388 0.0653 -0.0293 0.0294 -

1.6736 1.3122 1.6104 1.6397 0.6212 2.0054 1.6307 0.9263*

1.6771 1.4782 1.5117 1.6461 0.5837 2.0080 1.6621 1.5199

0.0035 0.1660 -0.0987 0.0064 -0.0375 0.0026 0.0313 0.5935

1.7517 1.4077 1.6570 1.7088 0.7016 2.0242 1.6627 1.0599*

1.7446 1.5580 1.5743 1.7017 0.6704 2.0447 1.6924 1.5584

-0.0071 0.1503 -0.0827 -0.0071 -0.0312 0.0205 0.0297 0.4985

0.0421 0.5986

1.4244 1.1870*

1.4876 1.6378

0.0632 0.4509

1.5159

1.5444 1.7031

0.0286 0.3961

* Data excepted from calculation .

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The most significant and decreasing the swelling degree is here the influence of molar volume Vм, the value R between lg (Sм-102) and Vм is equal to 0.569, and exception of this member from equation (3) destroys the correlation at all: R of threeparameter equation obtained gets infeasible low value of 0.572. Obviously, the larger is the size of solvent molecule, the more difficult it will be penetrate into polymer structure. The other members of equation have only certain correcting effect, at that the members with f(n2) and B, which characterize nonspecific and specific solvation of polymer molecules containing electronegative fluorine atoms, promote an adsorption of solvent by polymer. At the same time the increasing of solvent cohesion diminishes adsorption degree, probably, due to necessity of input of energy for tearing off a solvent molecule from the structure of liquid. Adequacy of equations (2), (3) and of all consequent ones is confirmed by coordination of the values of Fisher criterium, obtained for them, with table values for corresponding number of points at the degree of reliability α = 0.95. In Table 1 the values of lg (Sм-102), calculated by means of equation (3) and also their deviations from experimental values - Δ lg (Sм-102) are shown. As one can see the majority of deviations (with the exception of the results for three solvents, excepted from calculation) are within the limits of mean-root-square error s = ± 0.139 or are only slightly larger than it, not exceeding the value of 2s. Similar results were obtained at generalization of the data on swelling of copolymer Aflas FA 150 P at 45С and 60С (Table 1). The values of R of the equations, summarizing the data for all solvents, solvents which must be excluded from calculations for achievement of acceptable values of R, six- and fourparameter equations, adequately summarizing dependences of lg (Sм-102) on properties of organic liquids, are shown below. At 45С R of sixparameter equation for 21 solvents is equal 0.894. After the exclusion of the data for cyclohexane and dymethylacetamide adequate equations (4) and (5) were obtained: lg (Sм-102) = 1.433 + (10.456±1.983)f(n2) - (0.188±1.164)f() + (1.761±0.479)·10-3B + (0.019±0.038) ET – (5.918±0.892)·10-32 – (0.013±0.002) Vм (4) N = 19, R = 0.953, s = ± 0.131. lg (Sм-102) = 2.146 + (9.728±1.476)f(n2) - (1.732±0.413)·10-3B – (5.441±0.600) 10-32 –(0.013±0.001) Vм

(5)

R = 0.951, s = ± 0.134. Accordingly, at 60С for 21 solvents R=0.918, and after the exclusion of the data for cyclohexane and dymethylacetamide we have: lg (Sм-102) = 1.567 + (10.187±1.668)f(n2) - (0.028±0.979)f() + (1.585±0.403)·10-3B +(0.016±0.032) ET – (5.762±0.750)·10-32–(0.012±0.001)Vм (6) N = 19, R = 0.965, s = ± 0.100.

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lg (Sм-102) = 2.202 + (9.550±1.260)f(n2) + (1.594±0.352)·10-3B - (5.294±0.513)·10-32 – (0.013±0.001) Vм (7)

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N = 19, R = 0.962, s = ± 0.114. As one can see, in both cases signs and significances of individual members of equations are similar with those in the equations revealed at 25С . The maximal degree of swelling was determined in works [11-15] by constructing of the curves S vs time  before achievement of equilibrium, what allowed to calculate the proper coefficients of diffusion D from the slopes of the linear parts of adsorption curves, and also, as the process of adsorption is formally described by the first order equation of kinetics, to define the rate constants of penetration of solvents into polymer structure k. Diffusion of substances in homogeneous medium was interrogated and described by the known Fick, Einstein-Smolukhovsky and others’ formulas, however the relations between the characteristics of both membrane-making polymer and penetrating substance and coefficient of diffusion (i.e. its rate), were not obtained with the reliable quantitative decision until now [19, 20, 21]. So, in the case of diffusion of esters into ethylene-propylene membranes we observe n = 0.5 in expression Q/Q =kn (where Q and Q are the amount of adsorbed solvent during the time  and at saturation; k and n are constants, relatively), i.e. submission to the Fick law. However, for films from other materials (natural and synthetic rubbers, polyurethane) n  0.6, i.e. we deal with partial transition into the region of so-called anomalous transport conditioned by different influences of separate areas of polymer chain on adsorption processes [21]. Such deviations at diffusion of aromatic hydrocarbons or dymethylformamide into polyurethane membrane are yet more noticeable [19, 22]. It is due to the fact that between diffusing solvent and polymer structure the solvation interactions take place influencing on the energetics and the rate of the process of diffusion. As well as in the case of swelling only semiempirical account of them is possible on the basis of the concept of free energies linearity (LFE). The similar approach is used at generalization of the data on the effects of solvents on the rate of chemical reactions, solubility of gases, distribution of substances between two phases and so on. With this respect the Koppel-Palm equation, taking into account the effects of polarity and polarizability of solvents, as well as their capacity for acid-base interaction seems to be the most widespread. In the case of swelling of polymers and diffusion of liquids into their structure the additional account of geometric factor is necessary since the larger is a molecule of a solvent, the more difficult will be its penetration into a polymer structure, while solvatation of some areas of polymer structure by a solvent will facilitate diffusion. This supposition follows logically from the Einstein-Smolukhovsky formula: D = RT/N·6r,

(8)

in accordance to which the diffusion coefficient D in homogeneous media is inversely proportional to the radius r of diffusing particle. Judgements similar to foregoing ones were expressed earlier as well [23, 24].

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Table 2. Logarithms of coefficients of diffusion of solvents into copolymer Aflas FA150P at 30, 45 and 600С according to [11-15] and values, calculated by means of equations (10), (12) and (14)

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N 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Solvent Hexane Heptane Octane Isooctane Nonane Decane 1,2-Dichlorethane Chloroform Trichlorethylene Tetrachlormethane 1,1,1-Trichlorethane Tetrachlorethylene Tetrachlorethane Diclormethane Acetone Methylethylketone Acetonitrile Tetrahydrofuran 1,4-Dioxane Dimethyl acetamide Cyclohexanone Cyclohexane

lg (D107) 30ºС

45ºС 7

60ºС

exp.

calc.

lg (D10 )

exp.

calc.

0.2455 -0.1024 -0.5528 -0.3188 -0.9586 -0.9208 0.2355 0.7016 0.8363 0.3598 0.4502 0.4757 -0..1938 0.9745 0.6866 0.5527 -0.3372 0.7482 0.1492 -0.2924 -1.3010* -0.7696*

0.3255 -0.0722 -0.4698 -0.2492 -0.8287 -1.2168 0.3875 0.6183 0.5031 0.6186 0.6468 0.2767 -0.0364 0.7272 0.5836 0.4597 -0.1320 0.7681 0.1480 -0.3190 -0.1181 0.5588

0.0799 0.0301 0.0831 0.0695 0.1299 -0.2960 0.1520 -0.0832 -0.3332 0.2588 0.1965 -0.1990 0.1574 -0.2960 -0.1030 -0.0929 0.2053 0.0199 -0.0012 -0.0266 1.1830 1.3284

0.4900 0.3263 0.0828 -0.0605 -0.2518 -0.6576 0.6405 0.7839 0.8927 0.4014 0.4669 0.7267* 0.0086 0.7612 0.6170 -0.1135 0.8451* 0.2041 -0.0809 -0.5528 -0.0605*

0.5532 0.2576 -0.0533 0.1270 -0.3327 -0.6242 0.7657 0.9427 0.5230 0.5107 0.4690 -0.0251 -0.0092 0.6325 0.5542 -0.0786 0.2255 0.2482 -0.1557 -0.3719 0.3998

lg (D107) 0.0632 -0.0687 -0.1361 0.1875 -0.0809 0.0334 0.1253 0.1588 -0.3697 0.1093 0.0021 -0.7519 -0.0178 -0.1287 -0.0628 0.0349 -0.6196 0.0441 -0.0748 0.1809 0.4603

exp.

calc.

lg (D107)

0.5502 0.3820 0.2455 0.3032 0.0374 -0.0506 0.8235 0.9513 0.9415* 0.5729 0.4969 0.8169* 0.0334 0.7973 0.6355 0.0607 0.8531* 0.2227 0.0492 -0.3979 0.0453

0.6163 0.4261 0.2180 0.3642 0.0355 -0.1489 0.0877 0.9603 0.4269 0.3297 0.3911 0.4269 0.0549 0.7018 0.6615 0.0794 0.0335 0.2082 0.0498 -0.3165 0.2495

0.0661 0.0441 -0.0275 0.0610 -0.0020 -0.0983 0.0543 0.0090 -0.5146 -0.2432 -0.1059 -0.5146 0.0215 -0.0955 0.0261 0.0187 -0.8196 -0.0145 0.0006 0.0814 0.2042

* Data excepted from calculation.

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In Table 2 the values of diffusion coefficients of solvents into polymer Aflas FA 150 P at 30, 45, 60С D-107(cm2/s-1) are given (from [11-15]). They may be generalized by means of equation of the type (1) with acceptable exactness as well. For 22 solvents at 30С the value of R is equal 0.825, but after the exclusion of the data for cyclohexane and cyclohexanone we obtain the adequate equation (9): lg (D-107) = 5.015 + (1.815±2.183)f(n2) + (1.751±1.445)f() + (0.074±0.479)·10-3B – (0.045±0.046) ET –(5.548±1.154)·10-32–(0.022±0.002)Vм (9) N = 20, R = 0.956, s = ± 0.168. And after exception of parameters of little significance: lg (D-107) = 5.957 + (2.235±1.311)f() – (0.065±0.039) ET – (5.157±1.032)·10-32 – (0.022±0.002) Vм

(10)

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R = 0.954, s = ± 0.171. In contrast to generalization of the data on Sм parameters B and f(n2) appear to be insignificant here, although positive signs at these parameters show, that possible formation of interaction between electronegative atoms of fluorine and electrodonor solvents may hamper their penetration into a polymer. The effect of the factor of electrophilic solvation is also little significant – the value of R falls down to 0.947 at the exception of parameter ET. As well as in the previous case, the increase of solvents cohesion and of their molecule sizes reduces their capacity for penetration into polymer structure. The single factor promoting the process of diffusion into polar copolymer structure is polarity of solvents. Similar to the case of Sм, the values of lg (D-107), calculated by equation (10), and their deviations with the experiment results are given (Table 2). In this case most of deviations do not exceed the region of s ± 0.171 as well. The values of lg (D-107) for temperatures 45 and 60С may also be generalized by means of the equation of type (1). However, for these temperatures is insignificant the effect of only one parameter – polarizability f(n2). The effect of the factor of basicity is negative, possibly, because the process of penetration of the basic solvent into the polymer structure is slowed as a result of bonding of basic solvent by electronegative atoms of fluorine, however, in general this factor is relatively insignificant. At the same time, electrophilicity of solvents promotes their diffusion. In both cases the density of the cohesion energy and molar volume of a solvent have the most essential effect on the process. So, R of sixparameter equation for 21 solvents at 45С is equal 0.848, and after exclusion of the data for cyclohexane, trichlorethylene and tetrahydrofuran we get the adequate equation (11): lg (D-107) = 0.047 + (1.204±1.839)f(n2) – (3.184±1.260)f() – (0.707±0.459)·103 B+(0.143±0.044)ET –(7.567±1.017)·10-32 –(0.015±0.002)Vм (11) N = 18, R = 0.953, s = ± 0.138.

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Regularities of Organic Solvents Penetration…

161

lg (D-107) = 0.705 – (2.780±1.113)f() –(0.784±0.450)·10-3B+(0.128±0.039) ET – (7.305±0.946)·10-32 – (0.015±0.002) Vм (12) R = 0.952, s = ± 0.139. The subsequent exception of the parameter of basicity leads to equation with R = 0.943. For 21 solvents at 60С R = 0.786 and after exception of the data for trichlorethylene, tetrahydrofuran and tetrachlorethylene we obtain the adequate equation (13): lg(D-107) = –2.774 + (0.728±1.280)f(n2) – (4.604±0.828)f() – (0.887±0.317)·10-3B + (0.218±0.028) ET – (7.648±0.672)·10-32 – (0.008±0.001) Vм (13) R = 0.965, s = ± 0.094. lg (D-107) = –2.425 – (4.387±0.742)f() – (0.923±0.314)·10-3B + (0.211±0.025) ET – (7.517±0.638)·10-32 – (0.008±0.001) Vм (14)

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R = 0.964, s = ± 0.095. As well as in the previous case, the exclusion of basicity results in decrease of the value of R to 0.946. On the basis of the data on lg Sм and lg D the authors [11-15] calculate the values of the effects of enthalpy of absorption of solvents at swelling Н (kJ/mol) and activation energies of the process of diffusion ЕD (kJ/mol) (Table 3). These data can not be consider as fully reliable, because they were defined using the values only for three temperatures. Indeed, at generalization of 21 values of Н for the receipt of adequate equation with R  0.95 it is necessary to exclude from consideration the data for 5 solvents, that is 25% of their common number, and all parameters in resulting equation appears to be significant at the simultaneously extremely low values of pair correlation coefficients of separate terms with Н – about 0.15-0.30. The best results were got at generalization of the data on activation energies of the process of diffusion ЕD. In spite of the fact that R is equal only 0.825 at generalization of the data for 21 solvents, exception of the data for only three solvents (cyclohexane, dichlorethane and chloroform) allows to get the adequate eqution (15): ЕD = 64.26 – (300.59±74.92)f(n2) + (66.80±46.83)f() – (2.85±1.60)·10-2B – (3.81±1.73) ET + (0.25±0.04)2 + (0.61±0.05) Vм (15) N = 18, R = 0.965, s = ± 4.98. The factor of molar volume (r = 0.82) has the defining effect here too, that can be explained logically – the larger is the size of molecule, the more is the expense of energy for its penetration into polymer structure, i. e. the activation energy of the process. The equation can be simplified to fourparameter one:

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Table 3. Values of enthalpy, activation energy and logarithms of rate constants of the process of diffusion of solvents into copolymer Aflas FA 150P at 30˚C according to [11-15] and calculated by means of equations (16) and (17)

N

Solvent

H

exp. ED

calc. ED

 ED

exp. lg (k-103)

calc. lg (k-103)

lg (k-103)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Hexane Heptane Octane Isooctane Nonane Decane 1,2-Dichlorethane Chloroform Trichlorthylene Tetrachlorethane 1,1,1-Trichlorethane Tetrachlorethylene Tetrachlorethane Dichlormethane Acetone Methylethylketone Acetonitrile Tetrahydrofuran 1,4-Dioxane Dimethyl acetamide Cyclohexnone Cyclohexane

11.68 12.06 11.98 10.67 11.70 16.81 13.69 7.54 7.63 9.40 5.26 10.93 17.86 11.18 13.20 14.34 3.70 6.91 25.04 16.92 14.77

19.7700 31.6600 51.9100 39.9400 63.5600 56.8300 38.0300* 16.0000* 6.8000 14.2800 2.9500 22.1200 13.6800 7.6600 4.4400 31.4700 6.8300 4.7400 21.8600 22.8700 52.5500*

24.0639 34.0768 44.7810 39.5000 54.6109 65.2269 -1.1306 -4.1227 4.3870 7.9458 4.9618 18.5788 16.5198 4.7200 6.5789 26.9686 6.1880 19.0154 20.9678 24.2784 16.6218

4.2939 2.4168 -7.1290 -0.4400 -8.9491 8.3969 -39.1606 -20.1227 -2.4130 -6.3342 2.0118 -3.5412 2.8398 -2.9400 2.1389 -4.5014 -0.6420 14.2754 -0.8922 1.4084 -35.5500

0.4314 0.0682 -.0.3098 -0.2147 -0.7447 -0.6990 0.2405 0.7672 0.8248 0.3636* 0.3243 0.4249 -0.2218 0.8645 -0.1549 0.6721* -0.1549 -0.4685 -0.5850*

0.6147 0.4330 0.1673 0.2330 0.0212 -0.5229 0.5933 0.5999 0.6637* 0.3096 0.3284 0.5185* -0.0757 0.8306 0.5539 -0.0177 -0.1675 0.0969

0.6675 0.6542 0.4456 0.3598 0.2856 0.1367 0.7093 0.8825 0.7513 0.4183 0.3979 0.6730* -0.0177 0.6722 0.7067 0.3598 -0.0655 0.4232

* Data excepted from calculation.

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Regularities of Organic Solvents Penetration…

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ЕD = 11.29 – (225.69±65.61)f(n2) – (2.02±0.73) ET + (0.21±0.04) ) 2 + +(0.62±0.06)Vм (16) R = 0.955, s = ± 5.62. Along with a molar volume the factor of cohesion energy of liquid also influences on the value of ЕD substantially, promoting it. At the same time the solvation factors reduce the value of ЕD. However, they are relatively insignificant. In Table 3 along with the experimental values of ЕD, the values calculated by the equation (16) and their deviations from experimental data are shown. As it was mentioned above, the relative rate constants of the process of penetration of liquid into polymer structure k-103 (min-1) were determined in [11-15]. These values are also represented in Table 3 and may be generalized adequately by the equations of type (1) after the exclusion of the data for 1-3 solvents. In all cases the factor determining and reducing the penetration rate of liquid into polymer structure is the molar volume of a solvent (r about 0.50-0.60). Polarity and density of cohesion energy of liquids reduce the penetration rate. At the same time their capacity for electrophilic solvation increases the values lg k; the factors of polarizability and capacity to nucleophilic solvation are insignificant. The simplified fourparameter equations, generalized data on lg k at three temperatures, the values of R and s and also the solvents excluded from calculation are represented below. At 30С after exception of the data for cyclohexane, methylethylketone and tetrachlormethane we obtain equation (17):

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lg (k-103) = 5.16 – (2.72±1.66)f() + (1.66±4.65)10-2 ET – (3.35±1.15)10-32 –(2.19±0.23)10-2 Vм (17) R = 0.933, s = ± 0.186. At 45С after exception of the data for tetra- and trichlorethylene we get (18): lg (k-103) = 1.052 – (3.36±1.00)f() + (0.16±0.03)10-2 ET –(7.067±0.745)10-32 – (0.908±0.128)10-2 Vм (18) R = 0.949, s = ± 0.112. At 60С after exception of the data for tetrachlorethylene we obtain (19): lg (k-103) = – 2.213 – (5.59±0.84)f() + (0.194±0.025)10-2 ET – (5.509±0.020)10-32 – (0.626±0.109)10-2 Vм (19) R = 0.944, s = ± 0.094. Thus, consideration of the data from [11-15] show, that the processes of swelling and diffusion of solvents into the structure of tetraflurethylene-propylene coolymer can not be described quantatively using only one characteristic of a solvent. In all cases a molar volume

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of liquid has the determining effect, hampering its penetration into the structure of polymer. However, the effects of other factors – both solvation and density of cohesion energy of solvents – are significant. Adequate quantitative generalization of abovementioned processes can be obtained on the basis of free energies linearity concept by using of linear multiparameter equations taking into account the effects of different factors.

REFERENCES [1] [2] [3] [4] [5] [6] [7] [8]

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[9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22]

Richards R. B. // Trans. Faraday Soc. 1946. V. 42. N 1. P. 20-28. Briston G. V., Watson W. F. // Trans. Faraday Soc. 1958. V. 54. N 11. P. 1731-1741. Mc Kenna G.B., Flynn K. M., Chen Gihong. // Polymer. 1990. V. 31. N 10. P. 19371945. Aminabhavi T. M., Harogoppad S. B., Khinnavar R. S. et al. // IMS – Rev. Macromol. Chem. Phys. 1991. V. C31. N. 4. P. 433-448. Jonquieres A., Roizard D., Luchon P. J. // J. Appl. Polymer Sci. 1974. V. 54. N 11. P. 1673-1682. Makitra R., Pyrig I., Zagladko E. et al. // J. Appl. Polymer Sci. 2001. V. 81. P. 31333140. Makitra R. G., Zagladko Е. A., Midyana G. G., Protsaylo L. V. // Zhurn. fiz. khim. 2001. V. 75. N 12. P. 2283-2287. Koppel I. A., Palm V. A. // Advances in Linear Free Energ y Relationships / Ed. Chapman N.B., Shorter J. London; New York: Plenum Press, 1973. P. 203-280. Makitra R. G., Zagladko Е. A., // Zhurn. fiz. khim. 2002. V. 76. N 10. P. 1797. Makitra R. G., Zagladko Е. A., Turovsky A. A., Zaikov G. Е. // Zhurn. prikl. khim. 2004. V. 77. N 2. P. 324. Aminabhavi T. M., Harlapur S. F., Balundgi R.H. et al. // Polymer. 1998. V. 39. N 5. P. 1067-1074. Aminabhavi T. M., Harlapur S. F., Balundgi R.H. et al. // J. Appl. Polym. Sci. 1996. V. 59. P.1857-1870. Aminabhavi T. M., Harlapur S. F., Balundgi R.H. // J. Polym. Eng. 1996/1997. V. 16. N 3. P. 181-202. Aminabhavi T. M., Harlapur S. F. // Chem. Eng. Proc. 1997. V. 36. P. 363-370. Aminabhavi T. M., Phayde H. T. // J. Chem. Eng. Data. 1996. V. 41. P. 813-818. Makitra R. G., Pyrig Y. M., Kivelyuk R. B. // Major characteristics of solvents, using in LFE equations. М. 1986. 34 P. – Dep. in VINITI 26.03.86. N 628-В86. Abboud J. L. M., Notario R. // Pure Appl. Chem. 1999. V. 71. N 4 P. 645. Recommendations for Reporting the Results of Correlation Analysis in Chemistry // Quant. Struct.-Act. Relat. 1985. V. 4. N 1. P. 29. Aithal U. S., Aminabhavi T. M. // Indian J. Technology. 1990. V. 28. N 10. P. 592. Charlesworth J. M., Riddel S. Z., Mathews R. J. // J. Appl. Polymer Sci. 1993. V. 47. P. 653. Khinnavar R. S., Aminabhavi T. M. // J. Appl. Polymer Sci. 1992. V. 46. P. 909. Aithal U. S., Aminabhavi T. M. // Polymer. 1990. V. 31. N 9. P. 1757.

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[23] Kotyukov I. М. // Fizicheskaya khimiya. Tomsk: Sibirskaya nauchnaya mysl. 1993. V. 2. P. 713–725. [24] Gerasimov Ya. I., Dreving V. P., Eryomin E. I. at al. // Kurs fizicheskoy khimiyi. М.: Khimiya. 1979. P. 239-246.

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INDEX

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A absorption, x, 115, 120, 121, 123, 125, 126, 137, 161 absorption spectra, x, 115, 121, 125 absorption spectroscopy, 120 accessibility, 27 acetone, vii, ix, 14, 31, 33, 34, 35, 36, 37, 38, 39, 41, 44, 55, 61, 62, 69, 71, 72, 76, 80, 106, 112, 115, 117, 118, 119, 124, 125, 127, 128, 130, 132, 134, 136, 143, 144, 145, 148 acetonitrile, vii, ix, 9, 14, 31, 33, 34, 35, 36, 37, 38, 39, 41, 42, 44, 48, 49, 51, 53, 55, 61, 62, 68, 69, 71, 72, 76, 80, 84, 85, 110, 115, 117, 118, 119, 123, 125, 128, 130, 131, 133, 134, 136, 137, 138, 139, 142, 143, 144, 145, 147, 148, 150, 151 acid, vii, viii, ix, 11, 20, 26, 27, 29, 31, 32, 33, 35, 38, 39, 43, 44, 46, 47, 49, 50, 51, 52, 53, 54, 55, 60, 61, 78, 85, 95, 96, 98, 99, 100, 103, 105, 106, 107, 108, 109, 110, 111, 113, 114, 129, 154, 158 acrylate, 111, 112 activation energy, 161, 162 active site, 83, 117, 147, 149 active transport, 90 activity level, x, 116, 147, 148 acylation, viii, 32, 47, 51, 52, 53 adaptation, ix, 25, 89, 93, 94, 96, 101 adaptations, 101, 102 additives, 7, 21, 24, 52, 60, 84 adenovirus, 18 adhesion, 95 adsorption, 9, 24, 42, 157, 158 adsorption isotherms, 42 advantages, 15, 32, 58, 113 aerobic bacteria, 113 agar, 94, 95 aggregation, 2, 8, 20, 26, 71, 73, 74, 86, 120 alanine, 5, 6

albumin, viii, 9, 16, 25, 29, 57, 59, 60, 61, 65, 67, 68, 71, 72, 73, 74, 75, 77, 78, 81, 86, 87 alcohols, viii, 2, 7, 13, 14, 18, 21, 53, 54, 58, 61, 73, 74, 83, 92, 95, 101, 103, 108, 113, 147, 155 alkane, 95, 97, 102 amines, 7, 92, 155 amino acids, vii, 1, 2, 7, 8, 14, 23 ammonium, 9, 16, 25 antibiotic, 103 antibiotic resistance, 103 antibody, 11, 24, 25 antigen, 10, 12, 25 antioxidant, 47, 54 antioxidative activity, 33, 47, 50, 52, 54 apples, 55 aqueous solutions, 15, 21, 23, 45, 46, 54, 136, 151 arginine, 8, 10, 11, 17, 26, 28 aromatic compounds, 95, 103, 117 aromatic hydrocarbons, 92, 95, 102, 158 aromatics, 92 ascorbic acid, 47, 55 atoms, 96, 157, 160

B Bacillus subtilis, 96 bacteria, vii, ix, 19, 25, 89, 90, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 105, 106, 107, 108, 109, 110, 111, 113 bacterial strains, 90, 94 bacteriophage, 18 bacterium, 99, 100, 102 base pair, 20 basic research, 2 basicity, 154, 160, 161 benign, 106 benzene, viii, 14, 50, 58, 61, 62, 65, 68, 69, 71, 72, 76, 80, 84, 93, 94, 95, 99, 100, 101, 102, 130, 148 biocatalysts, 58, 90, 97, 116

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168

Index

biochemistry, 61 biocompatibility, 91, 100, 107 bioconversion, 90, 91, 92, 93, 94, 101, 102 biodegradation, 90, 97 biodiesel, 95 biodiversity, 90 biological activity, 20 biological systems, vii bioluminescence, 91, 92 bioremediation, ix, 89, 90, 100, 103 biosynthesis, 96, 98, 103 biotechnology, 8, 58, 61, 90, 93, 98, 117 bonds, ix, 58, 61, 63, 80, 83, 84, 118, 137, 138, 139, 140, 146, 147 brain, 7, 23, 24 bridges, 83, 147 bronchitis, 17, 28 butadiene, 154

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C calcium, 22 calibration, 34, 44, 60 calorimetry, 85, 87 capillary, 50 capsule, 93 carbohydrate, 52, 93 carbohydrates, 17, 54 carbon, 44, 46, 47, 65, 93, 94, 95, 96, 101, 154, 155 carbon atoms, 96 carbon tetrachloride, 65 catalysis, 21, 27, 58, 86, 116, 149 catalyst, 27, 49 catalytic activity, x, 44, 83, 92, 93, 116, 131, 132, 134, 146, 147 cation, 22, 61, 109 cell culture, 17 cell surface, 95, 109 chaperones, 24 charge density, 3, 7 chemical properties, 2, 154 chemical reactions, 13, 154, 158 chlorobenzene, 101 chloroform, 14, 27, 161 chromatograms, 35 chromatography, 3, 8, 9, 10, 11, 19, 22, 24, 25, 26, 28, 29 chymotrypsin, viii, ix, 27, 57, 60, 61, 65, 66, 68, 69, 70, 73, 74, 75, 76, 77, 78, 80, 81, 82, 83, 86, 87, 115, 116, 117, 120, 121, 123, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 136, 139, 147, 148, 149, 150, 151 class, 22 cleaning, 18

cleavage, 92, 95, 100 clusters, x, 116, 146 CMC, 45, 46, 47 CO2, 106 collagen, 21 color, iv complications, 120 composition, x, 2, 79, 83, 94, 95, 96, 103, 116, 129, 141, 145, 146, 147 compounds, ix, 2, 4, 6, 14, 22, 25, 85, 90, 93, 94, 95, 96, 97, 99, 101, 103, 105, 113, 116, 117 condensation, vii, 31, 32, 33, 35, 38, 39, 42, 43, 47, 49, 51, 53, 54 configuration, 101 consumption, 95, 113 contaminant, 17 contaminated soils, 102 contamination, 17, 28 contour, x, 59, 67, 115, 137, 139 coordination, 157 copolymers, 154 correlation, 6, 14, 39, 51, 59, 76, 92, 95, 107, 122, 128, 154, 155, 157, 161 correlation coefficient, 122, 155, 161 correlations, 41, 78, 92 cosmetics, 32, 47 cost, 58, 95, 111, 116 coxsackievirus, 17 crude oil, 53, 95, 102 crystallization, 6, 16 crystals, 16 culture, 17, 19, 97, 101 culture media, 17 cyclohexanone, 160 cyclopentadiene, 111, 112 cytochrome, 85

D damages, iv danger, 4 decomposition, 109, 138, 139 deficiency, 6 degradation, 48, 53, 93, 94, 95, 100, 101, 102, 103 degradation rate, 95 dehydration, viii, ix, x, 15, 16, 58, 60, 68, 74, 83, 84, 85, 86, 96, 116, 147, 148, 151 denaturation, viii, ix, 4, 6, 11, 15, 21, 22, 23, 24, 58, 61, 74, 76, 79, 84, 87, 110, 115, 117, 148 deoxyribonucleic acid, 29 derivatives, 11, 26 desorption, 2 destruction, 79 destruction processes, 79

Organic Solvents: Properties, Toxicity, and Industrial Effects : Properties, Toxicity, and Industrial Effects, edited by Ryan E. Carter, Nova Science

Index detection, 10, 24, 50, 99, 101 deviation, 3, 37, 122 dialysis, 5 diarrhea, 18 dichloroethane, 155 dielectric constant, vii, 7, 19, 22, 31, 40, 41, 48, 49, 51, 63, 64, 76, 91, 92, 118 differential scanning, 87 differential scanning calorimetry, 87 diffusion, x, 153, 154, 158, 159, 160, 161, 162, 163 dimethylformamide, 61, 62 dimethylsulfoxide, 26, 117 disinfection, 17, 28 distribution function, 55 diversity, 101 DMFA, 61, 62, 63, 74, 76, 80, 82 DNA, 2, 3, 4, 20, 29, 94 donors, 61, 118 double helix, 3 Drosophila, 20 drug resistance, 103 drugs, vii, 1, 2 drying, 14, 16, 55, 60, 65, 129 DSC, viii, 57, 58, 60, 79, 82, 83 dyes, 10

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E E.coli, 19, 20 ecosystem, 100 editors, 99 effluent, 42, 44, 48 electrodes, 129 electrolyte, 60 elucidation, 13 emulsifying properties, 32 emulsions, 55 endothermic, 79, 80 endurance, ix, 89, 90 energy consumption, 113 engineering, 21, 93, 106 entropy, 141, 144, 145, 146 environmental conditions, 18 environmental factors, 112 environmental issues, 106 enzymatic activity, 148 enzymes, ix, x, 7, 15, 16, 22, 25, 27, 52, 58, 59, 84, 85, 87, 91, 99, 105, 115, 116, 117, 149 equilibrium, vii, viii, 5, 15, 31, 32, 33, 34, 35, 37, 38, 39, 40, 41, 46, 49, 51, 58, 59, 65, 79, 119, 123, 148, 154, 158 ester, 22, 26, 27, 35, 40, 46, 50, 53, 55, 129, 131, 132, 133, 134, 136

169

ethanol, viii, 3, 4, 5, 6, 10, 14, 16, 18, 20, 23, 28, 29, 50, 51, 58, 61, 63, 68, 70, 71, 73, 76, 83, 84, 106 ethers, 92 ethylene, 10, 11, 13, 154, 158 ethylene glycol, 10, 11, 13 evaporation, 44, 50 exchange rate, 14, 27 exclusion, 9, 24, 25, 155, 157, 160, 161, 163 exothermic effects, 82, 84, 147 exothermic peaks, viii, 58, 79 experimental condition, 13, 120 experimental design, 55 exposure, 14, 23, 26, 92, 94, 101 extinction, 120, 137, 138, 139 extraction, ix, 20, 105, 110, 113

F fatty acids, 32, 43, 47, 53, 55, 95, 96, 102, 103 fermentation, ix, 98, 105, 106, 112, 113, 114 fibers, 29, 114 fibrinogen, 22 films, 59, 87, 158 filtration, 9 financial support, 97 flexibility, 15 flocculation, 97 fluctuations, 151 fluorescence, 13, 94 fluorine, 157, 160 fluorine atoms, 157 folding intermediates, 27 food additives, 52 food industry, 33 formamide, 13, 39, 60 fragments, 20, 24, 26, 29 free energy, 3, 5, 6, 23, 63, 64 freezing, 13 frequencies, 20, 67, 138 fructose, vii, 31, 33, 34, 35, 36, 37, 38, 39, 46, 47, 51, 52, 54 fruits, 47, 55 FTIR, v, viii, x, 57, 58, 59, 85, 86, 115, 136, 137, 151 FTIR spectroscopy, x, 85, 86, 115, 137, 151 fullerene, 102 fungi, ix, 105, 107, 109, 113

G gel, 9, 24, 60 gel permeation chromatography, 9 Germany, 86 Gibbs energy, viii, 58, 76, 119, 141, 143, 144, 146

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170

Index

glass transition, 85 glucose, 33, 34, 35, 36, 37, 47, 48, 51, 53, 55 glucoside, 55 glycerol, 7, 10, 13, 24, 85, 95 glycine, 5, 6, 10, 23 glycol, 9, 10, 11, 13, 20, 25, 29, 50 glycoside, vii, 31, 33, 47, 48, 49, 50, 51, 54 growth temperature, 103

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H heat capacity, 79, 80 heat treatment, 17 heating rate, 60 height, 42, 44, 45 herpes, 18, 28 herpes simplex, 18, 28 heterogeneity, 94 hexane, 48, 49, 79 HIV, 18, 28 HIV-1, 18, 28 human immunodeficiency virus, 28 hydrocarbons, 92, 93, 95, 98, 101, 102, 103, 107, 108, 113, 158 hydrogen, viii, ix, x, 12, 13, 50, 57, 58, 61, 62, 63, 64, 68, 71, 74, 75, 76, 77, 78, 79, 80, 82, 83, 84, 115, 116, 118, 130, 137, 139, 140, 141, 145, 146, 147, 148 hydrogen bonds, ix, 58, 61, 63, 80, 83, 84, 118, 137, 139, 140, 146, 147 hydrolases, 85, 149 hydrolysis, 15, 26, 32, 83, 85, 129, 131, 132, 133, 134, 147, 148 hydrophilicity, viii, 49, 58, 64, 76 hydrophobicity, viii, ix, 22, 23, 32, 51, 52, 61, 63, 64, 76, 95, 97, 106, 109, 115, 117, 118, 155 hydroquinone, 55 hydroxide, 18, 129 hydroxyapatite, 9, 25 hydroxyl, 35, 47, 48, 49, 51 hydroxyl groups, 35, 47, 49 hypothesis, 23, 79 hysteresis, 147

I ideal, 8, 119, 140 immersion, 59, 83 immobilized enzymes, 105 immunodeficiency, 28 immunoglobulins, 29 incubation period, 93 inhibition, 87, 101, 110, 117, 130

inhibitor, ix, x, 83, 115, 116, 117, 120, 126, 128, 130, 147, 148, 150 initial state, 65, 120, 136 interface, 4, 6, 110 intermolecular interactions, 86, 136, 150 interphase, 114 ion-exchange, 19, 25, 29 ionic strength, 9, 16, 17, 19, 20 ionization, 50, 96, 103 ions, 2, 6, 16, 21, 23, 28 IR spectra, 71, 137, 138 isolation, 29, 90, 93, 97 isomers, 35, 91 isoprene, 154 isothermal calorimetry, 85 isotherms, 41, 42, 148

J Japan, 1, 31, 52, 54, 105, 129

K kerosene, 106 kidney, 23 kinase activity, 11 kinetic curves, 131, 132 kinetic parameters, 49, 50, 55, 130 kinetics, 13, 14, 15, 21, 50, 51, 83, 113, 150, 154, 158

L lactate dehydrogenase, 10, 23 lactic acid, vii, ix, 105, 106, 107, 108, 109, 110, 111, 113, 114 ligand, 5, 11, 12, 15, 25 linearity, x, 153, 158, 164 lipases, 52, 53 lipid oxidation, viii, 32, 33, 50, 52 lipids, 17, 96 liquid chromatography, 24, 25 liquid phase, 60, 65, 101, 110, 119 liquids, vii, viii, ix, x, 57, 58, 59, 62, 63, 64, 76, 77, 84, 105, 106, 109, 110, 111, 113, 114, 116, 117, 149, 154, 157, 158, 163 low temperatures, 13 Luo, 102 lysozyme, 21, 79, 87

M mAb, 11 macromolecules, vii, 1, 2, 3, 5, 7, 21, 60, 116, 147 magnesium, 26

Organic Solvents: Properties, Toxicity, and Industrial Effects : Properties, Toxicity, and Industrial Effects, edited by Ryan E. Carter, Nova Science

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Index magnetic relaxation, 54 majority, 11, 94, 97, 157 maltose, 52, 54 manipulation, 11 manufacturing, 10, 29 mass spectrometry, 96, 103 measles, 17, 28 media, viii, ix, 11, 17, 21, 22, 27, 53, 54, 57, 58, 59, 84, 85, 86, 89, 90, 98, 99, 111, 114, 115, 116, 117, 148, 149, 150, 158 melanoma, 55 melting, 13, 29, 44 melting temperature, 13 membranes, 15, 18, 19, 109, 114, 154, 155, 158 memory, 16, 27 metabolic pathways, 90 metabolism, 94, 95 metal salts, 20 methanol, viii, 11, 12, 13, 14, 26, 27, 35, 48, 50, 58, 61, 63, 68, 70, 71, 73, 75, 76, 83, 84, 95, 110, 111, 112 methodology, 13 Mg2+, 3 mice, 29, 55 microbial cells, ix, 89, 91, 97, 110 microcalorimetry, 150 microorganism, 98, 106, 107 milligrams, 35, 47, 49 mixing, 18, 114, 140 model system, ix, 89 modification, 34, 90, 93, 110 moisture, 14 molar volume, x, 38, 63, 64, 76, 118, 153, 154, 157, 160, 161, 163 mole, 61, 62, 65, 118, 119, 120, 127, 128, 137, 140, 148 molecular biology, 61 molecular mass, 38 molecular structure, 91, 107 molecular weight, 20, 54 molecules, x, 2, 3, 4, 7, 10, 15, 20, 29, 46, 65, 74, 83, 84, 90, 96, 110, 116, 137, 140, 145, 146, 147, 148, 157 monitoring, viii, 13, 57 monoclonal antibody, 11, 24, 25 Moscow, 86, 150 mutant, 99, 103 mycobacteria, 96

N Na2SO4, 16, 17, 19 NaCl, 9, 10, 17, 19, 20, 28 NADH, 26

171

narcotics, 100 nitrogen, 14, 48 nitrogen gas, 48 NMR, 13, 14, 27, 48 nucleic acid, 8, 20, 25 nucleotides, 23 nutrients, 110

O octane, 93, 97 ODS, 34, 44, 48 oil, 53, 93, 95, 99, 101, 102, 103, 109 oligomers, 9 optical density, 60, 120, 137 optimization, 113 organic chemicals, 92, 100 organic compounds, ix, 94, 105 organism, 106, 107 osmolality, 17, 19 overlap, 138, 139 overlay, 93 oxidation, viii, 27, 32, 33, 47, 48, 49, 50, 51, 55, 96, 102

P Pacific, 99, 101 parallel, 137 partition, 39, 63, 64, 76, 106, 118 pasteurization, 28 pathways, ix, 89, 90 PCA, 92 peptide synthesis, 116 peptides, 23, 25, 27 percolation, 85, 146, 148 performance, 8, 24, 25 permeability, 96 permittivity, 154 pH, 11, 13, 14, 16, 17, 18, 20, 21, 22, 25, 27, 60, 120, 121, 122, 123, 124, 125, 129, 130 pharmaceuticals, 24, 32 phase behavior, 102 phenol, 20, 24, 27, 95, 101 phenylalanine, 133, 134 phospholipids, 95, 96, 102, 103 physical properties, 18 physicochemical properties, 92, 100 physiology, 94 plasma proteins, 4, 6, 16, 23 plasmid, 19, 20, 29 platform, 10, 11, 25 polar groups, 6, 76, 83, 147

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172

Index

polarity, viii, ix, 15, 32, 39, 51, 54, 58, 61, 92, 95, 110, 115, 117, 154, 155, 158, 160 polarizability, 154, 158, 160, 163 pollution, 106 polycyclic aromatic hydrocarbon, 95, 102 polymer, x, 106, 153, 154, 155, 157, 158, 160, 161, 163, 164 polymer molecule, 157 polymer structure, 154, 155, 157, 158, 160, 161, 163 polymerase, 24 polymerization, 22 polymers, 90, 154, 158 polyphenols, 52, 55 polyurethane, 158 positive correlation, 38 precipitation, 2, 4, 16, 19, 20, 22, 23, 28, 29 Principal Components Analysis, 92 probability, 55, 146 probability distribution, 55 production costs, 113 productivity, vii, ix, 31, 46, 89 project, 97 propylene, x, 153, 154, 155, 158, 163 protease inhibitors, 16 protein crystallization, 6, 16 protein folding, 13, 15, 27, 59, 86 protein structure, viii, 3, 13, 15, 16, 57, 58, 59, 71, 73, 77, 83, 147 protein synthesis, 94 protein-protein interactions, 9 protons, 12, 13 pumps, ix, 89, 93, 95, 97, 103 pure water, 61, 65, 119, 123, 131, 132, 134, 136, 137, 138, 146 purification, 7, 9, 10, 11, 16, 17, 18, 19, 20, 24, 25, 26, 29, 44, 48, 86 purity, 58, 116

R reaction mechanism, 15 reaction medium, 27, 53, 116 reactions, 13, 32, 54, 58, 83, 87, 100, 105, 116, 147, 150, 154, 158 reagents, vii, 1, 2, 10, 18, 58, 113 recommendations, iv, 60 redistribution, 125 refractive index, 154 regioselectivity, 32 regression, 50, 91, 92, 155 regression analysis, 91, 92 regression equation, 155 rehydration, 68 relative toxicity, 92

relaxation, 54 relevance, ix, 89, 90 reliability, 123, 130, 142, 157 resistance, 94, 96, 97, 102, 103 resolution, 9, 26, 27, 59, 137 respiratory syncytial virus, 19, 29 rights, iv RNA, 19, 20, 24 room temperature, viii, 13, 41, 42, 50, 58, 79, 83 Russia, 57, 86, 87, 115, 153

S salts, vii, 1, 2, 3, 4, 6, 7, 8, 9, 10, 16, 17, 18, 19, 20, 21, 22, 23, 60, 95, 110 saturated fat, 32 saturated fatty acids, 32 saturation, 96, 106, 107, 158 scanning calorimetry, 87 scattering, 151 screening, 90, 113 SEC, 9 secretion, 24 sediment, 93, 95, 99, 101 selectivity, 11, 58, 113 serum, viii, 9, 16, 25, 28, 57, 59, 60, 61, 65, 67, 68, 71, 72, 73, 74, 75, 77, 78, 81, 86, 87 serum albumin, viii, 9, 25, 57, 59, 60, 61, 65, 67, 68, 71, 72, 73, 74, 75, 77, 78, 81, 86, 87 shape, x, 46, 50, 96, 115, 123, 132, 137 signs, 155, 158, 160 sludge, 100 sodium, 18, 20, 60 sodium hydroxide, 18 solid phase, 53, 83, 147 solidification, 44 solubility, vii, 1, 2, 3, 4, 5, 6, 7, 8, 14, 23, 25, 35, 36, 38, 41, 43, 44, 48, 49, 51, 52, 58, 60, 64, 65, 86, 91, 97, 106, 154, 158 solvation, x, 7, 150, 153, 154, 155, 157, 158, 160, 163, 164 solvent molecules, x, 3, 4, 110, 116, 145, 146, 148 sorption, 83, 87, 139, 147, 148 species, 9, 17, 20, 99, 101, 109 Specord M, 60 spectrophotometer, 59, 60, 120, 137 spectrophotometric method, 123 spectroscopy, x, 27, 86, 115, 120, 136, 151 stabilization, 8, 21, 23, 24, 28 stabilizers, 87 standard deviation, 37, 122 storage, ix, x, 7, 85, 115, 116, 117, 129, 132, 134, 145, 147, 148, 149, 150 stretching, x, 115, 137, 138, 139, 140

Organic Solvents: Properties, Toxicity, and Industrial Effects : Properties, Toxicity, and Industrial Effects, edited by Ryan E. Carter, Nova Science

Index structural changes, viii, 57, 58, 71, 76, 83, 84, 147 substrates, vii, 10, 15, 31, 32, 33, 35, 38, 42, 48, 49, 51, 91, 94, 96, 97, 105 sucrose, 7, 10, 23, 52, 53 Sun, 99, 101, 103 supported liquid membrane, 113 surface properties, 19, 95 surface tension, 3, 4, 23, 43, 46, 54, 97 surfactant, vii, 31, 32, 43, 46, 51, 54 surrogates, 28 survival, 101 sustained development, 97 swelling, x, 153, 154, 155, 156, 157, 158, 161, 163 swelling process, 154 swelling processes, 154 synthesis, vii, 27, 31, 32, 33, 34, 35, 37, 40, 43, 46, 49, 51, 52, 53, 54, 55, 58, 94, 99, 116 synthetic rubbers, 158

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T tannins, 54 temperature, viii, 6, 7, 13, 16, 17, 21, 26, 35, 38, 41, 42, 43, 46, 48, 50, 58, 60, 65, 79, 80, 83, 86, 103, 112, 114, 119 tension, 3, 4, 23, 44, 45, 46, 54, 97 tensions, 32, 43, 45, 46 terpenes, 95, 103 tetrahydrofuran, ix, 61, 62, 68, 71, 72, 76, 115, 117, 118, 126, 135, 150, 160, 161 tetrahydrofurane, 69 thermal stability, 15, 21 thermodynamic properties, 140 thermograms, viii, 57, 58, 60, 79 thermostability, 28, 58 tissue, 7 titanium, 60 toluene, 91, 93, 94, 95, 97, 98, 99, 100, 101, 103, 107, 109 toxic effect, 93, 97, 107, 110

173

toxicity, iv, vii, ix, 23, 91, 92, 93, 94, 95, 97, 98, 100, 101, 105, 106, 107, 110, 111, 113 trace elements, 95 transesterification, 32, 52 transformations, 27 transport, 61, 90, 154, 158 trypsin, 26, 149 tryptophan, 5, 6 turgor, 96 turnover, 7 tyrosine, 129, 131, 132, 133, 134, 136

U Ukraine, 153 urea, 11, 18, 22, 27 UV, 48, 123

V vaccine, 17 vacuum, 60, 65 validation, 93, 97 vapor, 59, 65, 68, 70, 73, 106, 111, 119 variations, x, 91, 115, 124 versatility, 99 vibration, 137, 138, 139, 140 viruses, vii, 1, 2, 4, 8, 16, 17, 18, 19, 20, 28

W water activity, vii, x, 2, 15, 27, 31, 38, 41, 51, 64, 65, 116, 117, 119, 123, 124, 125, 128, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148 water desorption, 2 water sorption, 87 water vapor, 65, 68, 70, 73

Y yeast, 24, 25, 100

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