CARBOHYDRATES OP THE COFFEE BEAN;

ISOLATION OP A KANNAN

DISSERTATION

Presented In Partial Fulfillment of the Requirements

for the Degree Doctor of Philosophy in the

Graduate School of The Ohio State

University

By

MURRAY LANE LAVER, E. S. A.

The Ohio State University

1959

Approved by:

Adviser Department of Chemistry ACKNOWLEDGMENT

The author wishes to express his sincere thanks to

Professor M. L. Wolfrom for his continued interest, counsel and encouragement during the course of this work.

The assistance of Dr. Alva Thompson pertaining to the development of the chromatographic techniques and his general laboratory guidance is gratefully acknowledged.

This work was generously supported by the Nestle

Company, White Plains, New York, under contract with

The Ohio State University Research Foundation (Project

530) and continued by Westreco, Inc., Stamford, Conn., under Project 878 with The Ohio State University Research

Foundation.

Acknowledgment is also made to Dr. A. R. Mishkin and the staff of Westreco, Inc., Marysville, Ohio, for supplying starting material and offering counsel throughout the work.

11 TAELS OP CONTENTS

Page

I. INTRODUCTION AND STATEMENT OF PROBLEM 1

II. HISTORICAL REVIEW 2

A. Carbohydrates of the Coffee Bean 2

3. Hannans 11

1, Ivory Nut Hannans 13

III. EXPERIMENTAL 23

A. Isolation of the Polysaccharide Fractions of Roast Coffee 23

1. Nature of the Starting Haterial 23

2. Extraction with 80/20::Ethanol/ Water 23

3. Extraction with 2/1::Penzene/ Ethanol 2k

k, Extraction with Water 25

5. Extraction with Dilute Ammonlum Oxalate 25

6. Holocellulose Preparation 26

7. Extraction of the Holocellulose of Roast Coffee with 10,2 Potassium Hydroxide 27

B. Isolation of the Polysaccharide Fractions of Specially Extracted Spent Coffee Grounds 27

1. Nature of the Starting Material 28

2. Extraction with Absolute Ethanol 28

iii lv

Page

3. Extraction with 2/1::Bensene/ Ethanol 29

4. Extraction with '/later 29

5. Extraction with Dilute Ammonium Oxalate 30

6. Holocellulose Preparation 30

7. Extraction of the Holocellulose of Specially Extracted Spent Coffee Grounds with 10/® Potassium Hydroxide 31

C. Hydrolysis of the (10.® Potassium Hydroxide)-Insoluble Holocellulose of Green Coffee „ 32

1, Hature of the Starting Material 32

2. Hydrolysis with ?2,£ Sulfuric Acid 33

3, Hydrolysis of Cellulose with Anhydrous Sulfuric Acid 36

4. Hydrolysis of (10/o Potassium Hydroxide)-Insoluble Holocellulose of Green Coffee with Anhydrous Sulfuric Acid 41

D. Derivatives of the Constituent Sugars of the (10,« Potassium Hydroxide)- Insoluble Holocellulose of Green Coffee 46

1. Isolation of Galactaric (Hucic) Ac id 46

2, Isolation of Penta-(2,-acetyl^?-D- ^alactopyranose 4?

3. Isolation of Tetra-£-acetyl-L- arabinose Diethyl Dithioacetal 48 V

Page

a. Partial Hydrolysis with 1.5,-3 Sulfuric Acid ^8

b. Synthesis of Tetra-0.- acetyl-L-arabinose Diethyl Dithioacetal ^9

E, Quantitative Assay of the Constituent Sugars of the (10/£ Potassium Hydroxide)-Insoluble Holocellulose of Green Coffee 51

P. Separating Homogeneous Polysaccharides from the (10/£ Potassium Hydroxide)-Insoluble Holocellulose of Green Coffee 56

1, Acetylation v.lth Trifluoracetic Anhydride 56

a. De-acetylatlon and Hydrolysis of the Chloro­ form-insoluble Acetate 58

b. De-acetylation and Hydrolysis of the Chloro­ form-soluble Acetate 59

2. Extraction w ith 18,» Sodium Hydroxide 60

a. Determining an Hydrolysis Procedure for Praction-A 62

b. Hydrolysis of Praction-A with Pormio Acid 63

o. Purification of Fraction -A by He-precipitatlon from Sodium Hydroxide 6?

d. Purification of Fraction -A by Complexing with Fehling Solution 69 vi

Page

e. Isolation of D-mannose PhenylhyIrazone from Praction-A 71

f. Hydrolysis of Fraction- E with Formic Acid 71

g. Hylrolysis of Fraction- C ivith Formic Acid 72

h. Hydrolysis of Fractlon- D with Formic Acid 72

G, Determining the Structure of Praction-A 75

1. Ilethylation of Praction-A 75

2. Hydrolysis of lie thy la ted Praction-A in Formic Acid 78

3. Attempting to Isolate a Crystalline Derivative of 2,3,6-Tri-£-me thy1-D- mannopyranos e 81

a. Attempting to Prepare 2 ,3,6-tri-Q.-methyl-£- phenyl-D- mannopyranosylamine 81

b, Attempting to Prepare the Phenylhydrazide of 2,3,6- Tri-£-me thy1-D- mannonolac tone 83

IV. DISCUSSION 85

A. Isolation of the Polysaccharide Fractions of Roast Coffee 85 vil

Page

B. Isolation of the Polysaccharide Fractions of Specially Extracted Spent Coffee Grounds 85

C. Hydrolysis of the (10/* Potassium Hydroxide)-Insoluble Holocellulose of Green Coffee 93

1. Hydrolysis with 72.i Sulfuric Acid 93

2. Hydrolysis of Cellulose with Anhydrous Sulfuric Acid 9^

3. Hydrolysis of (10,t Potassium Hydroxide)-Insoluble Holocellulose of Green Coffee with Anhydrous Sulfuric Acid 97

D. Derivatives of the Constituent Sugars of the (10* Potassium Hydroxide)-Insoluble Holocellulose of Green Coffee 98

E. Quantitative Assay of the Constituent Sugars of the (10/S Potassium Hydroxide) -Insoluble Holocellulose of Green Coffee 99

F. Separating Homogeneous Polysaccharides from (10/S Potassium Hydroxide)- Insoluble Holocellulose of Green Coffee 103

1. Acetylation with Trifluoroacetic Anhydride 103

2. Extraction with 18/S Sodium Hydroxide 105

a. Determining an Hydrolysis Procedure forFraction-A 106 vill

Page

b. Hydrolysis of Praction-A with Formic Acid 108

c. Purification of Fraction-A by Re­ precipitation from Solium Hydroxide 109

d. Purification of Fraction -A by Conplexing with Fehling Solution 110

e. Isolation of D-Kannose Phenylhydrazone from Fraction-A 111

f. Hydrolysis of Fraction- 3 with Formic Acid 112

g. Hydrolysis of Fraction- C with Formic Acid 112

h. Hydrolysis of Fractlon- D with Formic Acid 113

1. General Discussion Pertaining to the Fractionation of the (10,£ P o ta s s ium Hyd ro x ide)- Insoluble Holocellulose of Green Coffee by Extraction with 18,2 Sodium Hydroxide Solution 11^

G# Determining the Structure of Fraction-A 118

V. SUMMARY 122

A. Isolation of the Polysaccharides of Roast Coffee 122 ix

Page

3. Isolation of the Polysaccharides of Specially Extracted Spent Coffee Grounds 122

C. Characterization of the (10,2 Potassium Hy 1 r o x 1 de} - In s o 1 uh 1 e Holocellulose of the Green Coffee Eesn 122

D. Separating Homogeneous Polysaccharides from the (10/a Potassium Hydroxide)-Insoluble Holocellulose of Green Coffee 123

E. Determining the Structure of Fraction-A 125 LIST OP FIGURES

Figure Page

1. Chromatogram of (10;£ Potassium Hydroxide)- Insoluble Hollocellulose Hylrolyzate of the Green Coffee Bean 37

2. Hydrolysis of Cellulose with Anhydrous Sulfuric Acid ^+0

3. Hydrolysis of (10/S Potassium Hydroxide)- Insoluble Hollocellulose with Anhydrous Sulfuric Acid ^3

Analytical Chromatogram Processed for Direct Photometry 52

5. Standard Curves for Galactose and Hannose

6. Standard Curves for Glucose and Arabinose 55

7. Hydrolysis of Praction-A in Formic Acid 65

8. Hydrolysis Curves in Formic Acid 68

9. Hydrolysis of Practlon-C in Formic Acid 73

10, Hydrolysis of Fractlon-D in Formic Acid 7^

11. Hydrolysis of Ilethylated Fraction-A in Formic Acid 79

x LIST 0? TABLES Table

X Hg Values of Methylated Sugars 82

2 A Compilation of the Data on the Fractionation of Green Coffee, Hoast Coffee, and Specially Extracted Spent Coffee Grounds {dry weight basis) 88

xi LIST OF CHARTS

Page

I Flow Sheet for the Fractionation of the Polysaccharides of Roast Coffee (g./lOO g., dry weight basis) 86

II Flow Sheet for the Fractionation of the Polysaccharides of Specially Extracted Spent Coffee Grounds (g./lOO g., dry weight basis) 87

III Flow Sheet for the Extraction of (10/» Potassium Hydroxide)-Insoluble Holocellulose of Green Coffee viith 18% Sodium Hydroxide 107

xli I. INTRODUCTION AND STATEMENT OP PROBLEM

Comparatively little fundamental chemical knowledge of coffee is available even though this food is widely used and is of great economic importance. The chemistry of the roasting process is little understood, and the chemical compounds responsible for the desirable aroma and flavor of roasted coffee are not definitely established.

Since carbohydrates constitute 50-60$ of the coffee bean, it would be expected that these substances would play a significant role in the roasting process. Therefore a fundamental study of the chemical nature of the carbohydrates was begun some years ago by this laboratory and this is a continuance of that work.

1 II. HISTORICAL R3VI3W

A j Carbohydrates of the Coffee Bean

Payen (1) was the first investigator to report on the

(1) A. Payen, Corapt. rend., 22, 233 (1846).

carbohydrates of the green coffee bean. He found glucose

(probably sucrose), starch, dextrin, and vegetable acids to comprise 15.5^ of the green coffee bean. The most notaole early work was that of Schulze and associates (2,3,4) who

(2) 3. Schulze, E. Steiger and W. Maxwell, Hoppe-

Seyler's Z. physiol. Chem., ^4, 227 (1890).

(3) E. Schulze, Hoppe-Sezler1s Z. physiol. Chem.,

387 (1892).

(4) E. Schulze, Chem. Ztg., 2Sl» 1263 (1893).

partially hydrolyzed the ether hot alcohol -, and 0.2,£ sodium hydroxide - extracted residue of the green coffee bean with sulfuric acid. Prom the sulfuric acid - soluble hydrolyzate Schulze isolated D-galactose and D-mannose. The

D-galactose crystallized as the free sugar and was further characterized as galactaric (mucic) acid, as the oxime and 3 as the phenylhyirazone. D-raannose was obtained by precipitation with lead acetate an! characterized as the phenylhyirazone. This contradicted the work of Reiss (5)

(5) H. Heiss, Ber., 22, 609 (1889).

who had isolated the same compound from ivory nut but considered it different from mannose and called It "seminoDe".

Schulze's results showed that "seminose" was in reality mannose. In his first paper (2) on the green coffee bean

Schulze and co-workers found insufficient evidence for pentoses.

In his second paper (3) Schulze worked with the residue from the 5/« sulfuric acid hydrolysis. He hydrolyzed this with

75/2* sulfuric acid and the resulting sirup remained slrupy for months but finally D-mannose crystallized. D-Mannose was also obtained by precipitation with lead acetate and characterized as the phenylhyirazone and as the oxlme. The presence of glucose in the sirup was shown by oxidizing it to the potassium hydrogen D-glucarate (saocharate). However, Schulze reported that the amount of the potassium salt of D-glucaric acid obtained from the green coffee bean was much less than that obtained from other seeds. Since L-gulose gives the seme

aldaric acii as D-glucose, Schulze attempted to precipitate

all the D-mannose in the sirup as D-mannose phenylhyirazone and then heated the remaining sirup with phenylhydrazine and

obtainei a phenylosazone identical with glucose phenylosazone.

However, mannose also gives the same phenylosazone. Therefore,

it appears that Schulze found very little glucose in the

difficultly hyirollzable fraction of the green coffee bean and did not obtain a confirmatory derivative of it. He did not

Isolate any galactose or pentose from this fraction.

Swell (6) reported the finding of a considerable pentose

(6) E. S. Swell, Am. Chem. J., U±, k?3 (1892).

content in the green coffee bean by the furfural method.

This caused Schulze to reinvestigate the green coffee bean and he reported a pentose content of 6.7# by the method of

Chalmot and Tollens (7).

(7) G. de Chalmot and B. Tollens,Ber. 2^, 69^ (I89I).

In conclusion Schulze reported that the green coffee beans contained, besides sucrose, a soluble carbohydrate 5 portion from alcohol extraction (not investigate!), a pentosan, a galactan, and a mannan. The last three substances were present in the water-insoluble portion of the coffee bean, Gilson (8) claimed to have isolated a

(8) S, Gilson, La Revue "La Cellule" T. 9» Ze fascicule, S, 429, cited by E, Schulze, Chem, Ztg., 70.

1263 (1893).

mannan by extracting green coffee beans with cuprammonia solution, passing carbon dioxide through the extract and thus precipitating the mannan. However, this procedure was very exacting and there is no record of anyone having repeating it.

There has been little significant work reported since

Schulze's but for a more complete historical review reference is made to the work of tfolfrom and Plunkett which is reported in the Ph. D. dissertation of Plunkett (9).

(9) Ph. D. dissertation of R. A. Plunkett, Department of Chemistry,'The Ohio State University, August, 1955.

Wolfrom and Plunkett undertook a systematic study of 6 the carbohydrates of the coffee bean and also reported non­ carbohydrate components when they were encountered. The free sugars and readily soluble components were identified by extracting finely ground green beans 'with 80/20:‘.ethanol/ water and fractionating the extract on a clay column using graded aqueous ethanol developers of concentration 95/5»

80/20, and 50/50::ethanol/water, successively. The 95/5u ethanol/water fraction contained sucrose (5*5^ of the dry green bean), chlorogenic acid, isochlorogenlc acid, caffeic acid, caffeine and some fats. The sucrose and caffeine were crystallized, the chlorogenic and isochlorogenlc acids were determined by the ultraviolet absorbtion method of Moores,

McDermott, and Wood (10) and the remaining components were

(10) H. G. Koores, D. L. McDermott and T. R. Wood,

Anal. Chem., 2£i, 620 (19^+8).

identified by paper chromatography which also indicated a trace of glucose. The chromatograms viere run simultaneously with known substances.

The 80/20::ethanol/water fraction contained chlorogenic acid, isochlorogenlc acid or caffeic acid or both, 7

trigonelline, a small amount of sucrose and seven amino

acids: glycine, -alanine, -amino butyric acid, proline,

valine, leucine (or isoleucine), and tyrosine. Chlorogenic

acid was isolated crystalline whereas the other components

were identified by paper chromatography simultaneously run

with known substances.

The 50/50::ethanol/water fraction contained trigonelline

and six amino acids: aspartic acid, glutamic acid, serine,

asparagine, -alanine, and -amlnobutyric acid. These

components were identified by developing them on paper

chromatograms simultaneously with known substances.

Wolfrom and Plunkett also carried out the same type of

separation on finely ground roast coffee. The 95/5* :ethanol/ water fraction contained succrose, glucose, fructose,

chlorogenic acid, isochlorogenlc acid and caffeic acid or both, and caffeine. These substances were identified by paper chromatography with simultaneously run known compounds.

The 80/20: :ethanol/ivater fraction contained chlorogenic acid, isochlorogenlc acid or caffeic acid or both, and trigonelline all of which were identified by paper chromatography with simultaneously run known substances. The

50/50::ethanol/water fraction could not be resolved by paper chromatography and probably contained polymeric material. The work on roast coffee was complicated by dark brown

decomposition products which caused poor resolution of the

paper chromatograms. No free amino acids were found in

roast coffee as they viere in green coffee.

Wolfrom and Plunkett Isolated and characterized to

some degree the polysaccharide fractions of the green coffee

bean. They fractionated the finely ground bean by successive

extractions with 80/20::ethanol/water, 2/1::benzene/ethanol, water, 0.5» aqueous ammonium oxalate, acidified aqueous

sodium hypochlorite, and 10,£ potassium hydroxide. The water-

soluble polysaccharide material gave rhamnose (or apios),

and two unidentified carbohydrates on hydrolysis. The

ammonium oxalate extract gave galactose, arablnose, mannose,

rhamnose (or aplos) and two unidentified carbohydrates on

hydrolysis. The (10^ potassium hydroxide)-soluble fraction

gave rhamnose (or apiose), xylose, arablnose, galactose, a

trace of mannose and several unidentified carbohydrates

(probably oligosaccharides) on hydrolysis. No crystalline

derivatives of these hydrolysis products were isolated.

They were identified by paper chromatograms developed

simultaneously with known compounds.

The (10# potassium hydroxide)-insoluble residue gave 9

glucose, arablnose, galactose, mannose and one Inconclusively

Identified carbohydrate on hydrolysis. These compounds were

identified by paper chromatography with simultaneously run

known compounds. D-Mannose was also identified as the

crystalline pentaacetate and as the phenylhyirazone. D-

glucose was also identified as the crystalline pentaacetate.

The amount of D-mannose was estimated as ^5.8$ by preparation

of Its phenylhyirazone. The amount of arablnose was

estimated as 6.7« by the phloroglucinol method. The arablnose

was readily removed by refluxing with 1.5-2 sulfuric acid.

Natarajan, KhantharaJ Urs and Bhatia (11) extracted

(11) C. P. Natarajan, M. KhantharaJ Urs and D. 3.

Bhatia, J. Indian Chem. Soc., Ind. and News Ed., l£j 9 (1955)*

green and roast coffee for 5 rain, with boiling water, and

identified the soluble sugars by paper chromatography without

isolating crystalline derivatives. The green coffee extract

showed sucrose and a trace of glucose, whereas the roast coffee extract showed only traces of sucrose and glucose. A portion of the hot water extracts were inverted by heating

for 9 rain, at 6?° with ]£ hydrochloric acid. The results 10

showed the presence of glucose and fructose in the green

coffee extract, undoubtedly from hydrolysis of the suorose,

whereas the roast coffee extract showed only traces of

glucose and fructose. From this work it appears that the

only free sugars in green coffee are suorose and glucose

and that the quantity of these are reduced on roasting.

This is not entirely in agreement with Wolfrom and Plunkett

(9) who showed that the only free sugars in green coffee

were sucrose and glucose and that the amounts of these

sugars were decreased in roast coffee but they also showed

the presence of fructose In roast coffee, Indicating some

hydrolysis of sucrose during roasting.

Natarajan, KhantharaJ Urs and Bhatia precipitated the

polysaccharides from the water extracts of green and roast

coffee with four volumes of alcohol and hydrolyzed them

with $ hydrochloric acid for five hours. Paper chromatography

showed that these fractions contained galactose, arablnose, and mannose but the intensity of the spot for arablnose was

low.

According to Thaler (12) roast coffee contains two

(12) H. Thaler, Deut. Lebensm. Rundschau., ^ ^9 (1957) 11 groups of materials extraotable with coll and hot water, respectively. He fractionated these further by graded alcohol precipitation. The main fraction of the precipitate consisted of glucose, galactose, mannose and arablnose. At lower alcohol concentrations mannose and glucose were predominant; at higher concentrations the galactose content

Increased.

In another paper Thaler (13) extracted roast coffee

(13) H. Thaler, Z. Lebensra, -Untersuch, U-Forsch,,

123 (1957).

with hot water and precipitated the polymers with Fehling solution. The polysaccharide contained mannose and galactose only and Thaler suggested that It was a galactomannan. It would appear from these two papers by Thaler that he has obtained a mixture of water-soluble polysaccharides which he has been unable to separate into homogeneous polymers.

Bj.—Manaana

According to Aspinall (1*0 true mannans consist of 95^

(14) G. 0. Aspinall, Advances in Carbohydrate Chem., lit. 0000 (1959). 12

or more of mannose residues. Under this definition the only true mannans isolated from land plants are those from vegetable ivory (Phyteleohas macrooaroaK Claims (15,16)

(15) K, Hess and M. Ludtke, Ann,, 4-66. 18 (1928),

(16) E, Husemann, J. Prakt. Chem,, 155. 13 (194-0),

to have isolated mannans from spruce sulfite pulp and spruce holocellulose have not been substantiated by determination of the composition of the polysaccharides. Attempts to obtain true mannans by the fractionation of wood glucomannans has failed (17,18). The salep mannan isolated from tubers of

(17) J. K. Hamilton, H, W. Kirchener and N. S,

Thompson, J, Am. Chem. Soc., £§., 2508 (1956).

(18) T. £. Timell and A. Tyminski, Tappi, 4&, 519

(1957).

various orchids has not been proven to be composed solely of

D-mannose residues (19,20), A true mannan has been isolated

(19) H, Pringsheira and A, Genin, Hoppe-Seyler1s Z. physiol. Chem., 14-0. 299 (1924-). 13

(20) P. Klages and H. Kaurenbrecker, Ann., 521, 175

(1938).

from the seaweed alga Pornhvra umblllcalls and has been shown to contain chains of (1—►*(■) linked 0-D-raannopyranose residues (21).

(21) J, K. N. Jones, J. Chem. Soc., 3292 (1950).

JL> Ivory Nut Hannans

When Fischer and Hirschoerger (22) reported the discovery

(22) E. Fischer and J. Hirschberger, Ber., 21, 1805

(1888).

of mannose by oxidizing mannitol with nitric acid, they predicted that mannose would be found in the plant kingdom.

Their prediction was proven correct when Reiss (5) discovered mannan as a reserve material of seeds possessing thick walls, mainly in the endosperm of the seed of the tagua palm

(Phvtelenhas macrooaroa). commonly called vegetable ivory.

Although Reiss's polysaccharide on hydrolysis yielded a sugar which gave a phenylhyirazone and an oxlme identical 14

with mannose he considered that he had a new sugar because

it precipitated with neutral lead acetate whereas Fischer

and Hirschberger (23) reported that mannose did not. Heiss

(23) S. Fischer and J. Hirschberger, Ber., 22, 36?

(1889).

called the reducing sugar "serainoseUpon re investigation

of the action of lead acetate on mannose, Fischer and

Hirschberger (24) found that an Insoluble lead-mannose

(24) E. Fischer and J. Hirschberger, Ber., ,22, 1155

(1889).

compound could be obtained from a concentrated mannose

solution, and thus corrected their previous report to the contrary, With this result Heiss1s "seminose" was shown

to be mannose and the reserve materials which he had found

in seeds were mannans. Fischer and Hirschberger (24) showed that on acid hydrolysis ivory nut mannan yielded

D-mannose.

Ivory nut mannan could also be extracted with alkali

(25) and Baker and Pope (26) extracted ivory nut meal with 15

(25) S, W. Johnson, J. Am. Chem. Soo., Jjjl., 21^ (1896).

(26) J. L. Baker ani T. H. Pope, J. Chem. Soo., 77.

696 (1900).

cold $$ alkali ani precipitated the mannan as the cupric

hydroxide complex. Decomposition of the complex with

hydrochloric acid gave a polysaccharide having a specific

optical rotationfdjj-^.l0. Hydrolysis of the polysaccharide produced D-mannose in 90/2 yield by precipitation as mannose phenylhydrazone. The other 10,2 was considered to be fructose. 11 In 1927 Hess and Ludkte (27) showed that the mannan from

(27) K. Hess and M. Ludkte, Ann., 4^6, 201 (192?).

ivory nut was not homogeneous. Extraction with 5/2 sodium hydroxide solution and then with 10,2 sodium hydroxide solution yielded, on graded alcohol precipitation, two fractions which were termed mannan-A and mannan-B, respectively. The A-fraction was soluble in Schwelzer's reagent while the B-fractlon dissolved in this solvent only after the addition of ammonium carbonate. The two fractions apparently differed essentially with respect to molecular 16 size, the more Insoluble B-fractlon having the greater chain length*

The structural Identification of ivory nut mannan proceeded slowly because it was poorly soluble, difficult to acetylate or methylate and was quite resistant to acid hydrolysis. Patterson (29) found only tri-£-methyl-D-

(29) J. Patterson, J. Chem. Soc., 123. 1139 (1923).

mannose in the hydrolytic products of the methylated polysaccharide (J+2.6# methoxyl). Klages (30) hydrolyzed the

(30) P. Klages, Ann., 509, 159 (193*0.

water-soluble methylated mannan-A (raethoxyl, ^5*8#; calcd*

**5 .63#) and obtained 1 .**# of 2 ,3 >^|6-tetra-£-methyl-D-mannose and 79# of 2,3,6-trl-£-methyl-D-mannose. Since this left undecided which of the positions or 5 ) was involved in the glycosldlc linkage or in the ring structure, the hydrolysis was interrupted at the di and tri-saccharide stage, the mixture was methylated, hydrolyzed and 2 ,3 ,^16-tetra-^- methyl-D-mannose was isolated as the anlliie (m.p. 1^5“1**7°)• 17

This proved the presence of the (1— 4) glycosldic link

between mannopyranose units in the methylated

oligosaccharide and consequently made probable the same

conclusion for the polysaccharide. Klages and Kaurenbrecker

(20) showed that the glycosldic linkage was by applying

the superposition rules to the osazones of disaccharides.

The mannan-3 fraction, on methylation and hydrolysis, yielded the same products as those from mannan-A (30)*

(30) P. Klages, Ann., 512, 185 (1934).

Kolecular weight determinations of the methylated mannan-A through measurements of freezing point depressions

in benzene (31) yielded a value of 5080 or a degree of

(31) F. Klages and R. Niemann, Ann., 523. 224 (1936).

polymerization of 24 D-raannose residues. The value obtained cryoscopically was considered a minimum in consequence of the possible presence of low molecular weight contaminants.

Wolfrom and Baker (32) determined the degree of

(32) Ph. D dissertation of T. N. Baker, Jr., 18

Department of Chemistry, The Ohio State University, 19^1.

polymerization of mannan-A by a mercaptalatlon methoi which consisted of determining the percentage sulfur in raercaptalated mannan-A. The hyirolysis ani mercaptalatlon were carried out In fuming hydrochloric acid. An equation was derived showing the relationships between degree of polymerization, time ani rate of hydrolysis, 1-1 * do-1 e d do -kt, where i is the degree of polymerization at time t, do is the degree of polymerization at zero time and k is the reaction rate. This equation may also be written as

In d-1 s kt+ln*dQ~l* an:i therefore a plot of -ln(d-l)/d d ( do ) against t should yield a straight line, the slope of which is k and from the intercept, (do-1). the original degree ( do ) of polymerization may be calculated. The plot was indeed a straight line and the extrapolation to zero time gave a degree of polymerization of l6*5+-l. However, it was considered that this end group analysis was probably low due to the quick change in value of the ordinate as it approached zero, making exact extrapolation difficult the value found was four or five times smaller than the end group assay of Kages (33). The slope of the line 19

(33) F. Klages, Ann., 509, 189 (193*0.

showed a specific rate constant k (hours ”*) at 0° of -2 1.3**xl0 ani this was considered as a valuable indication of the energy of bond breaking during hydrolysis.

The structure of the mannans of ivory nut has been studied by using the techniques of chromatography (3*035,36).

(3*0 G. 0. Aspinall, E. L. Hirst, E. G. V. Perclval and I. R. Williamson, J. Chem. Soc., 318^ (1953).

(35) G. 0. Aspinall, R. B. Rashbrook and G. Kessler,

J. Chem. Soc., 215 (1958).

(36) T. E. Tlmell, Can. J. Chem., 21* 333 (1957).

Aspinall, Hirst, Perclval and Williamson hydrolyzed mannan-A and found the following sugars: D-mannose, 97.6#; D-galactose,

1.8#; D-glucose, 0.8$. After methylation and hyirolysis the amounts of methylated sugars were as follows: 2 ,3,*+»6-tetra

-£-methyl-D-raannose, 7*3#; 2,3,*o6-tetra-£-raethyl-D-galactose,

1.7#; 2 ,3,6-trl-&-methyl-D-mannose, 83.0#; 2,3,**-trl-£-methyl

-D-mannose, 6.8#; a dl-£-methyl-D-mannose, 1,2#. Mannan-B 20 was shown to be compose! of essentially the same proportions of sugars. Prom these results the workers conclude! that mannan-A and mannan-B both contained at least two types of molecules, one terminated by a D-msnnopyranose residue and the other terminated by a D-galactopyranose residue.

The majority of the manno-pyranose residues were linked through positions (l-*4) but some (1—>6) linkages were also present.

However, when the mannans-A and -B were partially hydrolyzed, no evidence of oligosaccharides containing the

(1—*6) D-mannose linkage was detected (35) and this linkage has not been confirmed. The main products of the partial hydrolysis were the 0 -D-tl-*^)-linked mannobiose and mannotriose (35)• In addition, small amounts of two other series of oligosaccharides were Isolated, One series contained D-glucose residues at the reducing end of the molecules which caused speculation as to whether they were of structural significance and accounted for the small portion of D-glucose in the purified mannans or whether they arose from epimerization of D-mannose residues. The second series of oligosaccharides oontained some (l-*4)-

1inked<*-D-mannopyranose residues and no evidence could be 21 obtain©! that these could have arisen from reversion or from acid-catalyzed anomerization at the glycosidic bond during acetolysis.

Although mannans-A and -B contain the same chemical features, they appear to be different in molecular weight.

Time11 (36) nitrated ivory nut shavings with a mixture of nitric acid, phosphoric acid and phosphorus pentoxlde and fractionated the resulting polysaccharide nitrates into two different molecular weight ranges with acetone and water.

Osmotic pressure measurements on mannan-A and mannan-B nitrates gave values for the number-average degrees of polymerization of 17-21, and 80, respectively.

It is not known whether two linear molecular species are present, terminated at the non-reducing end by D- mannopyranose and D-gslactopyranose residues or whether some mannan chains are linear while others carry the D- galactopyranose units as slde-chains. Assuming all the chains to be linear, whether terminated by D-mannose or

D-galactose, the methylation studies gave average chain lengths of 10-13 and 38-*H) for mannans-A and -B (3*0. On the other hand, assuming all chains to be terminated by D- mannose residues and the D-galactose residues to be attached 22 as slde-chains, the values for average chain length are

12-16 and 75-80.

For a more complete review of mannans up to the year

1953 reference is made to Whistler and Smart (37).

(37) H. L. Whistler and C. L. Smart, "Polysaccharide

Chemistry," Academic Press Inc., New York, N. Y. (1953)* Ill EXPERIMENTAL

Isolation of the Polysaccharide Fractions of Roast Coffee

1. Nature of the Starting Material

There was received from Westreco, Inc., Marysville,

Ohio, ca. 1^00 g. of finely ground roast coffee which was weighed into two Jars; one containing 800 g. and the other

600 g, An amount of 1 g. of absolute ethanol was added for each gran of coffee and the resulting mixture was boiled for 3-5 minutes in a water bath.

Three samples of the original roast coffee as received from Westreco, Inc. were weighed into aluminum dishes and o dried to constant weight at 70 in a vacuum oven. The average moisture content was This value was used in subsequent calculations.

2. Extraction with 80/20::Ethanol Alater

After the roast coffee had remained under ethanol for ca. 50 hr., 3110 ml. of absolute ethanol and 753 ml. of water were added to bring the concentration to 80/20:: ethanol/water (wt. ratio). The mixture was kept at room temperature for 96 hr. with frequent stirring and then was H filtered through a table top 3uchner funnel. The residue was washed with 2 liters of 80/20::ethanol/water.

The combined filtrate and wash liquors were concentrated

23 24 in a laboratory flash evaporator (Catalog No. 07965. The

Emil Greiner Co., 20-26N, Moore St., New York, N. Y.).

The concentrate was further reduced to eg,. 400 ml. under reduced pressure at 45-50^ and lyophilized. The lyophilized material was dark brown in color ani had a distinct odor of stale coffee; yield 124.3 g. This constituted 16.2.2 of the original roast coffee on a dry weight basis.

3. Extraction with 2/l::Benzene Ethanol

The residue (not dried) from the 80/20::ethanol/water extraction was placed in a l?rge modified Soxhlet and extracted with 2/1;: benzene/ethanol. The extraction -was continued for a sufficient length of time for over 50 changes of solvent to occur.

The 2/1: :benzene/ethanol solvent v;as diluted to 5 liters which insured a homogeneous solution. Two 10-ml. aliquots of the adjusted solution were removed and the nonvolatile solids determined by the quartz sand-vacuum drying method

(38). The values obtained were 0.246 g. per 10-ml. aliquot

(38) Official and Tentative Methods of Analysis of the

Association of Official Agricultural Chemists, 6th Ed., p.

557 (1945) 25 which corresponded to a total of 122,9 g, of material in

the 2/1:: benzene/ethanol extract or 16.02/2 of the original

roast coffee on a dry weight basis.

Extraction with Water

An amount of ^00 g. (iry wt.) of residue from the

2/1:: benzene/e thanol extraction was put in a 5-Hter bottle,

three liters of water were added and the mixture was shaken for 0.5 hr. at room temperature. The mixture was filtered through a layer of milk filter disks (Hapid-Flo filter disk, 6.5", single gauze faced, manufactured by Johnson and

Johnson, ^ 9 ^ 65th St., Chicago 38, 111.) and washed with water until the filtrate turned from dark amber to light grey in color. The filtrate was concentrated on the large evaporator and lyophilized; yield 65.9 g. or 11.2,£ of the roast coffee.

An amount of 258.8 g. (dry weight) of the residue from the first water extraction was extracted a second time with water for 1 hr. at room temperature and filtered. The filtrate was concentrated and lyophilized; yield 7.3 g. of a tan colored, fluffy solid. This constituted 1.6/2 of the roast coffee on a dry weight basis.

5. extraction with. Dilute Ammonium -Oxalate

An amount of 200.0 g. (dry weight) of residue from the 26 second water* extraction was added to 8 liters of distilled water containing 40.2 g. of ammonium oxalate (0*5% solution).

The mixture was stirred vigorously at 90 - 3° for 1 hr,, n filtered through a table-top Buchner funnel ani the insoluble residue washed with Qa. 3 liters of water. This residue was extracted with 8 liters of 0.5^ ammonium oxalate at 90 - 3° for 1 hr. and the mixture filtered. The dry weight of the residue from the second ammonium oxalate extraction was

186.4 g. or 51*3-o of the roast coffee on a dry weight basis.

6. Holocellulose Preparation

The method used in preparing the holocellulose was a modification of the method described by Whistler and co­ workers (37) as adapted by Wolfrom and Plunkett (9). An amount of 5*0 g. (dry weight) of the residue from the second ammonium oxalate extraction was stirred into 120 ml. of water in a flask submerged in a water bath at 75 - 1°. A steady stream of nitrogen was bubbled through the mixture. At 20- minute intervals, 10 drops of glacial acetic acid followed by 2.5 g. of sodium chlorite were added to the mixture until a total of 8.5 g. of sodium chlorite had been added. The total time allowed for the reaction was 1.5 hr. Isoamyl alcohol was also added at the beginning of the reaction to prevent foaming. The hot reaction mixture was rapidly 27 filtered through Whatman No. 1 filter paper and the residue was washed with 600 ml. of distilled water and 100 ml. of acetone. The filtrate and wash liquors were flushed down the drain. The holocellulose residue ivas dried for 12 hr. at 70° In a vacuum oven; weight 4.2 g. or 43.1,3 of the roast coffee on a dry weight basis.

2. Extraction of, the Holocellulose of Roast Co£fee_ _wjt_h Xm .Wflqalte To 3.0 g. of the holocellulose from roast coffee was added 30.0 g. of 10,3 potassium hydroxide solution. The mixture was stirred for 24 hr. at 28 - 1° under an atmosphere of nitrogen. At the end of this time the mixture was some­ what yellowish. The mixture was filtered through a fritted- glass funnel and the residue washed with 50 ml. of 10,3 potassium hydroxide solution followed by 100 ml. of water.

The yellow color passed into the filtrate.

The white residue was further washed with a few ml. of

10.3 acetic acid and several portions of acetone. It was air-dried for 10 hr. and then dried in a vacuum oven at 70° for 12 hr.; yield 2.4 g. or 34.5/3 of the roast coffee on a dry weight basis.

B. Isolation of the Polysaccharide Fractions of Specially

Extracted Spent Coffee Grounds 28

1. Nature of the Starting Material

There was received from Westreco, Inc., Marysville,

Ohio, ca. 1200 g. of finely ground, specially extracted spent coffee grounds. An amount of 1000.0 g. of this material was weighed into a Jar, 1000 ml. of absolute ethanol was added and the resulting mixture was boiled for

3-5 min. in a water bath.

Three samples of the starting material, as received from Westreco, Inc., were weighed into aluminum dishes and o dried to constant weight in a vacuum oven at 70 . The average moisture content was ^.5/2 and this value was used in subsequent calculations. The dry -weight of the 1000.0 g, of specially extracted spent coffee grounds which were extracted with ethanol was, therefore, 95^*8 g.

2. Extraction with Absolute Sthanol

After standing for several days in absolute ethanol, the mixture was filtered through coarse filter paper in a table-top Euchner funnel and the residue washed with 500 ml. of absolute ethanol. The combined filtrate and washings were concentrated under reduced pressure at ^K)0. Water was added to the concentrate and then removed under reduced pressure. This was repeated several times to remove the ethanol and the aqueous mixture was lyophilized; yield 170.0 g. 29

or 17.96 of tine specially extracted spent coffee grounds on

a iry weight basis.

3. Extraction with 2/1: :Benzene/Ethano_l

An amount of 478.0 g. (iry weight) of the ethanol-

insoluble residue was extracted with 2/1:ibenzene/ethanol

in a modified Soxhlet extractor. The extraction was

continued for a time sufficient for 50 or more changes of

solvent to occur.

The 2/1:ibenzene/ethanol soluble solution was increased

to 4 liters and three 10-ml. aliquots were removed by pipet

and the nonvolatile solids determined by the quartz sand-

vacuum drying method (38). The average value obtained 'was

0.236 g. per 10-ml. aliquot. This corresponds to a total

of 94.2 g, of material soluble in the 2/1:ibenzene/ethanol

or 16.2,6 of the specially extracted spent coffee grounds on a dry weight basis.

4. Extraction with Water

An amount of 367.8 g. (dry weight) of residue from the

2/1:ibenzene/ethanol extraction was stirred for 30 rain, in

1500 ml. of water at room temperature and the mixture was 0 then filtered through a large table-top Buchner funnel. The

filtrate was lyophilized; yield 60.3 g. of grey solids or

10.86 of the specially extracted spent coffee grounds on a 30

dry weight basis,

A second water extraction was carried out on 287*8 g*

(dry weight) of the residue from the first water extraction

by aiding 1500 ml. of water and shaking occasionally for 1

hr. at room temperature. The mixture was filtered and the

filtrate lyophilized; yield 6.9 g. of brown solids or 1.3-£

of the specially extracted spent coffee grounds on a dry

weight basis.

5. Extraction with Dilute Ammonium Oxalate

An amount of 215.5 g* (dry weight) of residue from the

second water extraction was added to 8 liters of 0.5/^

ammonium oxalate and the mixture stirred at 90 - 2° for 1 hr. N The mixture was filtered through a table-top Euchner funnel

and the residue was washed with 2 liters of water. The

insoluble residue from the first ammonium oxalate extraction was again extracted with 8 liters of 0.5* ammonium oxalate + Q solution at 90 - 2 for 1 hr. and filtered through a table-

n top Buchner funnel. The dry weight of the insoluble residue

from the second ammonium oxalate extraction was 202.7 g. or

51.91 of the specially extracted spent coffee grounds on a dry weight badla.

6. Holocellulose Preparation

An amount of 5*0 g. (dry weight) of residue from the 31

second ammonium oxalate extraction of specially extracted spent coffee grounds was stirred into 120 ml. of water in a flask submerged in a water bath at 75 - 1°« A steady stream of nitrogen was bubbled into the mixture and every

20 min. 10 drops of glacial acetic acid, followed by 2.5 g, of sodium chlorite, were added until a total of 8.5 g. of sodium chlorite had been added. Isoamyl alcohol was added at the beginning of the reaction to prevent foaming. The reaction was allowed to continue for a total time of 1.5 hr.

The hot reaction mixture was rapidly filtered through

Whatman No. 1 filter paper and the residue was washed with

600 ml. of water and 150 ml. of acetone. The filtrate and wash liquids were flushed down the drain (explosion hazard).

The residue or "holocellulose" was dried for 12 hr. at 90° in a vacuum oven; yield 3.9 g. or 40.5,2 of the specially extracted spent coffee grounds on a dry weight basis.

2, ■Extraction of the Holocellulose of .Specially Extracted

Spent Coffee Grounds with lQjg JPotasslumJaylroxlde

To 3.0 g. of holocellulose from specially extracted spent coffee grounds was added 30.0 g. of 10,2 potassium hydroxide solution and the mixture was stirred for 24 hr. in a water bath at 28 - 1° under an atmosphere of nitrogen.

The mixture was filtered through a fritted-glass funnel and 32 the resiiue was washed with 50 ml. of 10# potassium hydroxide followed by successive washings with 100 ml. of water, 10 ml. of glacial acetic acid and several portions of acetone. It was then air-dried and further dried in a vaccum oven at 70° for 12 hr., yield 2.4 g. or 32.4# of the specially extracted spent coffee grounds on a dry weight basis.

Hydrolysis of the (10# Potassium Hydroxide)-Insoluble

Holocellulose of Green Coffee

.Lj Mature. _of_ the .Starting Material

The (10# potassium hydroxide)-Insoluble holocellulose of green coffee was prepared by the method of Plunkett and

Wolfrom (9) by extracting the holocellulose of green coffee with 10# potassium hydroxide for 24 hr. at 25° under an atmosphere of nitrogen. The mixture was filtered and the white resiiue was washed with 250 ml. of 10# potassium hydroxide, followed by 200 ml. of water, 100 ml. of absolute ethanol and 100 ml. of acetone. After drying in the air for several days the residue was dried for 16 hr. at 50° in a vacuum oven.

The (10# potassium hydroxide)-insoluble material was qualitatively tested for nitrogen by the sodium fusion method and the soda-llme method but both tests were negative.

The Galbraith Laboratories, Box 32, Knoxville, Tennessee, 33

also ran a test for nitrogen and reported only a doubtful

trace.

A few milliliters of the solution from the sodium

fusion were tested for sulfur by adding a few drops of

dilute acetic acid followed by a few drops of lead acetate

solution. No black precipitate of lead sulfide formed and

it was concluded that the material did not contain sulfur.

The solution from the sodium fusion was tested for phosphorus by adding concentrated nitric acid, boiling for

1 min. and aiding ammonium molybiate reagent. No yellow

precipitate of ammonium phosphomolybdate separated and it was concluded that the material contained no phosphorus.

2. Hydrolysis. _with_72^_5ulfuric Acid

A modification of the method of Monler-Willlams (39)

(39) S. W. Monler-Willlams, J. Chem. Soc., 119. 803

(1921).

for the hydrolysis of cellulose was used. A 3 g* sample of

(10$ potassium hydroxide)-Insoluble holocellulose was added to 25 ml. of 72^ sulfuric acid at ice-bath temperature. The mixture was allowed to come to room temperature and was maintained at room temperature for 5 days. At the end of this 34

time the mixture had become lark and small particles of black material remained undissolvel.

Since this procedure obviously resulted in considerable degradation of the carbohydrate material, it was repeated with some modifications. A 1.7115 g* sample of (10,5 potassium hydroxide)-insoluble holocellulose of green coffee was added to 34.6 ml. of 72,5 sulfuric acid in a round- bottomed flask submerged in a. doslum chloride-ice-water bath at -2°. After stirring for 60 hr. at this low temperature, there was no evidence of charring but the material had not completely dissolved. The mixture v?as slowly diluted to ca.

400 ml. at such o rate that the temperature was kept below

10°. Upon dilution, a considerable amount of a white, amorphous substance, ttfhich resembled the starting material, coagulated.

The hydrolysis reaction was further diluted to 1225 ml., resulting in an acid concentration of 3/5 sulfuric acid, and refluxed. Upon refluxing, the reaction mixture turned amber in color and all but a small quantity of material went into solution. After refluxing for Zk hr., a 2- ml. aliquot of the hydrolysis reaction was removed and neutralized with a 3# solution of sodium hydroxide. A quantitative sugar assay by the Somogyl (40) method indicated a total sugar content of (40) M. Somogyi, J. Biol, Chem. , 16&, 61 (1945).

1353*1 mg. of glucose equivalent. The hydrolysis mixture

was refluxed for an additional 4.5 hr. and another Somogyi

determination yielded a total sugar content of 1401.9 mg. of

glucose equivalent. Since this increase in total sugar

content indicated that hyirolysis was not complete, the

refluxing was continued for an additional 10 hr., at the

end of which time a Somogyi determination yielded a total

sugar content of 1402.3 rag. of glucose. Since no further hydrolysis had taken place, it was considered that hyirolysis was complete.

The amber colored hydrolysis solution was neutralized with Duollte A-4 ion exchange resin (Product of Process Co.,

Redwood City, Cal.), which was then removed by filtration and washed well with water. The neutralized filtrate and washings were concentrated to £a. 2 liters and decolorized with carbon. The resulting colorless solution was concentrated to a sirup, and, upon addition of absolute methanol to remove the final traces of water, a white, amorphous precipitate separated. This precipitate was removed by a centrifuge and the decantate evaporated to a sirup once more. When more 36 absolute methanol was added, a second quantity of amorphous, white, precipitate separated. This precipitate was also removed by centrifuge and the process was repeated several times until no precipitate separated on the addition of absolute methanol to the sirup. The combined precipitates were dried in a vacuum oven at 40° and weighed; yield 115.^ mg, or 6.13^ of the starting material. This white, amorphous material was undoubtedly unhydrolyzed or partially hydrolyzed starting material.

The methanol-soluble material was concentrated to a sirup and was chromatographed on paper, using the method of

Jermyn and Isherwood (4l) with ethyl acetate/ acetic acid/

(^1) M. A. Jermyn and P. A, Isherwood, Biochera. J.

(London), J+4, kOZ (19^9).

water:: 3/1/3 as developer. The chromatogram (Pig. 1) was developed for 60 hr. and sprayed with aniline phthalate reagent. The chromatogram, run simultaneously with know compounds, showed the presence of glucose, mannose, galactose, and arablnose.

3. Hyiralysis of Cellulose with Anhydrous Sulfuric Acid

The anhydrous sulfuric acid was made by mixing equal 37

Figure t. CKroma+Qyram of ( \0% Rof*ssium Hydro*»de,)- Insolubfe Holocellulose Hydro lyce+e of fbe Green Coffee Bean 38 portions of fuming sulfuric acid (30# SO^) and concentrated sulfuric acid In a flask submerged in a sodium chloride- ice-water bath. Additional small aliquots of concentrated sulfuric acid were added until the anhydrous sulfuric acid had a melting point 7-9°* A 1.8218 g. sample of medicinal cotton (dried for 96 hr. at 75 - 2°) was added, with stirring, to 30.0 g. (16.3 ml.) of anhydrous sulfuric acid in a three- necked round-bottomed flask submerged In a sodium chloride- ice-water bath. Within minutes the cotton had taken up the acid and the result was a brown viscous mass which was vigorously stirred and protected from moisture by a Drierite drying tube.

The hydrolysis mixture was stirred for 4-8 hr. In the sodium chloride-ice-water bath and, at the end of this time, it was very black. Water was very slowly added from a capillary tube at such a rate that the temperature did not exceed 10°. The hydrolysis mixture was diluted to 1 liter

(3% sulfuric acid) and there remained only a few black particles of undissolved material.

The hydrolysis reaction was refluxed and at 3 hr. intervals a 2-ml. aliquot was removed, neutralized with sodium hydroxide solution and diluted to 50 ml. Somogyi

(40) values were done on these to determine the percentage 39 hydrolysis. A curve of the hydrolysis reaction (Pig. 2)

indicated that hydrolysis was complete after 15 hr. of

refluxing. The black undissolved material was removed by

filtration through a weighed fritted-glass funnel and dried

in a vaccum oven at 50°; weight 2^.8 rag. or 1.36# of the

starting material.

The filtrate was made almost neutral to litmus paper

by slowly adding a saturated aqueous solution of barium

hydroxide. The precipitate of barium sulfate was removed

by centrifuge and the resulting decantate was made slightly

acid to methyl red indicator with 0.1 sulfuric acid and was concentrated to 500 ml. under reduced pressure at

HO0. Throughout the concentration care was taken to keep

the solution slightly acid to methyl red. The solution was neutralized to litmus paper with saturated barium hydroxide

solution. A white precipitate of barium sulfate separated which was removed by gravity filtration through S and S hard filter paper (Product of Schleicher and Schuell Co., New York,

N. Y.). The filtrate was decolorized with carbon and concentrated to a sirup, which was refluxed with three consecutive ^00-ml. portions of absolute methanol. The methanol dissolved the sirup but precipitated the inorganic

salts which were removed by gravity filtration. ^0

too

"oSO

o.

4 6 12 16 2 0 Reflux Time in Hour*

Figure 2. Hydrolgeis of Cellulose w*+h An^drous

'Sulfuric Acid 41

The methanolic filtrate was concentrated to a sirup

which was dissolved in water and diluted to 250 ml, in a

volumetric flask, A quantitative sugar assay by the Soraogyl

(40) method indicated a glucose content of 1785.7 mg. which

constituted a yield of 90,3/^ of the theoretical. The total

solids content of the solution was obtained by the method

of Cleland and Fetzer (42) which consisted of drying portions

(42) J. E. Cleland and W. H, Fetzer, Ind. Eng. Chem.,

Anal. Edition, 12, 858 (1941).

of acid-washed Cellte to constant weight In aluminum drying

dishes. Aliquots of the glucose solution were then-pipeted

into each of the drying dishes and dried to constant weight

in a vacuum oven at £&. 50°; yield 1.7755 g. ov 88.6/2 of the

theoretical,

4. Hydrolysis of (10.2Potassium Hydroxide)-Insoluble

Holooellulose of Green Coffee with Anhydrous Sulfuric Acid

A 2.0295 g* sample of (10/2 potassium hydroxide)-

insoluble holooellulose of green coffee was added to 16.3 ml.

of anhydrous sulfurlo aoid in a flask submerged In a sodium

chloride-ice-water bath at -2°. The resulting mixture

became dark in color. After 24 hr. there was still some 42

undissolved material, and an additional 8.2 ml. of anhydrous

sulfuric acid was added, making a total of 24.5 ml. After a

total time of 54 hr., all the material, except a few black

particles, had dissolved. The solution was stirred

vigorously and water was added dropwlse from a capillary

tube at such a rate that the temperature was kept below 10°.

When diluted to 1500 ml. to give a sulfuric acid

concentration the hydrolysis solution exhibited a clear,

amber color and no insoluble material could be detected.

The solution was refluxed for several hours. Aliquots

of 2-ml. each were removed from the reaction flask after 6

hr., 12 hr,, 15 hr., and 18 hr. of reflux time. These

samples were neutralized with sodium hydroxide solution and

a total reducing value determined by the Somogyi copper

reduction method (40). The hydrolysis curve (Pig. 3) showed

that the reducing values did not increase after 15-18 hr. and therefore hydrolysis was considered complete and the

refluxing was stopped after 18 hr.

The 2-*ml. aliquots were concentrated to sirups and electrochromatographed with borate buffer at pH 10, but the

coloring in the sirups caused streaking and no resolution of

the electrochromatograms was realized.

During refluxing some insoluble material had coagulated J

^3

100

» 8 0 -

7 0 -

2 0 Reflux Time in H o u r*

F iju rt 3. Hydroli/sis o f ( 10% Aof**s*um H^dro/ndoJ-

Iniolublt Holocellufoso w ith An h y d ro u s S ul fur** c Acid 44 and this was removed by filtration through a fritted-glass crucible; weight 58 mg. or 2.8/« of the starting material.

The filtrate was neutralized to litmus paper with a saturated solution of barium hydroxide and the resulting precipitate of barium sulfate vias removed by centrifugation. The decantate was kept slightly acid to methyl red with 0.1 N sulfuric acid and was concentrated under reduced pressure at 40-50° to about 600 ml., and then carefully neutralized to litmus paper with barium hydroxide solution. The resulting small precipitate was filtered through S and 3 hard filter paper under gravity. The filtrate was decolorized with a very small amount of carbon and was once more filtered under gravity. The carbon residue was refluxed with methanol in an attempt to determine if any of the hydrolysis products had become adsorbed on it. The total solids content of the methanol extract, as determined by the Cellte drying process of Cleland and Fetzer (42), amounted to a negligible 35 mg.

The clear, colorless filtrate from the carbon treatment was concentrated under reduced pressure and then diluted to

250 ml. In a volumetric flask. A 200-ml. aliquot was carefully removed for further concentration. A Somogyi (40) reducing value on the remaining 50 ml. showed a glucose equivalent of 1.5405 E» or 71.03^ of the theoretical. The 45

total solids content, as determined by tbe method of Cleland

and Fetzer (42), was 1.8875 g* or 87.03^ of the theoretical.

The 200-ml* aliquot was concentrated to a sirup at

40-50° under reduced pressure. The sirup was refluxed with

200 ml. of absolute methanol to extract the carbohydrate material and precipitate the inorganic salts. The resulting white precipitate was removed by gravity filtration and the

methanollc filtrate was concentrated to a sirup which rapidly

dissolved in methanol at room temperature, indicating that

the inorganic salts had been completely removed. The

solution was again concentrated to a sirup which was dissolved

in water and diluted to 200 ml. The 3omogyl reducing value

(40) of this solution was 1.5275 £* or 70.4* of the

theoretical. The total soliis content as determined by the method of Cleland and Fetzer (42) was 1.7332 or 85*4^ of

the theoretical. Paper Chromatograms of the sirup, developed by the method of Quick (43) showed the presence of glucose,

(43) a. II. Quick, Anal. Chem., 2£, 1939 (1957).

galactose, mannose and arablnose when run simultaneously with known compounds. 46

D. Derivatives

Potassium Hydroxide)-Insoluble -Holooe.llulose of Green Coffee

Paper chromatograms (see Fig. 1) of the hyirolyzate of

(10,$ potassium hydroxide)-insoluble holooellulose of green coffee indicated the presence of glucose, mannose, galactose and arabinose. Wolfrom and Plunkett (9) Isolated crystalline derivatives of glucose and mannose and this left galactose and arabinose to be isolated.

1. Isolation of Galaotarlc (Kuclc) Acid

The method of van der Haar (44) was used to oxidize

(44) A. W. van der Haar, Biochem. Z., 81, 263 (1917).

the galactose in the hyirolyzate of (10$ potassium hydroxide)

Insoluble holooellulose of green coffee to galactaric acid.

A sample of 0,5798 g. of the hyirolyzate sirup and

0.4202 g. of sucrose was dissolved in 60.0 ml. of 25,$ nitric acid and heated on a steam bath to a weight of 20.0 g. The solution was filtered through a cotton plug and, when cool, was seeded with a few crystals of galactaric (mucic) acid and put in the icebox. After a few days, a considerable amount of a crystalline substance had precipitated; m.p,

220-222° unchanged on admixture with authentic material, 47 accepted 222°; x-ray powder diffraction data (interplanar spacing, Cuk radiation; relative intensity, estimated visually; vs, very strong; s, strong; m, medium; w, weak; vw, very weak) (identical with that of authentic material):

6.86 vw, 5.83 vw, 4.87 w, 4.53 vs, 4,14 w, 3.87 w, 3.62 w,

3.41 vw, 3.33 w, 2.90 vs, 2.71 vw, 2.59 s, 2.44 w, 2.39 s,

2.20 vw, 2.05 vw, 1.66 vw.

2. . Isolation of Penta-O-acetyl-l-D-^alac topyranos_e

A large portion of the hydrolysis sirup was aided in a streak across the top of a 22.5 x 18.25 inch Whatman No. 3

MM filter paper. Nine such papers were prepared and developed with 1-butanol/pyridine/water:: 10/3/3 for an accumulative time of 98 h r , The papers were removed from the chromatographic cabinet every 20 hr., air-dried, and returned to the cabinet for further developing. Indicator strips were cut from each side and from the center and sprayed with aniline phthalate solution. The paper chromatograms were pieced together again and the zones of the unsprayed portions corresponding to the movement of galactose were cut out and pulverized in a liter of water. The mixture was filtered through Whatman No. 1 filter paper and the filtrate was concentrated to a sirup, A small portion of the sirup was electrochromatographei on paper and showed 48 the presence Qf galactose only* The remainder of the sirup was acetylated by heating with anhydrous sodium acetate and acetic anhydride and poured into 100 ml. of ice water.

After standing in water for several hours, the acetylatlon mixture was extracted with chloroform which was then dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated to a sirup. The penta-2.-acetyl-.0-D- galactopyranose was crystallized and recrystallized from absolute ethanol; m.p. I4l°-l42° unchanged on admixture with authentic material,[o(]Q^+260 (c,0.97i chloroform); accepted:

142° and +25°. X-ray powder diffraction data (identical with that of authentic material) were as reported by Wolfrom,

Thompson and Inatome (45).

(45) M. L. Wolfrom, A. Thompson and K. Inatome,

J. Am. Chem. Soc., 22* 3868 (1957).

3. Isolation of Tetra-O-aoetyl-L-arablnose Diethyl

Dlthloacetal

a. Fartlal Hydrolysis_w_ith X . 5 A Sulfuric _Aold

The method developed by Wolfrom and Plunket (9) was used for this hydrolysis. A 1.0200-g. sample of (10# potassium hydroxide)-insoluble holooellulose of green coffee 49 was added to 150 ml. of boiling 1.5/* sulfuric acid in a

500 ml, round-bottomed flask. The hydrolysis mixture was refluxed for 5 mln. and then rapidly filtered. The residue was washed free of acid with water. The acid filtrate and washings were neutralized with Duolite A-4 ion exchange resin. After filtering the resin, the filtrate was concentrated to a sirup. A small portion of this sirup was electroohromatographed using borate buffer and when sprayed with aniline phthalate solution the chromatogram showed only one spot which moved the same distance as an authentic sample of L-arablnose.

b . Svnthe sis of Te tra-Q-acetyl-L-arab lnose _Qle thy_l

BHaioagfttel The method of Karabinos and Wolfrom (46) was employed for

(46) J. Karabinos and M. L. Wolfrom, J. Am. Chem.

Soc., £2, 500 (1945).

the synthesis of tetra^-aoetyl-L-arabinose diethyl dlthioacetal. A 50.0 mg. sample of the sirup described above (see Section III-D-3-a) was dissolved in 1 ml. of

12 JJ, hydroohloric acid at ioe-bath temperature, 1.5 ml. of ethanethlol was added and the solution was stirred for 1 hr. 50

The reaction solution was neutralized with concentrated

ammonium hydroxide and the resulting salt was evaporated to

dryness under reduced pressure. This salt was further dried

by adding, and then evaporating, several portions of absolute

ethanol. To the white salt was added 9 ml. of an acetylating agent consisting of 2/l::acetic anhydride/dry pyridine.

The salt dissolved in this reagent and the reaction was allowed to stand at room temperature overnight.

The acetylation mixture was poured into 50 ml. of water

in an ice bath. It was then extracted Ur times with 25-ml. portions of chloroform. The chloroform was ivashed 4 times with 25-ml. fractions of a saturated aqueous sodium bicarbonate solution and finally with water. After drying

over anhydrous sodium sulfate, the chloroform extract was concentrated to a sirup. The sirup was taken up in a few ml. of hot absolute methanol and filtered through a cotton plug. Water was slowly added to the methanol solution until it turned cloudy and the mixture was left in the refrigerator overnight. The arabinose derivative crystallized

in white, needle-like crystals and was recrystallized 4 times; m.p. 76° unchanged on admixture with authentic material,

E>0 Q2-26.ifo (a 7 chloroform); accepted: 79° and -29°.90; x-ray powder diffraction data (identical with that of 51 authentic material): 13*59 vw, 9.6l w, 7.13 vs, 6,19 vw,

5.54 vw, 4,82 n , 4,51 vw, 4,09 s, 3.88 m, 3.72 vw, 3*59 w,

3 .40W, 3.22 vw, 2,96 vw, 2.83 vw, 2.67 vw, 2,51 vw, 2.42 vw.

E. Quantitative Assay of the Constituent Sugars jfehft

(10$ Potassium Hydroxide)-Insoluble Ho-Locellulose of. Green

2 2 U 2 2 . Quantities of the aqueous solution of the hydrolyzate of (10/» Potassium hydroxide)-insoluble holooellulose of green ooffe, hydrolyzed with anhydrous sulfuric acid, (see

Section III-C-4), ranging from 0,015 ml. to 0.030 ml., were applied by a microburet (Aloe Scientific Division, A. S.

Aloe Company, St. Louis 12, Mo.) to paper chromatograms, which were then developed with ethyl acetate/acetic acid/ water::9/2/2 followed by 1-butanol/pyridine/water::10/3/3 a according to the procedure of Quick (43). The chromatograms were set up so that each contained its own standards as well as the solution to be analyzed. Each paper was 9* in. wide and contained five spots of varying amounts of a known mixture and three spots of the mixture to be analyzed after development, the chromatograms were air-dried, sprayed with aniline phthalate indicator, allowed to stand In the air for

15 rain, and then heated in an oven for 15 min. at 110-115°•

Figure 4 is a typical chromatogram processed in this manner. ?\ co c to o 3 - r* » * 0 * 0 0 5 ml.

o e X 3 $ ♦ — 3 ? >c 0.02 ml. 9 ??

The densities of the spots were read as quickly as possible with a Photovolt Electronic Densitometer, Mod. 525 (Photovolt

Corporation, 95 Madison Ave., New York 16 , N. Y.) using ordinary white light and a slit width of 6 mm. The most dense reading for each spot was used. Standard curves were derived from each chromatogram by plotting the logarithm of the sugar concentration of each of the five applications

(see Pig. 4) against the density. Figures 5 and 6 show typical standard curves for each sugar. The concentrations of each of the sugars in the mixture being analyzed were read from these standard curves. The results of the quantitative determinations in percentage of the theoretical hydrolysis yield were as follows: D-glucose, 17.8,2; D-mannose,

48*5#; D-galactose, 14,8#; L-arablnose, 2.8#; total yield,

83*9#. The total solids of the solution before separation into the individual sugars was 85.4# of the theoretical.

The strongly acidic conditions used for hydrolysis degraded arabinose, and this sugar was quantitatively determined in a separate sample by refluxing 1.0297 g. of

(10# potassium hydroxide)-insoluble holooellulose with 50 ml. of 1.5# sulfuric acid for 20 min. The mixture was filtered and the filtrate neutralized and evaporated to a sirup as described above (see Section C-4). The amount of 5^

200

100 9 0 6 0

H 60

T 5 0

3 0

3 4 3 6 7 8 9 10 II 12

Figure 5. Standard CurvO For GtUcfosi «nd Mtnoot* 55

30

20

O G lu c o it A A r « b m o « c

OtnJtfi/ * 10 F«jurt 6. 3+sodsrd Curves for Glucose end Ar«btr>o»« 56 arabinose in the sirup, as quantitatively determined by paper chromatography using direct photometry, was 6.0$ of the theoretical.

F. Separating Homogeneous Polysaccharides from.the 110$

fotegglwa fl.glQ9.eIMP.sg. ,

Wolfrom and Plunkett (9) acetylated the (10$ potassium hydroxide)-Insoluble holooellulose of green coffee with anhydrous acetic acid and trlfluoroacetic anhydride according to the procedure of Barclay, Bourne, Stacey, and

Webb (*1-7) • However, this reaction could not be repeated

(4?) K. S. Barclay, E. J. Bourne, M. Stacey, and

H. Webb, J. Chem. Soc., 1501 (195*0.

when dry carbohydrate material was used. Therefore the alkali extraction was used as an "activating" step to enhance the reactivity of the surface of the polymers. This was accomplished by extracting the holooellulose of green coffee

* with 10$ potassium hydroxide for 25 hr. at room temperature under an atmosphere of nitrogen. The white residue was removed by filtration and never allowed to become dry on the filter but was washed successively w ith distilled 57 water, acetone, absolute ethanol and finally with chloroform.

It was then stored In chloroform. When eg,, 300 mg. of this

"activated" material was added to 21 ml. of anhydrous acetic acid and 30 ml. of trifluoroactlc anhydride, the mixture became warm and the sample went into solution in 2 hr. The reactants were poured into petroleum ether (30-60°) and a brownish, amorphous material separated. When the petroleum ether was decanted and the residue was treated with boiling chloroform, it completely dissolved, leaving no chloroform-

insoluble acetate.

The acetylation was attempted again by adding the polysaccharide material to the anhydrous acetic acid and trifluoroacetlc anhydride in a reaction flask submerged in an ice-bath. After 2 hr. there still remained some unreacted material and the reaction was allowed to come to room temperature and after five additional hours it had completely reacted. The reaction mixture was poured into petroleum ether and a white, curdy precipitate separated. The petroleum ether was decanted and the residue was thrice extracted with hot chloroform. This time a white residue of chloroform- insoluble acetate remained. The chloroform extract was concentrated, leaving a brittle, yellow film of chloroform- insoluble acetate.

The acetyl content of the chloroform-insoluble acetate 58

as determined by the method of Chaney and Wolfrom (48) was

(48) A. C. Chaney and M. L* Wolfrom, Anal. Chera., 2 8 .

1614 (1956).

38.9^. This fraction was re-acetylated with anhydrous acetic

acid and trlfluoroacetic anhydride but the acetyl content did

not increase. The acetyl content of the chloroform-soluble

acetate was 29.81 and was not increased by a second

acetylatlon with trifluoroacetic anhydride and anhydrous

acetic acid.

a.«~- Oe-ao.fety.lation and Hydrolysis of the Chloroform-

insoluble Acetate

A few drops of chloroform were added to a 0.7311 g.

sample of the chloroform-insoluble acetate and although the

material swelled considerably it did not go into solution.

To this swelled material was added 25.0 ml. of sodium

methoxide prepared by reacting 0.25 g, of sodium with 50.0 ml.

of absolute methanol. The sample immediately lost its swelled, soft appearance and became hard and h o m y . The de-acetylation reaction was protected from the atmosphere

and stirred for 2 hr. It was then filtered and the residue was washed successively with water, absolute ethanol and 59 aoetone. The de-acetylsted material was dried for 16 hr. at 50° in a vacuum oven; weight 0.4239 g* Since the starting material contained 3B.9^ acetyl this represented a yield of 94.9,®.

The de-acetylated polysaccharide was hydrolyzed with

25 ml. of 72,£ sulfuric acii for 2 hr. at ice-bath temperature followed by 3 hr. at room temperature. The solution was diluted to 600 ml., to give a 3-£ sulfuric acid solution, and refluxed for 15 hr. Upon dilution a white precipitate formed which did not dissolve after 15 hr. of refluxing. The precipitate was removed by filtration; yield, 18.5-» of the starting material.

The hydrolysis solution was neutralized with barium hydroxide and the resulting barium sulfate precipitate removed by filtration. The filtrate was treated successively with Amberlite IR-120 and Duolite A-4 ion exchange resins and the resulting neutral solution was concentrated to a sirup. A paper chromatogram of this sirup developed with ethyl acetate/acetic acid/water::3/1/3 according to the method of Jermyn and Isherwood (41) showed the presence of glucose, galactose, arabinose and mannose.

b. De-ace,tylation and Hvdrolys 1 s. of the Chlftr.&fam-

? 9 Jteet ofct

A 500-mg. sample of the chloroform-soluble acetate was 60

de-acetylated and hydrolyzed in the same manner as that

described above for the chloroform-insoluble acetate. A

paper chromatogram of this sirup shox'Jed the presence of

glucose, galactose, arabinose and mannose.

2. Extraction with 1 & & Sodium Hydroxide

An amount of 68,5 E* of freshly lyophillzed (10,«

potassium hydroxide)-insoluble holooellulose of green coffee was extracted with 1 liter of 18£ sodium hydroxide solution

for 8 hr. at 25-3°. Nitrogen was continuously bubbled

through the extraction mixture. The mixture was filtered

through a fritted-glass funnel and the residue was washed

free of base with water. The combined filtrate and washings were clear and colorless and the residue was white. The

residue was suspended In water and lyophillzed; yield, 5^*1 g, or 79.0^ of the starting material.

The extract was acidified to a pH of 5 with $0,t acetic acid. A white precipitate immediately formed and the mixture was stored at refrigerator temperature for one week to insure complete precipitation. The solids were removed by centrifuge and dialyzed overnight to remove traces of acid and inorganic salts. The resulting suspension was neutral to litmus. The solids were removed by centrifuge, lyophillzed, and dried over phosphorus pentoxlde for several 61 days in a vacuum desiccator; yield 8.8 g, This amounted to

12.8# of the starting material or 61.1# of the (18^ sodium hydroxide)-soluble portion. This material was designated

Praction-A.

The decantate resulting from the removal of Practlon-A was concentrated to c&, 2 liters in a large laboratory flash evaporator and dialyzed for 4 days to remove the sodium acetate. During dialysis some solids separated and these were removed by centrifuge and lyophillzed; yield, 0.2 g. of fluffy, white material. This portion was designated

Praction-B and amounted to 0.3# of the starting material or I.**# of the (18# sodium hydroxide)-soluble extract.

The decantate resulting from the removal of Praction-B was concentrated to 1500 ml. in the large laboratory flash evaporator, and added to k liters of 95# ethanol, resulting in a 70# concentration of alcohol. A white precipitate immediately formed and the mixture was allowed to stand overnight. When the mixture was centrifuged, most of the white material was removed but some of it was too colloidal to settle. The solids which were removed by centrifuge were lyophilized; yield, 2.2 g. of white, fluffy material. This portion was designated Praction-C and amounted to 3*2# of the starting material and 15*3# of the 62

(18# sodium hydroxide)-soluble extract.

The decantate resulting from the removal of Fractlon-C was concentrated in the large laboratory flash evaporator to

-soluble extract.

The (18# sodium hydroxide)-insoluble residue plus

Fractions-A, B, C, and D amount to 99.7/2 of the (10/2 potassium hydroxide)-insoluble holooellulose used as starting material. Thus, all of the starting material was accounted for.

Determining an Hydrolysis Procedure for Fraotlon-A

An amount of 39.8 mg. of Fraction-A was refluxed for

16 hr. with 50 ml. of 3# sulfuric acid, and at the end of this time there was still a considerable amount of material which had not dissolved. Therefore, dilute sulfuric acid did not offer a good possibility for hydrolysis.

An amount of 123,2 mg. of Fraction-A was added to 25 ml. of oonoentrated (37#) hydrochloric acid at ice-bath temperature. After 0*5 hr. the material had not dissolved so it was allowed to come to room temperature. But, after 63 an additional 0,5 hr., it still had not dissolved, Water was then added to give a 5# hydrochloric acid concentration and, when the mixture was refluxed, Fraction-A dissolved to give a homogeneous solution. Therefore, dilute hydrochloric acid appeared as a possibility for hydrolyzing Fraction-A,

A considerable amount of success in hydrolyzing mannose- rioh polysaccharides using formic acid has been described by

Andrews, Hough and Jones (4-9) and when Fraction-A was treated

(4-9) P. A, Andrews, L. Hough, and J, K, N. Jones,

J. Chem. Soc., 1186 (1953).

with formic acid, most of it went into solution almost at once. It completely dissolved after 2 hr. at room temperature.

Because of the success of other workers with formic acid and the mild conditions necessary, it was decided to use formic acid as the hydrolysis medium for Fraction-A.

Hydrolysis of Fraction-A with Formic Acid

To an amount of 494.7 mg. of Fraction-A was added 50 ml. of 90*6% formic acid. The material immediately swelled in the formic acid and a considerable amount of it went into solution within 5 min. After 2 hr. all the material had dissolved with the exception of a very few specks. The 64 22 rotation was[«

The hydrolysis solution was heated on a steam bath and the rotation was taken every hour. A rate curve for the hydrolysis (Pig. 7) showed that the rotation remained constant after heating for 3 hr. and it was considered that 22 o hydrolysis was complete. The final rotation was^^ +16 22 o which was very close to the rotationf*^ +18 for D-mannose under the same conditions which indicated that Practlon-A was composed largely of a mannan.

The formic acid was removed on a rotatory evaporator and four 50-ml. portions of water were successively added and evaporated in an attempt to remove all of the formic acid and to hydrolyze any formate esters which may have been formed. The resulting sirup was paper chromatographed using ethyl acetate/acetic acid/water::9/2/2 as developer.

After spraying with aniline phthalate indicator, the chromatogram showed four spots. The slowest moving spot was possibly an incompletely hydrolyzed oligosaccharide.

The second slowest moving spot was either glucose or 65

2 0 -

-20

H o u r \3 of H e a t in g

g u re 7. H ^ d r o lg n s oF Fracfion-A in Formic Acid 66

galactose or a mixture of the two (glucose and galactose

are not separate! by this developer)♦ The thirl slowest

moving spot was mannose and the fastest moving spot was

probably formate esters resulting from the formic acid

hydrolysis. In order to hydrolyze these formate esters,

25 ml. of 0*5 H (2.5^) sulfuric acid was added to the

sirup and the solution was heated on the steam bath for

2.5 hr. The hydrolysis solution was processed in the

usual manner (see Section III-C-3) ar*d the resulting sirup was paper chromatographed using ethyl acetate/acetic acid/ water::9/2/2 as developer. The spot corresponding to the

formate esters was not evident and it appeared that these

esters were hydrolyzed by the sulfuric acid treatment.

The sirup was then chromatographed on paper using

ethyl acetate/acetic acid/water::9/2/2 followed by 1- butanol/pyridine/water::10/3/3 according to the procedure

of Quick (43). The chromatogram showed three spots which

corresponded to mannose, galactose and a slow moving, unidentified material which was considered to be an

incompletely hydrolyzed oligosaccharide. The mannose spot was very strong, whereas the other two spots were very

faint. The raannose-to-galactose ratio was quantitatively determined by the densitometry method (see Section III-E). 67

The results showed that the polysaccharide was composed of

9 ^ mannose units and 2,2 galactose units (values corrected for 1,2 moisture).

Purifloation of Fractlon-A by He-precipitation from Sodium .Hydroxide

An amount of 6.17 mg. of Fraction-A was added to 25 ml. of 18,2 sodium hylroxide x*hich had nitrogen bubbling through it continuously. After 5 min. most of the material had gone into solution but even after 1 hr. there was still some undissolved material remaining so an additional 25 ml. of

18:2 sodium hydroxide was added. After stirring for 7 hr,, the material still had not completely dissolved but there was very little remaining. The undissolved material was removed by filtration and the clear, colorless filtrate was neutralized to pH 5 with 50,2 acetic acid and allowed to stand in the refrigerator for 1 week to precipitate. The resulting precipitate was removed by centrifuge, lyophllized and dried over phosphorus pentoxlde for 2 days; yield mg. or 70,2 of the starting material.

This re-precipitated material was hydrolyzed with formic acid. Figure 8 Bhows that the rate curve of hydrolysis was within experimental error for that of Fraotion-A. The hydrolysis reaction was processed as described for Fractlon-A 68

• 2 3 4 5 Hours of H e i f m j

D FV**c4*oi» - A Frscfior “A rc*pp«eipiUfcJ from 16% ionium hydroxide F o M m y ComplM I of Frscfion - A f f h l w j C o m plex U of Fr«cfion-A

Fiytiro 6. Hydrolysis Curves in Formic Acid 69

(see Section III-F-2-b). The resulting sirup was

chromatographed on paper according to the procedure of

Quick and showed a very weak spot for galactose and a

strong spot for mannose. Quantitative analysis of the sirup by the densitometry method showed the following results: mannose, 9^2, galactose, 3/». Thus it appeared that the re-precipltation did not remove the small amount of galactose in Fraction-A,

d. Purification of Fraction-A by Completing: with

Fehling Solution

An amount of 106,8 mg. of Fraction-A was aided to 50 ml. of 18,2 solium hydroxide which had nitrogen bubbling through it continuously. After several hours the material which had not dissolved was removed by filtration. To the filtrate was added 50 ml. of freshly prepared Fehling solution. This resulted in a gelatinous, blue precipitate which was removed by centrifuge and washed with more Fehling solution. The precipitate was suspended in water and dissolved with 2 ]£ hydrochloric acid to a pH of 2-3.

Although the blue copper complex was decomposed by the acid, a considerable amount of white precipitate remained. This precipitate was removed by filtration through a fritted- 70

glass funnel, dialyzed for three days and lyophlllzed;

yield 21,8 mg. This fraction was called Fehling Complex I

of Fraction-A and was hydrolyzed with formic acid. The

hydrolysis curve was within experimental error for the

hydrolysis curve for Fraction-A (Fig. 8 ), The hydrolyzate

was processed and chromatographed as described for Fraction-

A (see Section III-F-2-b). The chromatogram showed spots

corresponding to galactose (very weak) and mannose (strong),

A quantitative analysis by the densitometry method showed

mannose, 93# and galactose, 2#.

The filtrate after removal of Fehling Complex I was

added to three volumes of acetone. The solution turned

yellow and a turbidity developed. This mixture was allowed

to stand at -25° for several days to allow complete

precipitation. The resulting precipitate was removed by centrifuge, dialyzed for 3 days and lyophilized; yield,

62.1 mg. This was labeled Fehling Complex II of Fraction-A.

Fehling Complex I and II yielded a return of 83*9 mg. or 79# of the starting material.

Fehling Complex II was hydrolyzed with formic acid and

the hydrolysis curve (Fig. 8 ) was within experimental error of that for Fractlon-A. The hydrolysis mixture was processed and chromatographed as described for Fraction-A (see Section III-F-2-b)• The chromatogram showed a very weak spot for galactose and a strong spot for mannose. A quantitative analysis by the densitometry method showed mannose 94;S, galactose 3-»* Thus, it appeared that complexing Fraction-A with Fehling solution did not remove the galactose.

e . Isolation of P-Hannos_e Phenvlhydrazone f_r_om

Fraction-A

To a 20-ml. aliquot of the hydrolysate of Fraction-A was added 0.2 ml. of phenylhydrazine and 0.2 ml. of glacial acetic acid. The solution was placed in the refrigerator overnight and the resulting crystals of D-mannose phenlhydrazone was filtered through a weighed fritted-glass funnel; yield, 95/£ of the theoretical; m.p. 187-190°, unchanged on admixture with authentic material, [°dn6+25°

(

11.94 m, 5*60 m, 5*23 s, 4.43 vs, 3.96 vs, 3.^8 vw, 3.30 vs,

3.10 vw, 2.99 w, 2,79 vw, 2.68 vw, 2.41 m.

f. Hydrolysis of Fraction-B with Formic Acid

An amount of 78.4 g. of Fraction-B was hydrolyzed with

15 ml. of 9 0 .6# formic acid by heating on a steam bath for

5 hr. The rotation remained constant at +37*5° after one additional hour of heating and it was considered that 72

hydrolysis was complete. The formic acid hydrolyzate was

processed and chromatographed as described for Fraction-A

(see Section III-F-2-b), The chromatogram showed the

presence of mannose, galactose and arabinose. A quantitative

analysis by the densitometry method showed the following

results: arabinose, 8.9,®; mannose, 68,1,1; galactose,

16.3#.

_ Hydrolysis of Fraction-C with Formic Aold

An hydrolysis curve of Fraction-C in formic acid

(Fig, 9) Indicated that the material must be heated for

more than b hr, for hydrolysis to be complete. The

rotation after 4 hr, of heating remained constant a t M ^ + 960 .

An amount of 126,0 mg. of Fraction-C was dissolved in

25 ml, of 90,6,1 formic acid and heated on a steam bath for

7 hr. The hydrolysis solution was processed as described

for Fraction-A (see Section III-F-2-b). A paper chromatogram

indicated the presence of arabinose and galactose and a trace

of mannose. Quantitative analysis by the densitometry method

showed the following results: galactose, 63.5,1; arabinose,

19.OX.

Hi Hydrolysis of Fraction-D with _Formlc Acid

Figure 10 is a hydrolysis curve for Fraction-D in

formic acid. The specific rotation remained constant at 73

110

100

9 0

6 0

7 0

4 0

3 0 -

20

Hours of*

Pijur# 9. Hydrolysis of fraction-C in Formic 6cid 7^

100

9 0 ao

7 0

6 0

1 6 0

4 0

3 0

0 2 3 Hours «f

Flours IO« Hydrolysis of Frtcfion - D ♦n F o r m ic 4 c id 75

+89° after 5 hr, of heating and it was therefore considered that an hydrolysis reaction must be heated for this length of time to ensure complete hydrolysis.

An amount of 135.3 mg, of Praction-D was dissolved in

35 ml. of 90.6# formic acid and was then heated on a steam bath for 8 hr. The hydrolysis solution was processed as described for Praction-A (see Section III-F-2-b). A paper chromatogram indicated the presence of galactose, arabinose and a trace of mannose, Quantitative analysis by the densitometry method showed the following results: galactose, 65#; arabinose, 21#,

Q. Determining the Structure of .Fraction-A

X. MethYlation of Fraotlon-A

An amount of 1,38 g, of Praction-A was added to 50 ml. of 18# sodium hydroxide solution and the mixture stirred overnight, resulting in a thick, slurry-like suspension.

To this suspension was added 6 g. of solid sodium hydroxide pellets followed by the dropwise addition of 100 ml, of 30# sodium hydroxide and 50 ml. of dimethyl sulfate. The raethylatlon reaction was started at ice-bath temperature but after 3 hr. it was allowed to come to room temperature.

The reaction foamed considerably and 50 ml. of aoetone was added to prevent the foaming. Throughout the addition of 76

the reagents the reaction mixture was very strongly stirred.

After stirring a total of 48 hr., the mixture was somewhat

brown and a white, amorphous precipitate (sodium methyl

sulfate) had separated. The reaction mixture was made

almost neutral to litmus with 10% sulfuric acid. The

neutralization was carried out at ioe-bath temperature.

The reaction mixture was then dialyzed against running water

for 3 days.

The resulting neutral solution was concentrated to

ca. 200 ml. under reduced pressure at 35-^K)0 and

lyophllized. The sodium hydroxide, dimethyl sulfate

methylation was repeated once more. After the second

methylation the material was soluble in acetone and

tetrahydrofuran. Commercial tetrahydrofuran was dried over

potassium hydroxide pellets for several days and then

distilled at 64-65° from potassium hydroxide pellets.

The method of Falconer and Adams (50) was used to

(50) E, L. Falconer and G, A. Adams, Can. J. Chem.,

J*4, 338 (1956).

complete the methylation. The lyophllized product of the

second methylation was taken up In 150 ml. of dry 77 tetrahydrofuran and although the material appeared to dissolve the solution remained slightly cloudy. The reaction was stirred strongly and 10 g. of crushed sodium hydroxide was added, followed by the dropwise addition of

12 ml, of dimethyl sulfate. Six additional additions of the same quantity of sodium hydroxide and dimethyl sulfate were added over a period of 2 days. After the seventh addition, stirring was continued for 12 hr. Throughout the reaction, additional amounts of tetrahydrofuran were added to maintain fluidity. A heavy precipitate of sodium methyl sulfate was formed during the reaction. The reaction mixture was neutralized with 10$ sulfuric acid in an ice-bath and was then dialyzed against running water for 3 days. The resulting solution was concentrated to ca.

600 ml. under reduced pressure at 35-^° and extracted 3 times with 300 ml. portions of chloroform. The chloroform layer was concentrated under reduced pressure yielding a light brown solid. This solid was dissolved in acetone and filtered through a fritted-glass funnel. The filtrate was concentrated to ca. 50 ml. and poured into 200 ml. of petroleum ether (b.p. 30-60°). A white precipitate immediately formed which was allowed to stand in the refrigerator for several days to coagulate. The precipitate 78 was then removed by filtration, dissolved In water and lyophllized. The resulting white, fluffy solid was dried for several days in a vacuum dessicator over phosphorus pentoxide; yield, 595 rag.

The methoxyl content, as determined by the method of

Hoffman and Wolfrom (51), was ^5.**#. A suspension of the

(51) D, 0, Hoffman and M. L. Wolfrom, Anal, Chem.,

12, 225 (19^7).

methylated polymer in nujol did not show an absorption for hydroxyl groups by infrared absorption. The rotation of the methylated polymer was W 2^-70° (£ 0,6, formic acid) D showing that the linkages were primarily $ ,

2»Hydrolysis of Methylated Fraction-A in Formic Acid

The hydrolysis of methylated Praction-A in formic acid was followed by rotation and Figure 11 shows that hydrolysis appeared complete after 3 hr, of heating.

An amount of 221,7 mg. of methylated Fraotlon-A was dissolved in 35 ml. of 90.6# formic acid and heated on a steam bath for 3 hr. The formic acid was removed under reduced pressure and several successive portions of water were added and subsequently evaporated in an effort to 79

-30 -40

-60 - -70

Hour* of Hss+ing

Figure II* Hydrolysis of Msfhyla+ed

Fr«cfion - A in F o s m i c A c id 80

remove all traces of formic acid. The sirup was dissolved

in 15 ml. of 0,5 Ji sulfuric acid and heated on a steam bath

for 3 hr. The sulfuric acid was neutralized with saturated

barium hydroxide solution and the resulting barium sulfate

removed by gravity filtration. The filtrate was concentrated

to a sirup and extracted with refluxing absolute methanol.

The resulting insoluble inorganic material was removed by

gravity filtration and the filtrate was concentrated to a

sirup. There were some crystals in the sirup which were not

readily soluble in methanol but were soluble in water.

When some of this material was removed by spatula and held

in a flame an ash was left which indioated inorganic material. The sirup was dissolved in water and a small aliquot was removed and tested with sulfuric acid which resulted in a precipitate of barium sulfate. Thus the crystals which appeared in the sirup were probably barium formate which resulted from the incomplete removal of formic acid. In order to remove this substance the aqueous solution of the sirup was treated successively with Amberlite IH-120 and Duolite A-4 ion exchange resins. The resulting solution was concentrated to a sirup which readily dissolved in absolute methanol at room temperature with no indication of inorganic material being present. The methanolic solution 81 was concentrate1 to a sirup which wag dilute! to 200 ml. with water. A 25-ml. aliquot iras remove! to determine

the total solids of the solution by the method of Cleland

and Fetzer (^2); yield, 98/3 of the theoretical.

A paper chromatogram of the remaining solution was developed for one length of the paper using ethyl acetate/ acetic acid/water::9/2/2 as developer an! revealed five

spots, the Rg values of which are given in Table I (Rg is

the mobility of the sugar on the chromatogram relative to

2 ,3 ,^,6-tetra-2.-methyl-D-glucopyranose) .

The paper chromatogram indicate! a great predominance

of a trl-0.-methyl-D-mannose (see Table I), and thus Fraction

-A appeared to be a straight chain mannan.

3. Attempting to Isolate a Crystalline Derivative of 2.3.6- T r1-0-me thy1-D-mannopyranos e

a. Attempting- to prepare 2.3.6-trl-0-:nethyl-PhenYl- D-mannopy ran o sy lamlne

A 200-mg. sample of the sirup resulting from the hydrolysis of the methylated mannan was used in an attempt to prepare 2 ,3,6-tri-^-methyl-^-phenyl-D-mannopyranosylamine.

To an ethanol solution of the sirup 1 mole of freshly distilled aniline was added and the solution was refluxed for 5 hr. The reaction solution was then concentrated considerable and left in the refrigerator but no crystallization occurred. The solution was seeded but still TABLE I

Rg Values of Methylated Sugars

a Rg Value of Possible Compound Rg Value of unknown spot Possible Compound

0.98 b 2 ,3f4t 6-tetra-Q.-me thyl-mannose 0.96

0.92 b 2,3*4, 6-tetra-Q,-me thy 1-galactose 0.88

0.82 0 2»3 ,4,6-tr1-0-methyl-mannose 0.81

0.59 b 3, 4-di-0-me thyl-mannose 0,58

4,6-d1-0-methyl-mannose 0.57

2,3-dl-O-methyl-mannose 0.54

0.18 b 4-0,-me thyl-galac tose 0.16

6-0-methy1-galactose 0.18

£ E. L. Hirst and J, K* N. Jones, Discussions Faraday Soc., No.7 . 268 (19^9).

k A trace; meaning the spot was too weak to be recorded on a densitometer.

0 This spot was very strong. 83 the anilide ill not crystallize. Crystallization was attempted In various organic solvents such as ether and petroleum ether but still the derivative did not crystallize.

b. Attempting Mo. Prepare the Phenylhvdraz lie of

2. 3.6 -Tr 1-0 -me t hy 1-D-mannono-lac tone

Approximately 200 mg. of the sirup resulting from the hydrolysis of the methylated mannan were added in strips to eight sheets of Whatman No. 3 MM filter paper. These were developed one length with 1-butanol/pyrldlne/water::10/3/3 and side strips were cut off and sprayed with aniline phthalate indicator in order to looate the zones. The zone containing the heavy amount of sugar was cut out and eluted with water. The resulting aqueous solution was concentrated to a sirup which was extracted with boiling methanol to remove any material which washed from the paper. The methanolic extract was concentrated to a sirup which was dried in a dessleator and weighed; weight, 150 mg.

The method of Hudson and Isbell (5 2 ) for the

(52} C. S. Hudson and H. S. Isbell, J. Am. Chem.

Soc., 51, 2225 (1929). 84

preparation of calcium D-gluoonate was used to oxidize the

2,3,6-tri-Q,-raethyl-D-raannopyranose to the corresponding

acid lactone. The 150 mg. of sirup was dissolved in 10 ml*

of water containing 500 mg. of barium benzoate, 30 drops of

bromine were added and the solution was allowed to stand

for 6 days In the dark at room temperature. The reaction

mixture was processed as described by Hudson and Isbell

(52). The resulting sirup should have been 2,3,6-trl-gt-

raethyl-D-mannano-lactone which has been crystallized

(53»54), but this sirup resisted crystallization. An

(53) W, N. Haworth, 3. L. Hirst and H. R. L.

Streight, J. Chera. Soc., 1349 (1931).

(54) P. Smith, J. Am. Chem. Soc., ,20.1 3249 (1948).

attempt was made to obtain a rotation but the solution was

slighty yellow and no light passed through.

The sirup was dissolved in 5 ml. of methanol and 0.05 ml. of freshly distilled, colorless phenylhydrazine was added. The solution was refluxed for 45 min.

Crystallization was attempted from absolute ethanol but

even after seeding the material resisted crystallization. IV DISCUSSION

A j Isolation of the Polysaccharide Fractions of Roast Coffee

This work followed the procedure established by-

Wolf rora and Plunkett (9) for the Isolation of the polysaccharide fractions of the green coffee bean. It was carried out for comparative purposes In an attempt to determine the changes which take place in the green bean due to roasting. Chart I is a flow sheet for the

fractionation and the results are more fully discussed in

Section IV-B along with the results from the fractionation of the specially extracted spent coffee grounds.

£* Isolation of the Polysaccharide Fractions of Specially

Extracted Spent Coffee Grounds

As with the roast coffee, the procedure established by

Wolfrom and Plunkett (9) was followed. Chart II is a flow sheet for the fractionation. Table 2 is a compilation of the data on the fractionation of green coffee, roast coffee and specially extracted spent coffee grounds. The percentages for green coffee are taken from the Ph. D dissertation of R. A. Plunkett (9). Before the polysaccharides of coffee can be separated, the low molecular weight materials must be removed. Some of these materials are

85 CHART I

Plow Sheet for the Fractionation of the Polysaccharide of Roast Coffee (g./lOO g., dry weight basis)

100.0 g. (dry weight) of Ground Roast Coffee

80/20:: Ethan olA'Ja ter | j, Extract 16.2 g. Residue 83.8 g. 12/1::Benzene/Ethanol

I------— ; Extract l6.0 g Residue 67.8 g

Water Extraction (2 times) r 1 Extract 12.8 g Residue 55*0 g*

0.5,» Ammonium Oxalate Extraction (2 times) I Residue 51.3 g.

Acidified Sodium Chlorite f Extract 8.2 g. Residue 4-3.1 g. (holocellulose)

10,£ KOH Extraction r ~ — I Extract 8.6 g Residue 34-.5 g. CHART II

Plow Sheet for the Fractionation of the Polysaccharides of Specially Extracted Spent Coffee Grounds (g./lOO g., dry weight basis)

100.0 g. (dry weight) of Specially Extracted Spent Coffee Grounds

Absolute Ethanol i — ~ ...... ; Extract 17.9 g* Residue 82.1 g.

2/1::Benzene/Ethanol f—: \ Extract 16.2 g. Residue 65.9 g.

tfater Extraction (2 times) I------Extract 10.7 £• Residue 55 *2 5*

0.5/j Ammonium Oxalate Extraction (2 times)

Extract* 3*3 I — g* Residue 51*9 g*

Acidified Sodium Chlorite

Extract 11.4 g, Residue ^0.5 g. (holocellulose)

LO/S KOH Extraction

Extract 8.1 g. Residue 32.^ g. TABLE 2

A Compilation of the Data on the Fractionation of Green Coffee, Roast Coffee, and Specially Extracted Spent Coffee Grounds (dry weight basis)

Specially Extracted Fraction Extracted a by Green Coffee Roast Coffee Spent Coffee Grounds /» ,*

Ethanol/Water::80/20 15.9 16.2 17.9

Benzene/Ethanol::2/1 17.4 16.0 16.2

rfater 15.7 12.8 10.7

Ammonium Oxalate 3.0 3.7 3.3

Acidified Sodium Chlorite 6.3 8.2 11.4

10,S KOH 4.9 8.6 8.1

Alkali-insoluble Holocellulose 36.8 34.5 32.4 (final residue)

Total 100.0 100.0 100.0

a Extractions were made successively (from top to bottom).

b Adjusted from a 97.6^ actual summation for comparative purposes. 89 easily oxidized and interfere with the holocellulose preparation. Although a clean-out separation of one type of material from another is not accomplished, each of the fractionation procedures has a definite purpose, the total effect of which is to remove the low molecular weight materials.

Referring to Table 2, the percentage of materials soluble in 80/20::ethanol/water increases slightly for the roast coffee and still more for the specially extracted spent coffee grounds. The soluble extracts of the roast coffee and the specially extracted spent coffee grounds contain decomposition products, possibly from the elevated roasting temperature. These decomposed materials together with the removal of part of the coffee by commercial extraction to leave a more concentrated material, would account for the 2% increase in the alcohol-soluble portion of the specially extracted spent coffee grounds.

The benzene/ethanol extraction removes the lipids from the plant material, Table 2 shows a small decrease in the fat content of the roast coffee and the speolally extracted spent coffee grounds when compared with the green coffee.

Much of this decrease is probably due to decomposition during the roasting process. 90

After removing the lipids a water extraction at room temperature is possible. Table 2 shows a decrease of water-soluble material from the green coffee to the roast coffee, and a still further decrease in the specially extracted spent coffee grounds. Some of the water-soluble part of the green coffee undoubtedly underwent a change during the roasting process with some portions possibly volatilizing. It is to be noted that there is as much as

10.7? of the specially extracted spent coffee grounds soluble in water at room temperature. However, these fractionations are consecutive extractions; that is, the residue remaining from a prior extraction is used for the following extraction. The water extractions are performed on the material after the lipids have been removed by the

2/1::benzene/ethanol azeotrope. These water-soluble materials are probably bound into the fats so that the industrial extraction does not remove them. After the removal of the lipids, these compounds are free to dissolve in water at room temperature, resulting in the rather large percentage of water-soluble materials.

Dilute ammonium oxalate solution removes the water- insoluble pectin portion of plants. There is little variation in the amount of apparent^pectin in the green 91 coffee, the roast coffee, and the specially extracted spent coffee grounds. This extract probably contained trace amounts of pectlc substances grossly contaminated with other polysaccharide materials.

The strong oxidizing ability of chlorine dioxide removes the lignin and leaves the carbohydrate material behind. The apparent lignin content (acidified sodium chlorite extract) rises from the green coffee, to the roast coffee, to the specially extracted spent coffee grounds. This is probably due to decomposition of the carbohydrate material during roasting and extracting, thus making it soluble in the acidified sodium chlorite solution.

The (10,& potassium hydroxide)-soluble portion constitutes the "hemicellulose,, of the coffee. The increase in the hemlcellulose portion of the roast coffee and the specially extracted spent coffee grounds is probably due to a change in the holocellulose portion due to the roasting process.

Table 2 shows a decrease in the (lO# potassium hydroxide)-soluble holocellulose in roast coffee and specially extracted spent coffee grounds when compared to green coffee. This indicates water-solubilization of the polysaccharide material by the roasting process. It is 92

well established that pyrolysis of starches results in

water-solubilization with the final production of 1,6-

anhydro-D-glucopyranose (levoglucosan). Hudson and

co-workers (55) report that the mannan of tagua palm

(55) A. S. Knauf, R. >1, Hann, and C. 3. Hudson, J.

Am. Chem. Soc., 6^,, 1447 (1941).

seeds undergoes pyrolysis to form the water-soluble 1,6

-anhydro-j3-D-mannopyranose. It is probable that the

holocellulose of green coffee undergoes some pyrolysis, with

probable shifting (56) of linkages, during roasting,

(56) A. Thompson and M. L. Wolfrom, J. Am. Chem. Soc.,

££, 6618 (1958).

resulting in water-soluble materials. The increase in the

acidified sodium chlorite and potassium hydroxide

extracts for roast coffee and specially extracted spent

coffee grounds, as compared to green coffee, probably

arises from this decrease in the alkali-insoluble

holocellulose fraction. 93

Hydrolysis of the (IQ# Potassium Hydroxide)-.Ins.o.lub_lo

Holooellulose of Green Coffee

Schulze (3) In 1892 attempted to hydrolyze the

carbohydrate component of green coffee with 75% sulfuric acid but a small residue of undlssolved material always

remained, Wolfrom and Plunkett (9) also showed that there was some portion of the (10% potassium hydroxide)-insoluble holocellulose which was difficult to hydrolyze.

Upon the isolation of polysaccharide material from a plant source, that material should be qualitatively tested for the presence of nitrogen, phosphorus and sulfur. The

(10# potassium hydroxide)-insoluble holocellulose was tested for these elements by the usual basic tests and no evidence for nitrogen, phosphorus or sulfur was obtained,

JL. Hydrolysis with 72# Sulfuric Acid

Preliminary experiments indicated that allowing the material to stand in 72# sulfuric acid at room temperature until it had dissolved resulted in considerable degradation.

However, when the mixture was kept at lce-bath temperature the material did not completely dissolve and when the mixture was diluted to 3# sulfuric acid, considerable white precipitate came out of solution, Indicating that the starting material had not hydrolyzed, Although most of the coagulated precipitate dissolved when the 3# sulfuric

acid was refluxed, after the acid was neutralized with

Duolite A-*f ion exchange resin and the resulting neutral

solution concentrated to a sirup, the addition of

absolute methanol caused a white precipitate to form. This

precipitate was considered to be unhydrolyzed or partially

hydrolyzed (10# potassium hylroxide)-insoluble holocellulose.

It was therefore decided that 72# sulfuric acid was not a suitable hydrolyzing medium for a quantitative estimate of

the sugars in the (10# potassium hydroxide)-insoluble holocellulose of green coffee.

2l x HYdrclYais of. Cellulose with Anhydrous Sulfuric Acid

Since the (10# potassium hydroxide)-insoluble holocellulose of green coffee apparently did not completely hydrolyze in 72# sulfuric acid, it was decided to attempt the hydrolysis using anhydrous sulfuric acid. Anhydrous sulfuric acid is known to be a powerful solvent for organic compounds (57*58,59) and was applied to polysaccharides by

(57) A. Hantzsch, Z. physlk. Chem., 257 (1908).

(58) L. P. Hammett, "Physical Organic Chemistry,"

McGraw-Hill Book Co., New York, N. Y., 19^0, pp. ^5-^9,

53-56, 277-283. 95

(59) M. S. Newman, J. Am. Chem. Soc., 6^, 2431 (1941).

Wolfrom and Montgomery (60). The technique Intended was to

(60) M. L. Wolfrom and R. Montgomery, J. Am. Chem.

Soc., 22, 2859 (1950).

dissolve the (10^ potassium hydroxide)-Insoluble

holocellulose In anhydrous sulfuric acid and then slowly

add water to bring about the hydrolysis. Since this method

constituted a new procedure for hydrolyzing polysaccharides,

It was decided to experiment on cellulose to see if the method was feasible.

The cellulose was therefore dissolved in the anhydrous

sulfuric acid and water was slowly added with strong stirring. The resulting 3% sulfuric acid solution was refluxed and the hydrolysis followed by testing aliquots of the solution with Somogyl (40) reagent. The hydrolysis curve (Pig. 2) shows a drop in reducing value after 15 hr. of refluxlng. This could be due to two factors:

(a) Reversion; however, this is unlikely because the sugar concentration was only 0*Z# and Thompson, Wolfrom and Quinn (61) showed that for^glucose concentrations below 96

<6l) A. Thompson, M. L. Wolfrom ana E. J. Qjuinn,

J. Am. Chem. Soc., 2 h 3003 (1953)*

O'b# there was little or no reversion.

(b) Degradation. This was the more likely because with an acid concentration of 3/£ sulfuric acid the glucose could be degraded under prolonged refluxing; the products would be 5-(hydroxymethyl)-2-furaldehyde and finally levullnic and formic acid.

After hydrolysis was complete, the acid solution was neutralized with barium hydroxide. Some difficulty was encountered In removing the resulting precipitate of barium sulfate. Centrifuging was found to be the best way to remove the heavy precipitate. The resulting decantate was kept slightly acid to methyl red during concentration because Monler-W111lams (39) reported that the concentrate will become basic. After concentration to £&. 500 ml, the solution was neutralized to litmus with barium hydroxide and the resulting filtrate was conoentrated to a sirup which was refluxed with absolute methanol. The methanol dissolved the sirup but precipitated the inorganic salts.

These methanolio extractions were repeated until the sirup 97

completely dissolve! at room temperature with no

Inorganic salts left. In this way the barium sulfate was removed from the sirup.

The yield of 90.3# glucose compared favorably with

the 90.6,2 value reported by Monler-Williams (39)* Thus the anhydrous sulfuric acid appeared to be of comparable hydrolyzing ability to 722 sulfuric acid with the added «r advantage that it was a very pox^erful dissolving medium which would dissolve compounds not completely soluble in

72,2 sulfuric acid. h Hydrolysis. _of_ _(1Q# Potassium Hydroxide)-Insoluble

Holocellulose of Green Coffee with Anhydrous Sulfuric Acid

The (10,2 potassium hydroxide)-insoluble holocellulose of green coffee dissolved in the anhydrous sulfuric acid at -2° and did not precipitate upon dilution as it did when treated with 72# sulfuric acid (see Section IV-C-1). The hydrolysis reaction was refluxed and processed as described for the hydrolysis of cellulose. No carbohydrate material precipitated when the sirup was treated with absolute methanol as had occurred when it was hydrolyzed with 72# sulfuric acid. This was considered an indication that hydrolysis was complete and the total reoovery of 87.1# yield on quantitative assay of the individual sugars 98 appeared favorable because of the difficulty of hydrolyzing the material and the concomitant sugar destruction, established definitely with the pentose content. There would also be some loss due to adsorption on the heavy precipitate of barium sulfate which is formed by neutralizing the sulfuric acid,

Ha Derivatives of the Constituent Sugars of the (10%

Potassium Hydroxide)-Insoluble Holocellulose of Green Coffe_e

Paper chromatograms (see Fig. 1) of the hydrolyzate of

{10& potassium hydroxide)-insoluble holocellulose of green coffee indicated the presence of glucose, mannose, galactose and arabinose. Wolfrom and Plunkett (9) isolated crystalline derivatives of D-glucose and D-mannose and this left galactose and arabinose to be isolated. Galactose was first isolated as galactaric (muoic) acid but this being a meso compound did not show whether the galactose was D or

L. Therefore the galactose was separated on paper chromatograms and the resulting sirup acetylated, yielding crystalline penta-Q.-acetyl-rj3 -D-galactopyranose*

In order to isolate a crystalline derivative of arabinose advantage was taken of the preferential hydrolysis of arabinose with 1.5^ sulfuric acid. From the sirup of arabinose the crystalline derivative tetra-£.-acetyl-L- 99

arabinose diethyl dlthloacetal was isolated by the method

of Karabinos and Wolfrom (**6).

_ _auan.tltat.lve Assay of _the Constituent Sugars oXjthe

L10J6 Potassium Hydroxide)-Insoluble Holocellulose ofGreen

Coffee

The currently prefered method for quantitatively

determing the amount of each sugar in a solution of a

mixture of sugars is to aid a measured amount of the

solution to the center portion of a paper chromatogram,

develop the chromatogram, and then locate the zones by means of side indicator strips which are removed and

sprayed with an indicator. The zones corresponding to

the movement of the sugars are then cut out and the sugars

eluted from the paper with water. Some type of colorimetric or titration method is then used to determine the amount of

sugar in the eluent. This type of determination, using the method of Dubois, Gilles, Hamilton, Rebers, and Smith

(62), was used in this laboratory with little success.

(62) M. Dubois, K. A. Gilles, J. K* Hamilton, R. A.

Rebers, and Fred Smith, Anal. Chem., 2^, 350 (1956).

The elution technique has two major drawbacks. First 100

Is the location of the zones which are to be cut from the

paper. Although Indicator strips are cut from each side,

very often the solvent flows at a different rate in other

portions of the paper resulting in an arc or zig-zag zone

for the sugar, and it cannot be known whether all the

zone has been cut out. Separation of the sugars must be

good or the zones may overlap very slightly, causing discrepancies in the results. The second drawback is the elution of the sugars from the paper. Only seldom is 100#

recovery of the sugars realized. These two weaknesses in

the quantitative analysis of a mixture of sugars are eliminated by the use of direct photometry, as was used in

the quantitative determination of D-glucose, D-galactose,

D-mannose and L-arabinose in the (10# potassium hydroxide)-

insoluble holocellulose of green coffee. The method closely follows that developed by McFarren, Brand and

Rutkowskl (63) and was used also by Durso and Paulson (64)*

(63) E. F. McFarren, Kathleen Brand, and H. R.

Rutkowskl, Anal, Chem., 2£, 1146 (1951).

(64) D. F. Durso and J. C. Paulson, Anal. Chem.,

XL* 919 (1958). 101

The results of the quantitative determinations in percentage of the theoretical hydrolysis yield were as follows: D-glucose, 17.8a; D-matmose, 48.5a; D-gslactose,

14.8,5; L-arabinose, 2.85; total yield 83.91. This sum of the separate sugar percentages compares favorably with the

85.45 yield of a total solids analysis of the same solution.

Because the L-arablnose is easily cleaved by acid

(see Section III-D-3) it v;ould probably be removed at the beginning of the hydrolysis and therefore v:ould be subjected to a considerably more rigorous treatment than the other sugars and as a result would likely undergo appreciable degradation. Therefore this sugar was quantitatively determined in a separate sample by refluxing the (10,5 potassium hydroxide)-Insoluble holocellulose with 1.51 sulfuric acid for 20 min. The amount of arabinose in the resulting sirup as determined by direct photometry was 6.01. This increase in the L-arabinose yield through mild acid hydrolysis does not indicate that L-arablnose is any more acid-sensitive than the hexoses present but the degradation resulted from the fact that it was subjected to the acid treatment for a longer time as has been described above. The ease of cleavage of this sugar by 102 acid allowed it to be estimated and its degree of degradation (6.0-2.8 * 3,2) in the stronger acid could be determined. Such degradations probably accounts for

the lack of theoretical yield of total sugars on acid hydrolysis of the polysaccharides and justifies our corrections of these yields.

The results of the quantitative determinations in percentage of the theoretical yield, using 6,0/$ for

L-arabinose, were as follows: D-glucose, 17.8a; D-galactose,

14.8,$; D-mannose, 48.5$; L-arablnose, 6.0$. Total recovery,

87.1/$ of the theoretical. Correcting these values to 100.0,® resulted in: D-glucose, 20.4/$; D-galactose, 17.0,$; D- mannose, 55.7,$; L-arabinose, 6.9$. Considering the quanitity of arabinose as unity, this resulted in a molar ratio of:

L-arabinose, 1.0; D-glucose, 2.2; D-galactose, 1.8; and

D-mannose, 6.2. i The value of 6.0,$ L-arabinose obtained by direct photometry compares favorably with the direct pentose value of 6.7$ obtained by VJolfrom and Plunkett (9) and that reported by Schulze (4), This shows that all the pentose is L-arabinose and that all is easily acid-hydrolyzable.

The use of 6,7/$ for arabinose makes no significant change in the ratios of the sugars reported above. 103

The value of 46.3;o D-mannose obtained by precipitation

as Its phenylhydrazone Is slightly below the value of 48.5,£

obtained by direct photometry of paper chromatograms. This

is probably due to the Incomplete hydrolysis with72 the,i

sulfuric acid utilized in obtaining the former. The close

agreement obtained for the L-arabinose and D-mannose values

by direct methods and by paper chromatographic methods

substantiates the use of direct photometry of paper

chromatograms as a procedure for quantitative sugar mixtures

and supports the values obtained for the sugars in the (10,«

potassium hydroxide)-Insoluble holocellulose of green

c o ffe e ,

F. Separating Homogeneous Polysaccharides from (10 S

Potassium Hydroxide)-Insoluble Holocellulose of Green Coffee

1. Aoetylatlon w ith Trifluoroacetic Anhydride

The acetylation reaction with dry starting material was inconsistent, and although Wolfrom and Plunkett were

able to perform a homogeneous acetylation, the reaction was d ifficu lt to repeat. It is known that cellulose swells when treated with alkali (65) and that the surface also

(6 5 ) W. D. Nicoll, W. L. Cox and R. F. Conaway in

"Cellulose and Cellulose Derivatives," Vol. V, 2nd Edition, Part II, Edited by E. Ott, FI, M. Spurlin and Kildred W.

Grafflin, Interscience Publishers, Inc,, New York, N. Y.,

195^i P. 837.

tends to collapse when it is dried. Since the step

immediately preceding the acetylation was a 10# potassium hydroxide treatment, this was used as a means of

"activating" the surface of the (10/2 potassium hyiroxide)-

insoluble holocellulose. The method used was to never allow the material to become dry but to keep it under chloroform. When this "activated" material was treated with a large excess of anhydrous acetic acid and

trifluoroacetlc anhydride, it slowly reacted at room

temperature and chloroform-insoluble acetate was prepared.

The acetylation reaction must be started at Ice-bath temperature because, when glacial acetic acid and trifluoroacetic anhydride are mixed, heat is evolved, and this brings about an apparent degradation of the starting material and no chloroform-insoluble acetate is obtained.

The theoretical acetyl value of a fully acetylated hexose polymer is **5 .**■#* Although an attempt was made to fully acetylate both the chloroform-insoluble acetate

(acetyl value 38.9#) smi the chloroform-soluble acetate 105

(acetyl value 2 9 .8/0 , these fractions could not be further acetylated.

Both the chloroform-Insoluble an! chloroform-soluble acetates upon de-acetylation and hydrolysis showed the presence of glucose, galactose, mannose, and arabinose.

Although it had been anticipated that the acetylation would result in a fractionation of the various polymers in the (10# potassium hydroxide)-insoluble holocellulose, it appeared that if there was some fractionation it was no means complete because the fractions each contained the same sugar residues as had the starting material.

Therefore, due to the difficulty and inconsistency of the acetylation and the resulting incomplete fractionation, the acetylation reaction was abandoned as a method of fractionating the (10# potassium hydroxide)-insoluble holocellulose into homogeneous polysaccharides.

2. Extraction w ith 18# Sodium Hydroxide

It was observed by Hamilton, Kiroher and Thompson (17) that mannan-rich polysaccharides are more soluble in sodium hydroxide than in potassium hydroxide. Since the (10# potassium hydroxide)-insoluble holocellulose of green coffee contains 58# mannose it was considered probable that a fractionation of this material could be realized by 106

extraction with sodium hylroxide. It was decided to use

18# sodium hydroxide because Dutton and Hunt (6 6 ) used

(6 6 ) G. G. S. Dutton and K. Hunt, J. Am. Chem. Soc., M i 5697 (1958).

this concentration In extracting (10.2 potassium hylroxlde)-

insoluble holocellulose of Sitka Spruce, with good results.

Chart III is a flow sheet for the extraction -with 18,2

sodium hydroxide and the subsequent Isolation of the

various fractions. Nitrogen was continuously bubbled

through the extraction mixture to prevent oxidation and

alkaline degradation of the polysaccharides. The total

recovery of 9 9 *7# of the starting material indicates that

all of the (18# sodium hydroxide)-soluble material was

accounted for.

%x Determining an Hydrolysis Procedure for Praotlon-A

Although the (10# potassium hydroxide)-insoluble holocellulose of green coffee is very difficult to hydrolyze and was hydrolyzed finally with anhydrous sulfurio acid, this process is quite harsh and results in some degradation. Therefore, an effort was made to find a mild method of hydrolysis for Fraction-A. Dilute sulfuric acid 107

III Plow Sheet for the Extraction of (10$ Potassium Hydroxide)-Insoluble Holocellulose of Green Coffee with 18$ Sodium Hydroxide

68*5 g» (10$ KOH Insoluble)-holocellulose of green coffee

i NaOH, 8 hr,, 2$ H2 bubbled through r j, Residue Filtrate

Lyophillzed ph 5 5^,1 g. Precipitate 79.0$ of the starting material Centrifuged

” ^ Residue Decantate Concentrated to 2 liters Dialyzed Dialyzed Precipitate Precipitate Centrifuged Centrifuged r 1 \ Residue Decantate Residue Decantate jpyophllized Concentrated Lyophillzed Praction-B, 0,2 g, Hade up to 70$ IStOH 0,3$ of the starting material Fraction-A, 8,8 g. 1.4$ of the 18$ NaOH extract Preclp tate 12,8$ of the Centrifuged

starting material j------T 6l,l$ of the 18$ NaOH extract Residue Decantate Lyophillzed Concentrated ’ t Dialyzed Praction-C, 2,2 g, Lyophillzed 3.2$ of the starting material 15.3$ of the 18$ NaOH extract Fraction-D, 3.0 g. 4,4$ of the starting material 20,8$ of the 18$ NaOH extract 108

(3^) did not completely dissolve the material even after

16 hr. of refluxing and did not seem too acceptable.

Concentrated hydrochloric acid did not dissolve Praction-A

at room temperature, but when It was diluted to 5% and

refluxed the solution became homogeneous. This appeared

a possibility except that the removal of hydrochloric

acid involves several steps and a more convenient method

was found. Formic acid dissolves Praction-A within 2 hr.

at room temperature and since other workers (49) use it

with good results It was decided to attempt the

hydrolysis of Praction-A with formic acid.

Hzdrclysls of Fraction-A with Formic Acid

Fractlon-A dissolves In formic acid at room temperature

to give a rotation of£\]^2-22° which indicates that the majority of the glycosidlc linkages are ^-D (<<-L). After heating on a steam bath for 3 hr. the rotation remained

7 22 a constant at Hv +16 Indicating the ease of hydrolysis of

Practlon-A in formic acid. Formate esters formed on the hydroxyl groups during the hydrolysis with formic acid * but these were readily removed by 2,$$ sulfuric acid.

The resulting hydrolysis sirup contained mannose, galactose and a very slow moving spot which was considered to be an incompletely hydrolyzed oligosacoharide. However, 109 the spot was very weak and it was not considered a significant detriment to the formic acid method of hydrolysis. A quantitative analysis of the sirup by the densitometry method showed 9^$ mannose and 2$ galactose, indicating that Fractlon-A is a mannan.

o. Purification of Fraotlon-A by Re-preclPltatlon from Sodium Hydroxide

Although Fraction-A appeared to be quite pure in that there was very little galactose in it, an attempt was made to remove this galactose by re-precipitating the polymer.

The fact that the material did not completely dissolve in

18$ sodium hydroxide was not unexpected because the surface properties of polysaccharides often change when they are dried, making them less soluble. However, there was very little of the substance which did not dissolve and this was probably impure material and was better removed.

The fact that only 70$ of the starting material was recovered on re-precipitation was surprising because it was thought that Fraotion-A had been obtained in quite a pure state. Although some of the 30$ loss was due to the portion of Fraction-A which did not dissolve, there must have been considerable which did not re-precipitate. 1X0

Treatment of a polysaccharide with 18^ sodium hydroxide is a somewhat harsh process and perhaps some alkaline degradation occurred resulting In smaller molecular weight polysaccharides which did not readily precipitate.

Hydrolysis of the re-preclpitated Fractlon-A with formic acid resulted in an hydrolysis curve within experimental error of that of Fractlon-A {Fig. 8 ) which was considered an indication that the re-preclpltated material was the same polysaccharide as the original.

A quantitative analysis of the resulting sirup showed galactose and hence none of the galactose was removed by re-precipitation. Although the galactose was not reduced it is recommended that Fraction-A be re-precipitated from lB,i sodium hydroxide before reactions are performed on it because the appearance and surface properties of the re-precipitated material are better th^n the original

Fraction-A.

d. Purification of Fraction-A by Complexlng with

Fehllng Solution

The precipitation of cis-glycol-oontalning polysaccharides from a basio solution by complexing them with cupric ion is a well known method of purification.

The copper complex resulting from treatment of an (18^ I l l

sodium hydroxide)-solution of Fraction-A was decomposed with 2 ^ hydrochloric acid. Fraction-A is not water-

soluble and, therefore, it ttas surprising that 7*Jy» of the material recovered from the complex dissolved in the acid

solution (pH 2-3) and had to be precipitated with acetone.

This was interpreted as a polysaccharide degration into

smaller molecular weight components either by the

redissolving in sodium hydroxide, the complexing with cuprlc ion, or by the acid decomposition of the complex.

It was possibly a result of all three.

Hydrolysis curves in formic acid of both Fehllng

Complex I and II were within experimental error of that of Fraction-A (Fig. 8 ) which was considered an indication

4 that the complexes were the same polysaccharide as the original. Quantitative analysis of the constituent sugars showed 2-3;$ galactose which was the same amount as in the starting material and therefore complexing Fraction-A with Fehllng solution did not result in a fractionation,

e. lBO.lat.iQn.-Qf D-Mannose Phenylhvdrazone from

Fractlon-A

The isolation of D-mannose phenylhydrazone from the hydrolysis sirup of Fraction-A, considered with the 22 rotation of -22° of the original polymer in formic 112 acid, shows that Praction-A is a mannan composed mainly of 0 -D-mannose residues. The95,* yield of D-mannose by

the phenylhydrazone method compares favorably w ith the

yield by the densitometry procedure and substantiates the fact that Praction-A is a mannan.

f. Hydrolysis of Practlon-3 with Formic Acid

Fraction-B was hydrolyzed with formic acid and the hydrolyzate processed as described in Section III-F-2-b.

This fraction was still composed of 69/» mannose but contained more galactose (l6,a)» and some arabinose appeared (8.9/£). The arabinose and galactose were undoubtedly contaminants from other polysaccharides.

g. Hydrolysis of Practlon-C with Formic Acid

Although Fraction-C dissolves readily in formic acid and the hydrolysis curve (Fig. 9) reaches a steady maximum the quantitative yield (galactose, 6 3 .5,*; arabinose, 19.0«) is not high. The hydrolysis consisted of heating with formic acid, removing the formic acid under reduced pressure, refluxing the resulting sirup with 2 .5 & sulfuric a d d and neutralizing the sulfuric acid with barium hydroxide. The resulting precipitate of barium sulfate was filtered and the filtrate concentrated to a sirup.

This sirup was then refluxed with absolute methanol to 113 precipitate formed which, did not appear to be completely composed, of inorganic material. A small amount of It was taken on a spatula and held in a flame and some charring resulted although the inorganic material remained. This could be taken as an indication that the formic acid did not completely hydrolyze the polymer and this resulted in the lower yields. Although formic acid was used with good success on Practions-A and -B these are largely mannans while Praction-C contains only a trace of mannose units and is composed of galactose and arabinose. Therefore it is suggested that in the future one try hydrochloric acid for the hydrolysisof Practlon-C,

h. Hydrolysis of Fractlon-D with Formic Acid

As with Praction-G thequantitative results (galactose,

£>5$ I arabinose, 21^) of the hydrolysis are low. The same reasons and suggestions are offered as for Practlon-C. It appears that ?ractions-C and D are very similar. This was not unexpected because Fraction-C was isolated by precipitation vjith ethanol (see Section III-F-2) and all of the precipitate did not settle in the centrifuge but remained as an almost colloidal suspension. It was this milky suspension formed by ethanol addition which was dialyzed and then lyophillzed to yield Fraction-D. 11**

Therefore Fraction-D may be the same polymer as Fraction-C but did not coagulate enough In the 70% ethanol to be removed by centrifuge.

1 * f o w a l 9 ;scy;ssj.ori erta^n^g to, the £rac,UQ.mUS>n of the (IQi Potassium Hydroxide)-Insoluble Holocellulose of age_e.n_C_QXfee_b^__^tm.c_tlo.n .with 1.8^ Sodium Hydroxide Solution

The 3’oove results show that there Is a fraction (2 1 .0^) of the (10,t potassium hydroxide)-Insoluble holocellulose of green coffee which Is soluble in 18;* sodium hydroxide.

This solium hydroxide soluble material can then be readily separated into four fractions of which Fractlon-A is considerably the largest (6l.l^). Fraction-A is composed of D-mannose units and 2,* galactose units. This galactose could not be removed either by re-precipitation from 18,« sodium hydroxide or by complexing with Fehling solution. The polysaccharide is, therefore, a mannan containing a small amount of galactose, either as an impurity or as chemically linked in the polymer.

The latter possibility Is probable following the recent results in enzyme studies in which it has been shown that enzymes can convert sugars from one type of polysaccharide into another but will often not complete the conversion and leave some of the original monomer 115

moiety in the new polysaccharide. An example of this Is

the conversion of sucrose Into dextrans by various species

of Leuconostoc (6?, 68 ). Some of these dextrans appear

(6 7 ) E. T. Hehre, Proc. Soc. Exptl. Biol, 'led,,

18 (19*0).

(6 8 ) W. W, Carlson, C. L. Hosano and V. Whiteside

Carlson, J. Eacteriol., 65., 136 (1953).

to have D-fructose end groups which are introduced into

the molecule by sucrose acting as an acceptor to

initiate dextran synthesis. The fructans of grasses

likewise always contain some D-glucose,

There is a graded fractionation of the (18,$ sodium hydroxide)-soluble extract from Fraction-A through

Fractions -B, C and D, Fraction-A is a mannan whereas

Fractions-C and D contain.only a trace of mannose units and Fraction-E Is intermediate between the two. Fraction-A contains no arebinose and only 2,3 galactose. The ara'cinose and galactose increase through Fraction-3 until

Fractlons-C and D are composed wholly of the two. This separation reflects the solubility of the different types of polymers in water. The mannan (Fraction-A) is 116 insoluble In water, Praction-E Is precipitated by dialysis resulting in the removal of inorganic salts, and Practions-C and D are soluble in water and must be precipitated by ethanol.

It is not known whether Practions-C and D are arabogalactans in which the arabinose and galactose units are chemically linked or whether they are a mixture of an araban and a galactan. An effort might be made to separate the material into an araban and a galactan. This might be accomplished in two ways.

a. Graded ethanol precipitation. Arabans are very soluble in water and perhaps this might be used to fractionally precipitate the galactan with ethanolleaving the araban behind. To do this one would dissolve Fraction-

C in water and make the solution up to 50% ethanol and recover the resulting precipitate if one formed. Then the decantate from the precipitate could have the ethanol content increased to 60,2 and so forth. In this way the precipitates which form at the lower concentrations of alcohol should be rich in galactose.

b. Extraction with acetone, Hamilton and Partlow (69)

(6 9 ) J. K. Hamilton and E. V. Partlow, J. Am. 117

Chem. Soc., 8Q.* ^ 8 0 (1958).

have made a rather surprising report that arabans can be

extracted with acetone. They extracted red cedar shavings

with acetone and obtained an arabinose rich polysaccharide.

Then they extracted the acetone insoluble residue with

water and obtained a galactose rich polysaccharide. This

would seem directly applicable to separating Fractions-

C and D into an araban and galactan if Indeed they exist

as such. A review of arabogalactans and galactans has

been compiled by Aspinall (1*0.

The residue from the 18,2 sodium hydroxide extraction

has not been studied. It is evident that the difficultly

hydrolyzable portion remained in this residue because

Praction-A,B,C and D appear readily hydrolyzable.

Although the (10,5 potassium hydroxide)-Insoluble

holocellulose of green coffee contains 20% D-glucose# no

glucose residues appear In the material soluble In 18,5

sodium hydroxide. Whether this D-glucose is in the form

of true cellulose is not known at present.

The amounts of each sugar present in the residue may

be calculated by difference. The results are as follows:

D-mannose, D-galactose, 13.7,2; D-glucose, 25,7/2; 118

L-arabinose, Therefore, it is quite eviient that

the fractionation was by no means complete in any of the monomer sugars present. How this residue can be further

fractionated is not known at present. Hamilton (70)

(70) J. K. Hamilton and G. H. Q,uimby, Tappl, 4Q.,

781 (1957).

gives a method for exhaustive extraction with sodium hydroxide. Fractionation might also be attempted by acetylation once more now that part of the material has been removed. It is quite clear, however, that considerable work must be done before the complex carbohydrate material, the (10,* potassium hydroxide)-insoluble holocellulose of green coffee, is understood.

— determining the Structure of F_raotlon-A

The classical method of methylation vras the first step used in determining the structure of the mannan

(Fraction-A). Since Fraction-A is soluble in 18,£ sodium hydroxide, the Haworth (71) method of methylation was used

(71) W. N. Haworth, J. Chem. Soc., 107. 8 (1915) 119

firs t by adding dimethyl sulfate dropwise to the alkaline

solution. The method was repeated once more and the

resulting methylated polymer was then soluble in dry

tetrahydrofuran. The one-step methylation of Falconer

and Adams (50) was then used to complete the m ethylation.

The mannan appeared to methylate very readily under these

conditions to an essentially theoretical methoxyl content;

,calcd. ^5.6, found. ^5*5*

The methylated polymer was readily hydrolyzed with

formic acid and the paper chromatogram of the hydrolyzate

sirup showed a great predominance of one spot having an

Eg value very close to that of 2f3f6-tri-£-methyl-D-

mannopyranose. Therefore the mannan appears to be a

straight chain polymer. It had been considered that this

mannan was very similar to the mannans from ivory nut but

methylation studies of the mannans from ivory nut show a

small amount of branching (3^) and no such evidence of

branching was found for the coffee mannan.

The Eg values of the trace amounts of the other four

spots resulting from the methylation studies (see Table 1)

Indicate: 2,3»i*'*6-tetra-2.-methyl-D-mannopyranose which

would result from a non-reducing end group; 2f3»**»6-

tetra-^-raethylgalactopyranose also resulting from a 120 non-reducing end group and accounting for the small amount of galactose in the mannan; and a di-£,- methyl sugar which could result from a very small amount of branching or from the cleavage of some of the methyl ethers during hydrolysis* However, these four spots are very, very fa in t and indeed the chromatogram has to be almost overloaded with respect to 2, 3,6-trl-£.-m ethyl-D- mannopyranose in order to detect them at a ll.

Although the paper chromatogram indicated the presence of a predominant amount of 2 ,3,6-tri-Qrmethyl-D- mannopyranose a crystalline derivative of the compound could not be prepared even when seeds were available; two derivatives were tried. Although Hamilton and Partlow

(69) indicate that 2 ,3,6-tri-<2,-methyl-IJ-phenyl-D- mannopyrsnosylamire is not a good derivative, other workers

(53,5*0 have used it as a derivative with good success.

The method of Hudson and Isbell (52) for the oxidation of the 2,3,6-tri-£,«*me thyl-D-mannopyranose to the acid proved to have 9 step which brought about difficulty.

Barium benzoate is added as a buffer to take up the hydrobromic acid which is formed, although most of the resulting benzoic acid precipitates and is filtered, the 121 final amounts have to be removed by chloroform extraction. With unsubstitued sugars this is satisfactory because they are not chloroform-soluble; however, with methylated sugars they are somewhat soluble in chloroform.

Therefore it was difficult to obtain a clear separation of the benzoic acid and the tr1-0-methyl sugar acid.

Therefore it is recommended that in future the methylated sugars be oxidized with bromine water only, as is done by other workers (72,55,56). Hudson and Isbell (5^) developed the barium benzoate method to speed up the oxidation and increase the yields. However, bromine oxidation should be much cleaner.

Why the phenylhydrazlde derivative did not crystallize, even when nucleated, is not known. Although the spot corresponding to 2,3,6-trl-^-methyl-D-mannopyranose was eluted from paper chromatograms and should be pure, perhaps there was some 2,3,6-tri-^-methyl-D-galactopyranose also present and this might prevent the crystallization of derivatives. Even though the paper chromatograms of the hydrolyzate sirup from the methylated mannan showed ^ust one predominant spot, the problem may be more complex than was first supposed. V SUMMARY

A. Isolation of the Polysaccharides of Roast Coffee

1. Ground roast coffee has been fractionated by successive extraction with 80/20::ethanol/water, 2/1:: benzene/ethanol, water, 0,5% ammonium oxalate, acidified aqueous sodium hypochlorite, and 10,£ potassium hydroxide,

B. Isolation, of the Polysaccharides of Specially .Extracted

Sp.ent gq,flge .grgvmfe

1, Specially extracted spent coffee grounds have been fractionated by successive extraction with 80/20::ethanol/ water, 2/1::benzene/ethanol, water, 0.5$ ammonium oxalate, acidified aqueous sodium hypochlorite, and 10$ potassium hydroxide.

C^_ Characterization of the (10$ Potassium Hydroxide)-

Insoluble Holocellulose of the Green Coffee Bean

1, The (10# potassium hydroxide)-insoluble holocellulose of green coffee was tested for nitrogen, phosphorus and sulfur but no evidence for the presence of any of these elements was obtained,

2, The (10$ potassium hydroxide)-insoluble holocellulose of green ooffee was completely hydrolyzed with anhydrous sulfuric acid,

3, Paper chromatograms of the hydrolyzate run

122 123 simultaneously with known compounds showed the presence

of arablnose, mannose, glucose and galactose,

D-Oalactose was isolated as crystalline

galactarlc (muclc)aoid and as crystalline penta-£-acetyl-£

-D-galaotopyranose,

5. L-Arablnose was isolated as the crystalline

tetra-£-acetyl-L-arabinose diethyl dithioacetal.

6. A quantitative asssyof the constituent sugars

showed the following ratio: L-arablnose, 1.0; D-glucose,

2.2; D-galactose, 1,8; D-raannose, 6.2.

D. Separating Homogeneous Polysaccharides from the (10^

Potassium Hydroxide)-Insoluble Holocellulose of Green Coffee

1. The acetylation reaction with trifluoroacetic

anhydride and anhydrous acetic acid was studied and resulted

in the separation of (10$ potassium hydroxide)-insoluble holocellulose into a chloroform-insoluble acetate and a

chloroform-soluble acetate.

2. The ohloroform-insoluble acetate was de-acetylated and hydrolyzed. Paper chromatograms showed the presence of arablnose, glucose, galactose, and mannose.

3. The ohloroform-soluble acetate was le-aoetylated and hydrolyzed. Paper chromatograms showed the presence of 124 arablnose, glucose, galactose, and mannose.

4. The (10# potassium hydroxide)-insoluble holocellulose was extracted with 18# sodium hydroxide and separated Into an (18# sodium hydroxide)-Insoluble residue (79%) and an

(18# sodium hydroxide)-soluble portion (21#),

5. The (18# sodium hydroxide)-soluble portion was fractionated into Fractions A, B, C, and D.

6. Fraction-A was hydrolyzed with formic acid and paper chromatograms showed the presence of mannose (9b%) and galactose (2#).

7• Fraotion-A was re-precipitated from 18# sodium hydroxide but this did not remove the galactose.

8. Fraction-A was precipitated by complexlng with

Fehling solution but this did not remove the galactose.

9. D-Mannose phenylhydrazone was crystallized from the hydrolysis sirup of Fraction-A and showed that this fraction was composed of 95# mannose residues.

10. Fraotion-B was hydrolyzed with formic acid and paper chromatograms showed the presence of mannose,

(6 9 .1#), galactose (1 6 .3#), and arablnose (8 .9#).

11. Fraction-C was hydrolyzed with formic acid and paper chromatograms showed the presence of galactose

(6 3 .5#)* and arablnose (19.0#); a molar ratio of galactose 125 to arablnose of 3*1*

12, Practlon-D was hydrolyzed with formic acid and

paper chromatograms showed the presence of galactose

(65/S), and arablnose (21$),

B. Determining the Structure of Fraction-A

1, Praction-A was methylated with 18/S sodium

hydroxide and dimethyl sulfate (2 times) and the methylation was completed by dissolving it In tetrahydrofuran and

adding sodium hydroxide and dimethyl sulfate. The methoxyl

content of the final product was 45.4$,

2, Methylated Praction-A was hydrolyzed with formic

acid and paper chromatograms Indicated a great predominance

of a tri-Q,-methyl sugar with an Rg value equal to that of

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14- G. 0. Aspinall, Advances in Carbohydrate Chem., Jit, 0000 (1959). 11 AUTOBIOGRAPHY

I, Murray Lane Laver, was born on a farm near the village of Warkworth, Ontario, Canada, on March 7, 1932.

I received my elementary education in a rural school near my father's farm, and my secondary schooling at the

Warkworth High School, from which I graduated in June,

1951. In September, 1951# I enrolled at the Ontario

Agricultural College, Guelph, Ontario, Canada,from which

I received the Bachelor of Science degree in Agriculture in May, 1955* I enrolled in the Graduate School of The

Ohio State University in June, 1955. I was appointed a

Research Fellow on the Nestle^Company project with The

Ohio State University Research Foundation, project 530, until September 1958. In October, 1958, the project was was changed from the Nestle^Company to Westreco, Inc., '. and I was appointed a Research Fellow on this Ohio State

University Research Foundation project 878 from October

1# 1958, until the present.

132