Characterization of carbohydrates

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NAME AND ADDRESS

DATE

NORTHWESTERN UNIVERSITY

CHARACTERIZATION OP CARBOHYDRATES

A DISSERTATION SUBMITTED TO THE GRADUATE SCHOOL IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE DOCTOR OF PHILOSOPHY

DEPARTMENT OF CHEMISTRY

BY KENNETH MILTON GORDON

EVANSTON, ILLINOIS JUNE, 1942

P ro Q u e st N u m b e r: 10101451

All rights reserved INFORMATION TO ALL USERS The quality o f this reproduction is d e p e n d e n t upon th e quality o f th e c o p y subm itted. In th e unlikely e v e n t th a t th e author did not send a c o m p le te m anuscript an d th ere are missing p ag es, these will b e n o te d . Also, if m aterial h a d to b e rem o ve d , a n o te will in d ic ate th e deletion.

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ACKNOWLEDGMENT

The author wishes to express his grateful appreciations to Professor Charles D* Hurd for his capable direction of this investigation and his many words of encouragement and advice, to the Pabst Company for funds which made the investigation possible and for their cheerful cooperation.

TABLE OF CONTENTS Page INTRODUCTION HISTORICAL AND DISCUSSION OF RESULTS 1. The Methyl Ethers of Sugars History The Purdie and Irvine Method The Muskat Method Analyses of Synthetic Sugar Mixtures by the Method of Hurd and Cantor Analyses of Malt Syrup by the Method of Hurd and Cantor Treatment of the Monosaccharide Fraction from Malt Syrup Analyses Treatment of the Disaccharide Fraction from Malt Syrup Analyses

4

5 6

9 10 13 14

2* The Osazones History Osazones of Malt Syrup

16 17

3* Sugar Acetates History Acetate Formation on Malt Syrup 4 # Attempted Isolation of the Crystalline Free Sugars by Extraction Methods

19 19 21

5. Miscellaneous Derivatives The Benzoates The Triphenylmethyl Ethers

22 23

6 . The Propionates

History Preliminary Survey of the Propionates Preparation of the Sugar Propionates Attempts to Prepare the Isomeric Form of Maltose Octapropionate Distillation of the Propionates Propionylation of Malt Syrup Treatment of the Monosaccharide Propionates from Malt Syrup Treatment of the Disaccharide Propionates from Malt Syrup Analysis of Malt Syrup by the Method of Hurd and Liggett

24 26 26 28 29 30 31 31 32

Page 7. Separation of Reducing and Nonreducing Sugars The Distillation Method ^ Synthesis of Heptapropionyl-a -maltosyl Chloride. Application of Hudson*, s Rules Conversion of Heptapropionylmaltosyl Chloride to Methyl Heptapropionylmaltoside Synthesis of 1-Naphthyl Heptapropionylmaltoside The Oxidation Method Oxidation of Malt Syrup

35 36 37 39 40 41

EXPERIMENTAL 1, The Methyl Ethers of Sugars Methylation of Hydrol by the Haworth Method Methylation by the Purdie and Irvine Method Methylation by the Muskat Method Apparatus for Methylation in Liquid Ammonia Methylation of Methyl a-Blucoside Moih£hre’Determinations on Malt Syrup Protein Removal on Malt Syrup Analyses of Malt Syrup by the Method of Hurd and Cahtor Nitrogen Analysis on Residues from Malt Syrup Analyses Remethylation of the Residues from Malt Syrup Analyses «" Methylation of Maltose Hydrolysis of the Monosaccharide Fraction from Malt Syrup Analyses Hydrolysis of the Disaccharide Fraction from Malt Syrup Analyses Attempts to Obtain 2,3,6-Trimethylglucose from Octamethylmaltose 2.

3.

4.

5.

43 44 44 46 48 48 48 49 50 50 51 51 52 53

The Osazones Osazone Formation on Malt Syrup First Experiment Second Experiment Third Experiment Fourth Experiment

54 55 55 55

Sugar Acetates Acetylation of Malt Syrup Acetylation of Maltose Dextrin Removal on Malt Syrup Distillation of Acetates

55 56 57 58

Attempted Isolation of the Crystalline Free Sugars by Extraction Methods Attempted Maltose Isolation from Malt Syrup

58

Miscellaneous Derivatives Preparation of 3 ,5-Dinitrobenzoyl Chloride

59

Page Treatment of Maltose with 3,5-Dinitrobenzoyl Chloride Benzoylation with Benzoyl Chloride Benzoylation of Glucose with Benzoyl Chloride Preparation of Bis(triphenylmethyl)maltose Conversion of the Bis(triphenylmethyl)maltose to Hexaacetyl-bis(triphenylmethyl)maltose

59 60 61 61 62

6 . The Sugar Propionates

Distillation Apparatus Distillation of Sugar Propionates Analyses for Propionyl G-roups Preparation of the Sugar Propionates Glueose Pentapropionate Maltose Octapropionate L-Rhamnose Tetrapropionate L-Arabinose Tetrapropionate D-Xylose Tetrapropionate D-Fructose Pentapropionate D-Mannose Pentapropionate D~Galactose Pentapropionate L-Sorbose Pentapropionate Cellobiose Octapropionate Lactose Octapropionate Sucrose Octapropionate Gentiobiose Octapropionate Trehalose Octapropionate Melibiose Octapropionate Neolactose Octapropionate Attempt to Prepare the Isomeric Form of Maltose Octapropionate Propionylation of Malt Syrup Distillation of the Propionates from Malt Syrup Treatment of the Monosaccharide Propionates from Malt Syrup Treatment of the Disaccharide Propionates from Malt Syrup Identification of the Decomposition Liquid Analysis of Malt Syrup by the Method of Hurd and Liggett

63 64 66

67 69 70 71 71 72 72 72 73 73 73 74 74 74 75 75 76 76 76 77 78 79 80 80

7. Separation of Reducing and Nonreducing Sugars Preparation of Heptapropionylmaltosyl Chloride 82 Preparation of Methyl Heptapropionylmaltoside 83 Attempted Analysis of a Synthetic Mixture of Maltose and Sucrose 85 Synthesis of 1-Naphthyl Heptapropionylmaltoside 86 Attempted Separation of a Synthetic MaltoseSucrose Mixture 87 Comparison of the Crystalline Qualities of 1-Naphthyl Heptapropionylmaltoside and MaltoseOctapropionate 88 Preparation of Potassium Gluconate 89 Preparation of Potassium Maltonate 89

Page Action of Potassium Hypoiodite on Sucrose Oxidation of a Synthetic Sucrose-MaltoseMixture Oxidation of Malt Syrup Oxidation of Depropionylated Fractions from Distilla­ tions of Malt Syrup Propionates

90 90 91 92

SUMMARY

g4

VITA

96

INTRODUCTION The wide occurence of carbohydrates, their suitability as raw materials for a variety of uses, and our everyday de­ pendence on them both biologically and chemically, have been the impetus for a large number of investigations in the field of carbohydrate analysis.

Yet, the problem of complete carbo­

hydrate analysis is one that has never received a simple and satisfactory solution. The fact that carbohydrates usually occur not as single components but as mixtures from biological and chemical ori­ gins has laid emphasis on the close Interrelation of separa­ tion, characterisation, and estimation as phases of the problem. Because the closely similar structure of sugars results in a general similarity of their chemical reactions, and because of the difficulty attendant in crystallization of either the free sugars or their derivatives, characterization of a complex mixture is often Impossible without a preliminary separation into the various classes of mono-, di-, and trisaccharides. The chemical reactions used in the usual methods of estimation of sugars are exceedingly complex, and little is known concerning their stoichiometric relations; such highly empirical methods as oxidations with Fehling’s and permanganate solutions, fermen­ tation procedures, etc., and methods involving a combination of these with physical methods such as refractive indices, specific rotations, etc., all require a complete characterization

2.

prior to the operation.

Thus, the necessary sequence directed

toward complete analysis of a complex mixture is, of necessity, separation, characterization, and estimation. The time-honored methods generally used for separating and characterizing organic compounds, namely, fractional distil­ lation or crystallization, still seem to be the most logical approach,

Fractional crystallization, while not as promising

as fractional distillation, offers the possibility of simpli­ fication of a sugar mixture should suitable derivatives be found or techniques developed.

It is not to be expected that frac­

tional distillation on derivatives of sugars of the same classes would accomplish a separation, inasmuch as these compounds possess nearly the same vapor pressures for any one derivative; adequate boiling point differences should exist between deriv­ atives of the various classes, however, and fractional dis­ tillation should allow separation into these classes.

Fractional

crystallization methods could then be employed after this pre­ liminary simplification of the mixture. Along these lines should be mentioned the work of Hurd and Cantor1 , who first developed a successful method of separation of sugars Into their various classes by distillation of the methyl ethers of the sugars.

This method, while achieving good

separation according to the classes, does not allow separations by crystallization within the classes inasmuch as most of the methyl ethers of the sugars ere not crystalline.

Identification

of distillation fractions must therefore be made on the basis of specific rotations rather than melting points of crystalline 1. Hurd and Cantor, J. Am. Chem. Soc., 60, 2667 (1938)

3. solids*

Furthermore, transformation of uncrystalline methyl

ether derivatives to other crystalline derivatives is impossi­ ble without altering the parent structure of the sugar.

These

are obvious disadvantages. The aim of the present investigation, therefore, was to perfect crystallization or distillation techniques on established sugar derivatives, or discover new derivatives which would lend themselves well to such operations.

The success of the pro­

cedures would then be tested upon malt syrup, a complex syrup resulting from the enzymatic hydrolysis by diastases on sprouting barley grains.

It was /hoped that the method would supply def­

inite evidence as to the nature of the carbohydrates present in malt syrup. The following topics were studied in regard to the proposed operations: 1.

The methyl ethers; complete methylation as an aid to

crystallization* 2.

The osazones; purification of osazones resulting from sugar

mixtures. 3.

The acetates; either crystallization or distillation.

4.

Isolation of the crystalline free sugars from complex

mixtures by extraction methods. 5*

Miscellaneous derivatives such as the triphenylmethyl

ethers, the acetylated triphenylimethyl ethers, or the benzoates, from the stand-point of crystallization. 6.

The propionates; either crystallization or distillation.

7.

Separation of reducing and nonreducing sugars.

historical and discussion

I*

of r e s u l t s

The Methyl Ethers of Sugars , —

History The methyl ethers of sugars have been extremely Important in the field of carbohydrate chemistry.

No effort will be made

to review the vast amount of literature on this subject, but the principal methods of methylation will be indicated briefly. Fischer^. first found that hemiacetal formation took place when glucose was refluxed with 0.25$ hydrogen chloride In meth­ anol, but it remained for Purdie and Irvine^ to attain etherification of the alcoholic hydroxyls* repeated treatment of methyl iodide.

This was accomplished by

methylof-glueoside with silver oxide and

Because of the oxidizing action of the silver

oxide towards the aldehyde group of the free sugars, this method was applicable only alter partial methylation had been obtained. Some time later, Haworth^ found that methyl sulfate and sodium hydroxide could bring about methylation of both the hemi­ acetal and alcoholic hydroxyls of a sugar.

Thus, glycoside form­

ation took place at 35 °; the temperature was then raised to 70° and etherification of the alcoholic hydroxyl took place.

A

partially methylated sugar resulted, however, complete methyla­ tion being attained only after repeated application of the Purdie and Irvine method* 2. 3. 4.

Fischer, Ber., 28, 1151 (1895) Purdie and Irvine, J. Chem. Soc., 83, 1021 (1903) Haworth, ibid., 107, 8 (1915)

5. Muskat^ observed that potassium could replace all the active hydrogens of a methyl glycoside when the latter was treated in liquid ammonia solution.

Treatment of the potassium salt with

methyl iodide resulted in methylation. Hurd and Cantor^ have found two limitations to the simple methylation procedure.

In the first place, direct methylation

of glucose was shown by them to result in the formation of both -9-0M * KH3* KI Accordingly, another Muskat methylation wascarried

out on an­

other part of the monosaccharide portion from the Haworth methyl­ ation of Hydrol,

The above recommendations were heeded, and all

efforts made to maintain anhydrous conditions (See Fig. l).

Dia­

grams of the apparatus will be found in the experimental part. The product possessed a methoxyl content of 56*1%, a value even 10.

Muskat, J. Am. Chem. Soc. , 5(3, 2449 (1934)

8. lower then that obtained during the first trial. Inasmuch as Muskat used an all glass apparatus for main­ taining anhydrous conditions, it was decided to construct a simi­ lar one, making minor changes necessitated by the handling of larger quantities of material. part).

(See Fig. 2 in the experimental

An initial run on methyl- ar -glucoside with the new

apparatus showed the stopcocks of too small a bore to withstand the pressure generated by the evaporating liquid ammonia.

The

stopcocks were then changed to ones of larger bore, and another operation carried out on methyl - -glucoside.

The product was

found to possess a specific rotation of 155.5, 157.9, 160.30 (the rotation of methyl trimethyl-*-D-glucoside is 160.3°, and that of methyl 1btr ame thy 1- bv the Method of Hurd and Cantor. Malt syrup is the name given to a complex carbohydrate mixture obtained in the following ways Barley is allowed to germinate by steeping in water at 25°C. The germinated barley, or malt, thus obtained is subjected to the mashing process where it is agitated for about two hours in water at a temperature of from 50-65° C.

In this process, the starches

are broken down into dextrins, and hydrolysis of both the starches and dextrins to maltose takes place.

This liquor, or wort, is

then run through filter presses, and then concentrated to a thick syrup in vacuum ovens. In some quarters, this material is called ”malt extract”, the name ”malt syrup” being reserved for a malt extract to which sugar has been added.

This distinction is not recognized generally,

however, so the term ”raalt syrup” will be used throughout the dissertation*

No sugar was added to the syrup used in the present

work. By analogy to the fact that maltose is usually prepared by allowing malt diastases to hydrolyze starch, it is to be expected that the malt syrup would contain a high percentage of maltose. Furthermore, some enzymatic cleavage of maltose should take place and result in the presence of a certain amount of glucose.

To the

best of our knowledge, however, no glucose or maltose has ever been characterized as such from malt syrup. The malt syrup used in these analyses was of the type termed ”non-diastatic”, and was prepared by a reme.shing of the grains from a previous mashing, the temperature being maintained at 63-66° G for one hour in order to destroy any diastases, filtering, and

11. concentrating to a syrup.

Samples were generously provided by

the Pabst Brewing Company, At the time of its manufacture, the syrup contained

7.3% of

non-carbohydrate material such as proteins, acids, and ash.

Since

the percentage of carbohydrates was to be obtained by difference, it was necessary to carry out moisture determinations.

These were

performed by heating the syrup at 105° for periods of nine to sixteen hours.

The average of eight determinations was 21.9%,

a valiie in fairly close agreement with the 78.8% solids by spindle furnished by the Pabst Comp any. Since malt Syrups usually contain

4-5% proteins, the question

was raised if the presence of these materials would influence the results obtained from the methyl ether method.

Accordingly, one

sample, Run 12, was treated with phosphotungstic acid to precipitate the proteins.

By comparison of the results from this run with

those of Runs 9 and 10, which had not been de-proteinized, it is seen that proteins do not interfere in this method of sugar analyses, and their removal is therefore not essential.

The

results are summarized: Monosaccharides Disaccharides Trisaccharides

Run 9 7,9% 52.4$ 10.5$

Run 10 7.7% 51.6$ 11.5%

Run 12 8.5% 50.7$ 11.6$

Total

Average 8.0% 51.8$ 11.0 of maltose in the dextrin-free syrup. Extraction -of propionylation mixture from malt syrup should always he made with ether, as chloroform extracts formexceptional­ ly stable emulsions during washing. Distillation of the Propionates from Malt Syrup * Great emphasis is placed on the fact that separation,

to a

reasonable extent, of the proteins and dextrins in malt syrup must always precede propionylation if distillation of the resulting propionates is to be accomplished successfully. Therefore, 600 g. of the malt syrup was heated for one hour with 500 cc. of 95/& ethanol.

The solution was allowed to cool and

settle and the supernatant liquid was decantated. similarly treated with three more 500-cc. portions.

The residue was The alcohol

from the extract was then removed in vacuo s and the resulting syrup dried with two toluene distillations. Forty-three grams of this material was then shaken with 500 cc. of pyridine and 300 cc. of propionic anhydride, there being ob­ tained 77.9 g. of propionates. This amount was then placed in the 200-cc. distilling flask on the low pressure sppsratus and distilled.

At 155° (o.002 mm.),

78. some decomposition took place, and a light yellow fluid of low viscosity distilled.

Distillation of the monosaccharide fraction

began at 185° (0.008 mm.) and continued to 240° (0.03 mm.).

At

/

248

(0.003 mm.) , signs of severe decomposition were noticed,

distillate becoming quite fluid.

the

This fraction was collected in

the same receiver as was used to collect the first decomposition liquid.

When the distillate became more viscous, it was collected

again as the disaccharide portion. at a bath temperature of 280°.

The distillate was stopped

There was collected 20.2 g. as the

monosaccharide fraction, 4.6 g. as the disaccharide fraction, and a total of 16.6 g. of decomposition liquid (including that found in the cold trap). Treatment of the Monosaccharide Propionates from Malt Svrun The monosaccharide fractions from several such aforementioned distillations were combined and decolorized (to some extent) by dissolving in ether and heating the solution with norite. Rotation:

Subs., 0.5759 g.; ang. rot., 2.54°; [}*Jd=44 .10

Depropionylation was then effected on 22.3 g. of the material by dissolving in 500 cc. of methanol containing 0.4 g. of sodium. The mixture stood at room temperature for fifty-one hours.

The

methanol was removed in vacuo at temperatures below 50°, and 100 cc. of water and 100 cc. of ether were added to the residue.

After

separation of the two layers, evaporation of the ether showed only a small amount of residue.

The water layer was neutralized with

dilute acetic acid, and then reduced to dryness in vacuo at temper­ atures below 55°.

Toluene was then distilled twice from the residue.

The residue was dissolved in 500 cc. of pyridine, and 150 cc.

79. of acetic anhydride added. brown syrup.

There was obtained 10.8 g. of a light

This amount represents a yield of 57.7$ in the

conversion of the propionates to the acetates, ihe specific rotation was then measured: Rotation:

Subs., 0.5131 g.; ang. rot. 2 .22 °; C ° O d - 4 3*3°

Crystallization of the syrup occurred after solution in ethanol. After one re crystallization from ethanol, the melting point was 128-130°.

Since the crystals were soluble in hot water, the entire

body of crystals were recrystallized from that medium. Yield, 1.56 g. of crystals of melting point 131-132°. Rotation:

Subs., 0.4742 g.; ang. rot., 0.26°; £ > JD - 5.48°

Hudson and Dale^S list the values of 132° and 3.8°, respect­ ively, for the melting point and specific rotation of /^-glucose pentaacetate• After removal of the 1.56 g. of ^-glucose pentaacetate, the remainder of the material failed to crystallize.

Since this prob­

ably consisted of the two isomeric forms, a zinc chloride conversion to the of-form was attempted by heating the remaining material with fused zinc chloride and acetic anhydride for thirtyminutes 100 °.

at

No crystalline material was obtained.

Treatment of the Disaccharide Propionates from Malt Syrup Several disaccharide fractions, from which any crystalline maltose octapropionate had been removed, were combined and redistilled. Rotation:

Subs., 0.5885 g.; ang. rot., 3.51°; E®Oc=*59.6°

Depropionylation was effected in the same manner as above by dissolving 3.37 g. of the propionates in 100 cc. of absolute methanol which contained 0.15 g. of sodium.

The reaction mixture was allowed

to stand two days. 52.

Hudson and Dale, J. Am. Chem. Soc. 37, 1264 (1915)

80. Acetylation with 20 cc. of pyridine and 20 cc. of acetic anhydride was then carried out yielding 2.9 g. of a syrup; this amount represents a yield of 72.4$ in the ester conversion.

This

material, however, could not he induced to crystallize. Rotation:

Subs., 0.4255 g.; ang. rot., 1 .93 °; C* J d- 4 5 .3 °

Identification of the Decomposition Linuid This d.ecomposit ion liquid was that obtained during distillations of the propionates from malt syrup. was found to be negative.

A qualitative nitrogen test

The material was then distilled from a

modified Clalsen flask with a column 18 cm. long. the material distilled at 140-142°.

Almost all

A neutral equivalent was then

determined: Anal.

Subs., 0.2421 g.; cc. 0.1050 N base, 30.7 Calcd. for CgH^COOH:

eq.wt., 74.0

Found: €q.wrt. 75.2 An an/?lide was then prepared by mixing 2.5 cc. of the liquid with 5 cc. ofansline and 30 cc. of toluene, and refluxing for eight hours on the steam bath.

After one recrystallization from ethanol,

the an#lide melted at 103-104°.

Pure propionanilide melts at 104°.

Analysis of Malt Svruo bv the Method of Hurd and Liggett Dextrins in the malt syrup were first removed.

Directions were

kindly furnished by S. M. Cantor of the Corn Products Refining Company.

To 200 g. of the wet malt syrup was added 50 cc. of water

and the mixture shaken to an homogeneous syrup.

Twenty-five hundred

cubic centimeters of 95$ ethanol was then added and the mixture shaken for several hours.

The supernatant liquid was then decanted,

and the ethanol removed in vacuo.

The original residue was treated

with a total of three such extractions.

The extractable materiel

was then dried by toluene distillations, yielding 88.0 g. of dried material.

The residue was transferred with water to a 1-1 . flask,

the water removed in vacuo, and the material dried by toluene distillations.

There was obtained 68.9 g. of material as dextrins,

proteins, inorganic salts, etc. The reducing value of the residue was then determined by the method of Lane and Eynon32, 50 cc. of Fehling1s solution being equivalent to 0.4632 g. of maltose hydrate.

Results were:

(a) Solution containing 1.6046 g./ 50 cc.: 50 cc. and 25 cc. Fehling1s required 22.0 cc. and 11.0 cc; $ maltose hydrate, 65.5,65. (b) Solution containing 1.6109 g./50 cc.: 50 cc. and 25 cc. Fehling’s required 22.4 cc. and 11.2 cc; $ maltose hydrate, 64.7, 64 (c) Solution containing 1.7008 g./50 cc. Fehling*s required 20.9 cc; $ maltose hydrate, 65.0 (d) Solution containing 1.7006 g./50 cc.; 50 cc. Fehling*s required 21.0 cc.; $ maltose hydrate, 64.8 The lov^est of the above six values was discarded, and the remaining five averaged: 65.1$ A dextrin determination was then carried out on the malt syrup according to the directions In the A.O.A.C. “Methods of Analysis"^: (a) Sample w t ., 8.00 g.; w t . dextrins , 1.15 g., or 14.4$ (b) Sample wt., 8.37.; wt. dextrins, 1.13 g., or 13.5$ A reducing value could not be determined on these dextrins. Eighty-eight grams of the dried, de-dextrinized malt syrup was then propionylated with 300 cc. of pyridine and 350 cc. of propioniC' anhydride. 53.

There was obtained 165.5 g. of propionates.

Methods of Analysis- A.O.A.C. 4th Edition, n 489, 1935

About one-fourth of the malt propionates (42.99) was dissolved i Propyl alcohol.

crystalline material. and this distilled.

Crystallization occurred, yielding 9.6 g. of

The mother liquor was evaporated to a syrup The distillation occurred in a smooth manner,

the cut being taken at 250° (0.06 mm.)

The bath was taken to 310°.

There was obtained 6.7 g. as the nonosaccharide fraction, 11.1 g. as the disaccharide fraction, and 15.5 g. as the trisaccharide fraction (difference between the 42.9 g. and the weights of the crystalline maltose octapropionate, the mono- and di- fraction). Wt. of mono, propionates Wt. of di. propionates (11.1 * 9.6) 20.7 Wt. of tri. propionates (difference) 15.5 Eq-. wt. of free mono. 2.6 Eq. wt. of free di. 9.4 Eq.. wt. of free tri. 6.5 Percent mono. 14.1$ Percent di. 50.8 Percent tri. 35.1 Wt. of ale. soluble portion 88.0 g. Wt. of free mono. In soluble portion (.141 x 88.0) 12.4 Wt. of free di. in soluble portion (.508 x 88.0) 44.7 Wt. of free tri. in soluble portion (.351 x 88.0) 30.9 Wt. of ale. insoluble portion 88.0 Percent reducing sugars in insoluble portion 65.1$ Wt. of maltose hydrate in insoluble portion (65.1 x 68.9) 44.8 g. Total wt. of maltose hydrate In 200 g. malt syrup 89.5 Percent Percent Percent Percent percent

mono, in whole syrup (12.4/200) di in whole syrup (89.5/200) tri. in whole syrup (30.9/200) dextrins, proteins, salts, etc.(24.1/200) moisture

7.

6 .2$

44.8 15.5 12.1

21.9 100.5

Separation of Reducing and Nonreducing Surars

Preparation of Heotanropionvlmaltosvl Chloride ToYproduce this chloride, 20 g. of maltose octapropionate was dissolved in 80 cc. of absolute chloroform, and the r esulting so­ lution treated dropwise (with stirring) with 7 cc. of titanium

tetrachloride in 56 cc* of chloroform.

The clear yellow solution

was heated at 52° for one-half hour, 5 cc,

more of titanium

tetrachloride in 20 cc, of chloroform then added, and heating continued for one and one-half hours, followed by one hour heating at 73°, and a two hour period of standing at room temperature. The solution was then poured over cracked ice, the resulting mix­ ture extracted with chloroform, the extract washed free of acid, and then dried over anhydrous sodium carbonate. Evaporation of the solvent yielded 19.1 g. of syrup.

A small

portion was taken up in absolute ether and diluted with low petroleum ether.

No opalescence occurred.

readily from _i-propyl alcohol.

However, crystallization occurred Of 3.74 g. of syrup, there was ob­

tained 2.1 g. of cr?/-stals of melting 'ooint 91-92°. crystallized to a melting point of 99-100°.

This was re­

A sodium fusion test

for chlorine on the purified material was definitely positive. Rotation; Subs., 0.3331 g.; ang. rot., 4.29°; C**Jd =139.7° a Analysis for chlorine was carried out by means ofYParr bomb: Anal. Subs., 0.2537 g.; w t . of silver chloride, 0.0492 g. Calcd., for C^^H^q O^q CI: Cly 4.62 Found:

Cl, 4.80

Preparation of Methyl Keotaoronionvlmaltoside_ Fourteen and one-half grams (0.0187 mols) of the noncrystalline heptapropionylmaltosyl chloride was dissolved in 300 cc. of absolute methanol, 20 g. (0.0725 mols, or 0.145 eq-. w t.) of freshly prepared silver carbonate added, and the mixture shaken for tnventy-two hours. These Quantities are proportional to those used by Cantor9 in an analogous reaction on heptaacetylmaltosyl chloride, although the period of shaking was only fourteen to fifteen hours.

After filtering the solution and evaporating the solvent, there was left 7.43 g. of a light brown, fluffy solid that resembled dried malt syrup.

This is considerably below the theoretical yield

of 14.4 g., but closely apnroxiwates a yield based on the assumption of complete hydrolysis to methyl maltoside (b.85 g.).

The material

was found to be water soluble, and the water solution reduced Fehling1s solution. Acetylation of this material resulted in a syrup which crystal­ lized easily in i—prooyl alcohol. raised the melting point from 116°

Repeated recrystallizstions to 125-125.5°.

A specific ro­

tation was measured by dissolving the sample in chloroform, diluting to 2.42 cc., and observing in a 1 dm. tube. Rotation:

Subs., 0.107 0 g.; ang. rot., 2.31°; &Od-52.2°

Hudson and Sayre37 list the following constants for methyl heptaacetyl-^-maltoside:

IVI.p. 124-126°;

C * J d =53*3° (in chloroform).

In an effort to prevent hydrolysis of the ester groups, the time of shaking was reduced:

19.57 g. of heptaoropionylmaltosyl

chloride (syrup), 8 g. of silver carbonate, and 300 cc. of absolute methanol were shaken only twelve hours.

There was obtained 11.0 g.

of reaction product (19.4 g., theoretical yield); the product re­ duced Fehling^ solution strongly, and only 4.98 p;. was extracta.ble with hot chloroform. The chloroform-insoluble portion, amounting to 5.18 g. was then acetylated with 30 cc. of acetic anhydride and 40 cc. of pyridine. From the acetylation was obtained 6.88 g. of a syrup which crys­ tallized easily from jL-propyl alcohol.

After several recrystal­

lizations, there was obtained 1.4 g. of material of m.p. 123-124°.

65. ^

BPtation:

Subs., 0.5277 g.; ang. rot., 2.75°; G* 3d = 51*9°

Synthesis of methyl heptapropionylmaltoside was finally effected by reducing the time of shaking still further:

6.0 g.

(0.0078 mol©) of heptapropionylmaltosyl chloride, 2.15 g. (0.0078 mols) of silver carbonate, and 240 cc. of absolute methanol were shaken for only six and one-half hours.

There was obtained 5.66 g.

of syrup (theoretical yield, 5.98 g.) which still gave a slight test for chlorine and a weak reduction of Fehling1s solution.

The

product could not be induced to crystallize from iypropyl alcohol. The syrup was, therefore, transferred to the low pressure apparatus and distilled.

Some material started distilling at a

bath temperature of 230° (0.0008 mm.) and continued smoothly to a temperature of 296° (0.0008 mm.) observed.

No outstanding (icutn point was

The distillate crystallized readily from i-propyl

alcohol; several recrystallizations raised the melting point from 80° to 83-84°. Rotation:

Subs., 0.2839 g.; ang. rot., 1.23°; C ^ 3 d =43.3o

Anal. Subs., 3.336 mg.; mg. COg, b.701; mg. H 2 0 , 2.096 Calcd. for C34H52018: C, 54.53; H, 7.00, Found:

C, 54.78; H, 7.03

Attempted Analysis of a Synthetic Mixture of Maltose and Sucrose A 1:1 mixture of 10 g. of sucrose and 10.S g. of maltose hydrate was propionylsted with 200 cc. of anhydrous pyridine and 200 cc. of propionic anhydride.

Complete solution took place only

after two days in the refrigerator and twelve days at room temperature. After working up in the usual manner, there was obtained 45.6 of propionates.

86 • The propionates were then dissolved in 160 cc. of chloroform and treated with 25 cc. of titanium tetrachloride in 140 cc. of chloroform.

The reaction product weighed 37.7 g.

This amount of material was then shaken with 8 g. of silver carbonate and 300 cc. of absolute methanol. continued for twelve hours.

Shaking inadvertently

There was obtained 33.4 g. of reaction

product. To the small flask on the low pressure apparatus was then transferred 15.1 g. of the above material and distillation carried out.

ho ncutn point at all was observed, different fractions being

taken by chance alone.

The following analysis, therefore, is not to

be regarded as significant; Material collected as the maltose portion, g. Material collected as the sucrose oortion, g. Material in trap, g. Residue (difference) g. Weight of free maltose, g. Weight of free sucrose, g. Percentage of maltose Percentage of sucrose

4.24 g. 5.30 2.51 3.03 1.99 2.45 44.9 55.1

The fraction taken as methyl heptapropionylmsltoside failed to crystalli ze. Synthesis of 1-Nanhthvl Heptapropionylmsltoside Five grams (0.0065 mole) of heptapropionylmaltosyl chloride, 1.8 g. (0.0065 mole) of silver carbonate, and 1.87 g. (0.013 mole) of impure 1 —naphthol were shaken for nineteen hours in 100 cc. of absolute ether.

After washing the ether extract with water, removal

of the solvent left 5.0 g. of a dark brown oil (4.9 g. theoretical). This material failed to crystallize from i-propyl alcohol.

Because

the possibility exists for partial hydrolysis of the ester groups,

87. the material was propionylated with 50 cc. of propionic anhydride and 50 cc. of pyridine.

There was obtained 4.8 g. of a dark brown

oil which likewise failed to crystallize. In a second attempt, identical quantities as above were used. However, purified 1-naphthol was used and shaking continued for only eight hours.

The unused naphthol was removed by washing with the

ether solution with 5% sodium hydroxide solution. tenth grams of a syrup was obtained.

Four and one-

This crystallized easily in

i-propyl alcohol yielding 2.0 g. of melting point 95-96°. yield, 35%.

Percentage

Another recrystadlization raised the melting point

to only 96-97°. Rotation; Anal.

Subs., 0.4003 g.; ang. rot*, 5.62°;

£)-s*140°

Sub s,, Calcd. for

C > 5 9 *97 H >6 -56

Found:

C*

H*

Attempted Separation of a Svnthetic-IVlaltose-Sucrose Mixture. Thirteen and seven-tenths grains of sucrose octapropionate (partially crystallized syrup) and 20.1 g. of partially crystalline heptapropionylmaltosyl chloride were dissolved in 300 cc. of absolute chloroform.

Then 7.5 g. of purified 1-naphthol and 7.3 g. of silver

carbonate were added, and the mixture shaken for eight to nine hours. After filtering the solution and. washing with 5% sodium hydroxide, sxceptiona3.1y stable emulsions resulted.

The solution was centri­

fuged, the chloroform removed and replaced by ether, the latter being found to cause no emulsification.

After loss of some of the

material during removal of the ether, 22.3 g. of a syrup was obtained. A small portion of the syrup was induced to crystallize in iynropyl

83. alcohol.

These crystals of 1-naphthyl'heptapropionylmaltoside

melted at 90-92°. Tiie mixture was then subjected to distill at Ion on tiie low pressure apparatus but difficulty was encountered in lowering the pressure*

The bath temperature remained for about an hour at 180°.

Distillation was accomplished, however, over a range of 250-290° at a pressure of 0.1 mm.

The weight of the distillate, 18.7 g.,

indicated the distillation of some of the maltoside, although no "cut” point was observed. To see if 1-naphthyl hepta.rropionylmaltoside would distil, 11*5 g. of the material was subjected to a distillation.

During

the disti3_lation, decomposition fumes and darkening of the solution became apparent at 256° and the pressure changed from 0.03 to 1.5 mm.

The bath temperatures was, therefore, allowed to drop

and heating was resumed only after the pressure had lowered.

Even

at 255° (0.2 mm.), there was slow but appreciable distillation. Distillation continued to 305° (0.1 mm.) with the production of 4.4 g. of distillate.

The distillate could not be induced to cip/s-

tallize from I-rropyl alcohol, even though seeded. Comparison of the Crystalline Qualities of 1-baphthvl He ptapr op iony1:maltoside and Maltose 0cta p r o p i o n a t e ____ The tendency of 1 -naphthyl heptapropionylmaltoside to crystallise was compared with that of maltose octaoropionate by the following experiment.

Two mixtures were made, the first (A) containing 2 g.

of crystalline sucrose octapropionate (not purified by recrystal­ lization) and 3 g. of crystalline maltose octapropionate (m.p. 144— 144.5°) and the second (B) containing 3 g. of crystalline 1-naphthyl heotapropionylmsltoside(m.p. 95-96°) and 2 g* of the aforementioned

sucrose octapropionate.

In the case of mixture (A), crystallization

occurred easily after solution in 15 cc. of _i-propyl alcohol. Mixture (B) could not he induced to crystallize in 15 cc. of the same solvent Yfithin twenty—four hours, "but did after forty-eight hours when kept alternately at 0 ° and 25°. Preparation of Potassium Gluconate The method of Moore and L ink ^ was followed In every o detail.* except the quantities employed were twice those used "by them. Four grams of glucose was dissolved in 8 cc. of water on the steam hath and 50 cc. of methanol was added.

This solution was

- then poured into a three neck, one liter flask surrounded by a water bath at 40° and containing a solution of 11.4 g. of Iodine dissolved in 160 cc. of methanol.

There was then added In drop-

wise fashion, during, ten to fifteen minutes, 130 cc. of a solution of potassium hydroxide in methanol (prepared by dissolving 56 g. of the base and diluting to one liter).

Stirring was then con­

tinued for ten minutes, after which 100 cc. of the potassium hy­ droxide solution was ad_d.ed dropwise, and the reaction mixture stirred for a final ten minutes period.

At this stage, the potassium

gluconate had all precipitated, and the solution was colorless. The precipitate was collected, washed with small amounts of methanol and ether, and immediately removed from the filter inasmuch as it seemed to be somewhat hygroscopic.

After drying overnight in a

vacuum desiccator over phosphorus pentoxide, the potassium gluconate, which was white and nicely granular, was found to weigh 5.1 g. yield, 98^o. Preparation

of Potassium Maltonate

Eight grams of maltose hydrate was carried through the above

90. procedure, Identical quantities being used.

Originally, there was

obtained 15.5 g. of material from the oxidation, but this quantity dried to 8.8 g. after several days in a desiccator.

As the theo­

retical yield is 8.82 g., the reaction was practically quantitative. The rotation was measured In water; Rotation: Subs., 0.1740 g.; ang. rot., 1^36°; —

*- ^ubs., 0.5037 g.; wt . of potassium sulfate, 0.1161 g. Calcd. for ci 2H 21°12K: K, 9.82 Found:

K, 10.34

Action of Potassium Hvpoiodite on Sucrose Eight grams of sucrose was subjected to the same procedure as used to orepare potassium gluconate, Identical quantities being employed.

This gave rise to 1.6 g. of granular salt which, if

assumed to be potassium gluconate, represents hydrolysis of 2.34 g. of sucrose, or 29.9% hydrolysis. Oxidation of a Synthetic Sucrose-Maltose Mixture For this purpose, 8 g. of maltose hydrate and 8 g. of sucrose was subjected to the oxidation using the same quantities as used to oxidize glucose.

There was obtained 9.1 g. of granular material.

As the theoretical yield of potassium maltonate is 8.82 g. , there is seen to have been only slight hydrolysis of the sucrose (5 .5%). The filtrate from the above oxidation was then evaporated to dryness in.vacuo and the residue treated with 50 cc. of acetic anhydride and 60 cc. of pyridine.

After standing three days at room

temperature, the mixture was filtered, the filtrate poured into water, and the material so obtained was worked up in the usual manner.

By this process, 9.9 g. of syrup was obtained.

On solution

91. i"Pr°Pyl alcohol end seeding with sucrose octaacetate, crystal­ lization occurred to the extent of 5.2 g.

Theoretical yield from

8 g. of sucrose, 15.9 g.; actual recovery, 32.7%.

When recrystal­

lized from a very dilute ethanol solution, the crystals showed a melting point of 81-83°; when jL-propyl alcohol was used as the solvent, the melting point was found to he 71-72°•

These melting

points are in fairly close accord with the accepted values of 87° and 70° for the polymorphic forms of sucrose octaacetate^.

xt is

interesting to note that these two forms can be obtained from the same material by use of the appropiate solvent. \

Oxidation of Malt Svrun Eleven and three tenths grams of malt syrup (this quantity containing 8 g. of carbohydrates) was dissolved In 6 cc. of water, and 50 cc. of methanol was added. with charcoal.

The solution was then decolorized

The oxidation was run In the usual manner using

11.4 g. of iodine and 230 cc. of the solution of potassium hyd.roxide In methanol.

After drying the potassium salts for twenty-four hours

over phosphorus pentoxlde, they weighed 8.2 grams.

Ten grams of

barium iodide dihydrate in 50 cc. of methanol was then added, the precipitate allowed to settle, and the mixture centrifuged.

After

drying on the steam bath, there was recovered 5.1 g. of material as the barium salts. The filtrate from the above procedure was evaporated to dryness in vacuo, and about 20 g. of material obtained.

Acetylation was

then carried out with 50 cc. of acetic anhydride and 50 cc. of pyridine, the mixture beine shaken for sixty-eight hours. this was obtained 0.27 g. of a syrup which crystallized.

From Rccrystal-

lizatlon from 1-propyl alcohol yielded a minute amount of crystals R4

Linstead, Rutenberg, Dauben, and Evans, J.Am.Chem.Soc.62, 3260 (1940)

92. sufficient for a melting point:

148-149°.

flie oxidation was repeated with four times the quantities used in the previous oxidation.

Only 0.3 g. of acetylated material re­

sulted, however, and defied all efforts at crystallization.

Upon

decolonization of an alcoholic solution of the material with Norite, a clear solution resulted, hut turned dark hrown during the sub­ sequent evaporation of the alcohol.

The material smelled strongly

of iodoform. There was obtained 25.9 g. of potassium salts and 15.3 g. of barium salts.

Note that these quantities are not proportional to

those obtained from the oxidation of the smaller sample of malt syrup. Oxidation of Depropionvlsted Fractions from Distillations of Malt Syrup Propionates A depropionylation of the monosaccharide fraction was first attempted by subjecting 15.3 grams to the action of barium methoxide according to the method of Mitchell^S. no water soluble material.

This, however, produced

The resulting material, however, was

successfully depropionylated by dissolving in 150 cc. of absolute methanol containing 0.25 g. of sodium.

After standing for twenty-

four hours, the solution was poured into about 200 cc. of water and the water solution neutralized with dilute acetic acid.

The

solution was then reduced to a syrup In vacuo. This syrup, weighing 5.5 g., was then oxidized by the Moore and Link method, quantities being 15.7 g. of iodine in 220 cc. of 55.

Mitchell, J. Am. Chem. Soc. 63, 3534 (1941)

93. methanol, and 317 cc. of the potassium hydroxide solution.

No

weight of the potassium salts was recorded because of an accident. To the filtrate was then added 10.7 g. of barium bromide dih.yd.rste in o9 cc. of methanol and the barium salts thus precipitated. The filtrate was then evaporated to d.ryness and acetyl at ed / with 50 cc. of pyridine and 50 cc. of acetic anhydride. After completion of the process, there was obtained 0.5 g. of syrup. This was taken up in ether, decolorized with charcoal, and the ether solution allowed to evaporate in a slight current of air. Crystallization occurred yielding only enough material for a melting point: 122.5-123°. Thirty and nine-tenths grams of the disaccharide propionates was subjected to a depropionylation, likewise unsuccessful, by Mitchell's method.

Depropionylation was finally accomplished by

dissolving the 17.0 g. left in 200 cc. of absolute methanol con­ taining 0.4 g. of sodium.

After twenty-four hours, the solution

was poured into about 300 cc. of water and neutralization effected with dilute acetic acid.

Evaporation of the water and methanol

left 9.3 g. of syrup. This amount was oxidized with 12.9 g. of iodine dissolved in 180 cc. of methanol, and 265 cc. of the potassium hydroxide solution. To the filtrate, after removal of the potassium salts, was then added 8.8 g. of barium bromide dihydrate in 60 cc. of methanol. After removal of the barium salts, the filtrate was evaporated to dryness, and the residue acetylated with 50 cc. of pyridine and 50 cc. of acetic anhydride. material resulted.

Seven-tenths grams of acetylated

No crystalline material could be obtained.

94.

SUMMARY 1*

A study was made of* the various ways for attaining

complete methylation of sugars.

Inasmuch as most of the methyl

ethers of sugars are noncrystalline, they are regarded as poor derivatives. 2.

The osazones, acetates, triphenylmethyl ethers, and

"benzoates were found useless in characterization of complex carbohydrate mixture s. 3.

The propionic esters of sixteen sugars were prepared and

their physical constants recorded.

They were found to distil

smoothly under diminished pressure. 4.

The propionic ester of maltose is regarded as the best

derivative so far encountered for this sugar.

The propionyl

derivatives of cellibiose and gentiobiose are likewise good deriva­ tives.

Those of arabinose, xylose, sucrose, and trehalose, al­

though crystalline, do not possess the characteristics of good derivatives. 5.

By the use of the propionyl derivatives, glucose and

maltose were established as the main constituents, respectively, of the monosaccharides and dlsaccharides in malt syrup. 6.

The percentages of the various classes of sugars In malt

syrup were determined by two different approaches.

That involving

distillation of the propionic esters is regarded as being less accurate in this case than the method of distilling the methyl ethers.

By the latter method, the following results were obtained:

95. monosaccharides, 1 0 .2 $; disaccharides, 7 4 .0 $; trisaccharides, 15.8$. 7.

A procedure to characterize nonreducing sugars in the

presence of reducing sugars was developed. were found in malt syrup by this procedure.

No nonreducing sugars

V ITA

Kenneth Milton Gordon Born: Beardstown, Illinois, November 30, 1911 Education: Public Schools, Beardstown, Illinois, 1918-1929 University of Illinois, Urbans., Illinois, 1934-1938, A.B. Northwestern University, 1938-1942 Positions: Pabst Company Fellow, 1938-1942 Publications: Hurd, Liggett, and Gordon, J. Am. Chem. Soc. 63, 2556 (1941) Hurd and Gordon, ibid. 63, 2557 (1941) Portions of this dissertation were also presented as part of a paper entitled "The Separation end Identification of Carbohydrates" by Charles D, Hurd, K. M. Gordon, and H. W. Liggett before the Division of Sugar Chemistry at St. Louis, Missouri, April, 1941, and before the Chemistry Division of the Illinois Academy of Science at Evanston, Illinois, May, 1941. Societies: Sigma XI Phi Beta Kappa Phi Lambda Upsilon