EFFECTS OF TRANS -HYDROXYL GROUPS IN ALKALINE DEGRADATION OF GLYCOSIDIC BONDS

U.S.D.A., FOREST SERVICE RESEARCH PAPER FPL 188 1972

U.S. Department of Agriculture, Forest Service Forest Products Laboratory, Madison, Wis. ABSTRACT

The influence of ß-dihydroxyl groups in the alkaline hydrolysis of glucosidic bonds is examined. In deriva­ tives of ß-D-glucopyranosides, all hydroxyl groups are trans to each other, and the alkaline elimination of sub­ stituted methyl ethers in positions C-2 and C-4 can be explained, in part, by assistance from neighboring trans a-hydroxyls. The rate of degradation of ethyl 2-2-methyl-ß-D-glucopyranoside is two times slower than the corresponding 2-hydroxyl derivative, whereas the rate for ethyl 2-deoxy-D-glucopyranoside is 8 times slower in 2.5N NaOH at 170° C. EFFECTS OF TRANS -HYDROXYL GROUPS IN ALKALINE DEGRADATION OF GLYCOSIDIC BONDS

By R. M. ROWELL and J. GREEN, Chemist

Forest Products Laboratory 1 Forest Service U.S. Department of Agriculture

INTRODUCTION

Of the chemical pulp produced in the United tion has been directed to the effects of reactive States, 80 percent is produced by an alkaline hydroxyl groups adjacent to the glucosidic link- ; thus, determining the reactions ages. These ß-dihydroxyl groups contribute taking place when is subjected to aqueous significantly in degrading cellulose under alkaline alkali has been of great importance. Much of the conditions. work has dealt with the degradation caused by the It has long been recognized that linked hemi- endwise depolymerization (peeling) reaction that have much greater stability to dilute 2 gives rise to isosaccharinic (4, 13, 15, 22) . alkali than any of the polysaccharides (1, 21). Not only has the mechanism of this reaction been This results from the inability of the hydroxyl studied, but also a mechanism to modify the re- group on carbon atom 2 -hydroxyl) to form a ducing D-glucose end unit to stop or at least to carbonyl group necessary for the degradation to slow down this endwise degradation (16, 19). proceed at room temperature to saccharinic Of equal interest has been the study of the de- (22, p. 298). gradation of cellulose caused by the splitting of Several workers have studied the mechanism internal glycosidic linkages. Cleavage of these of alkaline glycosidic cleavage (2, 3, 6, 7, 11, 12, and bonds between internal D-glucose units gives rise 14), and the most widely accepted mechanism is to new end units that then undergo the endwise shown in figure 1. In the alkaline solution, the depolymerization reaction. Increases in cellulose hydroxyl oxygens will be ionized. As shown, the yield during alkaline pulping ultimately depend on C-2 oxygen anion is trans to the $-glycosidic retention of glycosidic bonds, linkage, and can easily displace the C-1 oxygen In the research on the alkaline degradation of bond to form an epoxide B between carbons 1 and cellulose, many environmental factors have been 2. This epoxide is then hydrolyzed to a glucosyl considered. The variables of temperature, type of anion, which degrades further. alkali, alkali concentration, addition of oxygen, and It can be seen from A in figure 1 that other various stabilizing chemicals have all been ex- trans arrangements are in the molecule. In fact, amined, and their individual and combined effects in the ß-D-glucopyranoside structure, all hydroxyl have been determined. Much has been written on groups are trans to each other. Thus, as easily these environmental effects; however, little atten- as C-2 hydroxyl can assist in the elimination of

1 Maintained at Madison, Wis., in cooperation with the University of Wisconsin. 2 Underlined numbers in parentheses refer to Literature Cited at the end of this report. Figure I.--Mechanism of alkaline glycosidic cleavage. (M 140 232)

Figure 2.--Alkaline degradation of 2-2-substituted glycosides. (M 140 231)

FPL 188 2 the C-1 hydroxyl, so C-2 can eliminate C-3; C-3, modification of the one originally given by Janson C-2; C-3, C-4; and even the C-6 hydroxyl is in and Lindberg (11). Pyridine (25 ml.) and acetic close proximity to eliminate C-1 or C-4 hydroxyls. anhydride (10 ml.) were added to 2-?-methyl- Lindberg and coworkers (6) studied the influence D-glucose (5 g.) (5, 9). The mixture was allowedto of cis-trans hydroxyl groups in model glycosides, stand overnight at room temperature, then con- and found that in all cases a trans hydroxyl in a centrated at 60° C. to a thick oil. The syrup was position alpha to the glycosidic bond hydrolyzed dissolved in acetic acid (20 ml,): then acetyl faster under alkaline conditions than did the cis bromide (25 ml.) was added. After cooling the configuration. They also found that by blocking solution to 10° C., water (0.5 ml.) was added the a-hydroxyl group, the rate of hydrolysis was dropwise over 15 minutes. The solution was slowed (11), but not stopped as might have been warmed to room temperature, and allowed to expected. These degradations were at 170° C. in stand for 2 hours. The mixture was then extracted 10 percent sodium ; under these condi- with chloroform (2 x 100 ml.), and the combined tions, blocking the C-2 hydroxyl did not offer very chloroform extracts were washed successively much protection. This not only results from the with sodium bicarbonate-ice solution and ice rather severe conditions but, as stated earlier, water. The chloroformwas then dried over sodium can result from the trans hydroxyl on C-3 aiding sulfate, and concentrated at 40°C. to a thick light- in eliminating the C-2 blocking group that in turn, yellow syrup. The syrup was dissolved in dry aided in eliminating the glycosidic linkage (fig. 2). benzene (7 ml.), and ethanol (30 ml.) was added. Since both the glycoside and the C-2 hydroxyl in To this sulfate (5 g.) and silver carbonate these experiments were substituted with methyl (6 g.) were added, and the mixture stirred over- groups, it was impossible to determine the origin night at roomtemperature. After filtration through of the methyl detected after the degrada- a Celite pad, the filtrate was concentrated to a tion. For this reason, ethyl 2-0-methyl-ßD-- syrup that showed one major product of R Sol- f glucopyranoside triacetate was prepared here-to vent A, 0.60. The syrup was dissolved in a small differentiate between the points of elimination. De- volume of chloroform, and applied to the top of grading this model determines whether the block- a chromatographic column (2.5 x 50 cm.) packed ing group is eliminated simultaneously with glyco- with Mallinckrodt SilicAR CC-4 (100-200 mesh). side hydrolysis or whether a different mechanism Elution with solvent A followed by combination is in effect. Of equal interest was the complete and concentration of the pure fractions gave 1.7 removal of the a-hydroxyl group. For this purpose, grams of I. Crystallization from hot ethanol gave ethyl 2-deoxy-D-glucopyranoside was synthe- sized. m.p. 96-97° C., -22.7°C. (c22 chloroform). To closely simulate a cellulose molecule, a Anal, calc. for C H O : C, 51.72; H, 6.94. model compound, ethyl 4-0-methyl-ßD--gluco- 15 24 9 pyranoside, has been synthesized. Degrading this Found: C, 51.78; H, 6.72. derivative will determine the mode of degradation Et h y 1 2-deoxy-α-D-glucopyranoside (II).--2- in the internal linkages of cellulose. Deoxy-D-glucose (10 g.) and BioRad AG 50 W-X8 (H+) resin (30 g.) were added toethanol (200 ml.). The mixture was stirred and refluxed for 1 hour, EXPERIMENTAL and the resin removed by filtration. Air evapora- tion of the ethanol gave a small yield of crystal- Purity of crystalline products was determined line product, and on further evaporation gave a by thin-layer chromatography on silica g e 1 thick syrup, Redissolution of the syrup in hot H-coated glass plates irrigated with either ethyl ethanol followed by air evaporation gave more acetate-hexane (1:1, v/v) or chloroform-ethanol crystalline product. Repeating this procedure four (4: 1, v/v). Components were located by spraying times, a total crystalline yield of 3.2 grams was with sulfuric acid (10 pct.) in ethanol, then char- achieved. Recrystallization from hot ethanol gave ring until permanent spots became visible. m.p. 124° C. +120° C. (c, 3.09, water). Ethyl 3, 4, 6 - tri -0-acetyl-2-0-methyl-ß-D- R solvent B 0.43. glucopyranoside (I).--The following procedure is a f

3 Anal. calc. for C H O : C, 50.00; H, 8.33. Table I.--Rate of alkaline degradation of model glycoside 8 16 5 Found: C, 49.94; H, 8.16. Ethyl 4-0-methyl-ß-D-glucopyranoside (III).-- Compound K (h-1) This compound was prepared as described (18).

Alkaline Degradation of Samples

Solutions of the above compounds (10 ml., 4 mg./ ml.) were degraded in 2.5N NaOH in stainless steel (316, 1/2 in. x 10 in.) reaction vessels. Oxygen was purged from the alkali solutionprior to addition with a stream of oxygen-free nitrogen, The vessels were sealed with threaded caps, and 1See Literature Cited (11). placed in an oil bath at 170° ±0.2° C. At various intervals, samples were collected by cooling and this reason, the stainless steel reactors were opening the containers. Aliquots of these solutions used. were treated with sulfuric acid, and analyzed spectrophotometrically as described by Scott Because of the possible leakage of gas, no quan- et al. (20). The optical density was read at titative significance was given to the ethanol and 322 nanometers for compounds I and III and at methanol production. However, assuming the rate 306 nanometers for compound II. The method de- and extent of evaporation, if any, was the same termines the hydroxy methyl produced for the two , the ratio of ethyl alcohol to when the unreacted glucosyl moiety is dehydrated methyl alcohol is given as an indication of the in sulfuric acid. Solutions of known concentrations extent of elimination of the two groups from the of the three compounds were used for preparing derivatives, standard reference curves. In the ethyl 2-0-methyl-ß-D-glucopyranoside, The amounts of ethanol and methanol produced the rate of degradation is just half that of the 2-2- by the degradation were determined by gas hydroxy compound. There is littleprotection of the chromatography (8) on a Porapak Q-S 80-100 mesh glucoside from the methyl group at 170° C. From (Waters Assn., Inc., Framingham, Mass.) column samples at 1, 12, 24, 48, and 136 hours, the ratio (1/8 in. x 6 ft.); carrier gas, N ;flow rate, 20 ml./ of methyl to ethyl alcohol was constant at 1.4. 2 If all the glycosidic bonds were broken by assist- min.; isothermal temperature, 130° C. ance from the 2-hydroxyl once the methyl group The amount of D-glucoisosaccharinic acid pro- was eliminated, the ratio would be 1. Since it is duced from III was determined as described (17). 1.4, about 30 percent of the glucosidic bonds are broken without the participation of the C-2 hydroxyl. This shows that more than one mech- RESULTS AND DISCUSSION anism is involved in the alkaline hydrolysis of 2-2-substituted glucosides. In ethyl 2-deoxy- α- D-glucopyranoside, the rate The first order rate constants determined from of degradation is 8 times slower than that in the the spectrophotometry data are given in table 1. 2-hydroxy derivative. This shows quite dramati- It was found that in the runs when both methanol cally the effect of the 2-position in the alkaline and ethanol were produced the stainless steel hydrolysis. The fact that this is ana-glycoside tubes were not able to contain the pressure dev- has no significance on the degradation, since the eloped, and part of the sample evaporated. In one 2-hydroxyl is absent. In this situation, there may experiment at 150° C., sealed Pyrex glass tubing be some assistance from C-6 hydroxy as in the was substituted, but the glass reacted with the reaction-forming levo-glucosan. sodium hydroxide and caused a drop in alkalinity In the degradation of ethyl 4-2-methyl-6-E- as follows: Time 0, N = 2.5; 22 h, N, 1.6; 48 h, glucopyranoside, two possible mechanisms might N, 1.25; 120 h, N, 1.0; and 170 h, N, 0.82. For be considered. In one, the ethyl glycoside is

FPL 188 4 eliminated as just described giving rise to 4-?-methyl ether bonds. The only major degrada- 4-2-methyl glucose. This, in turn, is degraded tion product, as determined by ion exchange by a ß-alkoxycarbonyl mechanism (10) that elim- chromatography, was D-glucoisosaccharinic acid; inates the 4-2-methyl group, and is followed by a however, the ratio of III/isosaccharinic acid was benzilic acid type rearrangement (17) to give D- 1.4, which showed that 30 percent of III goes into glucoisosaccharinic acid. In a second possible reaction pathways other than saccharinic acid mechanism, the 4-2-methyl group is eliminated formation. At this high-alkali concentration and independently from the glycoside with assistance temperature, enough energy is in the system to from the trans C-3 hydroxyl or C-6 hydroxyl. cause the epoxide intermediates to degrade by The rate of degradation of III is not significantly any number of pathways. On the basis of these slower than that of the C-4 hydroxy derivative, results, it is impossible to assign any one mech- which indicates that the rate-controlling step is anism of degradation. From earlier experiments, still the eliminating of the glycoside. The ratio of isosaccharinic acid formation seems favored at EtOH/MeOH was 1.6; this means that 40 percent lower temperatures. more glucosidic bonds are breaking than are the

5 CONCLUSIONS

It is obvious from these data that the breaking of glycosidic bonds is influenced by the a-hydroxyl group. The reactions of I have shown that the a-blocking ether can easily be eliminated to give rise to an active center that assists in the rupture of the glycosidic bond. By removing this reactive site in 11, the rate of degradation decreases by a factor of 12, which is significant. It is also apparent from the reactions of III that the major mode of internal degradation of cellulose is by alkaline hydrolysis of the glycosidic bond between C -O-R and not between C -O-R. From the results 1 4 of the ethyl alcohol to methyl alcohol data, it is obvious that no single mechanism can explain all of the pathways of degradative reaction.

ACKNOWLEDGMENT

The author thanks Dr. W. E. Dick, Jr., North- ern Region Research Laboratory, U.S. Depart- ment of Agriculture, Peoria, Illinois for furnish- ing part of 2-0-methyl-D-glucose.

FPL 188 6 LITERATURE CITED

1. Aurell, R., Hartler, N., and Persson, G. 9. Hodge, J. E., and Rest, C. E. 1952. N-(3,4,6-triacetyl- D - gluco- 1963. Alkaline stability of 2-0-(4-0- syl)-piperdine and its use in pre- methyl-a - D - glucopyrano- paring 2-substituted glucose de- syluronic acid)-D-xylopyranose. rivatives. J. Amer. Chem. Soc. Acta Chem. Scand. 17(2): 545-546. 74 1498-1500. 2. Best, E. V., and Green, J. W. 10. Ibell, H. S. 1969. Alkaline cleavage of glycosidic 1944. Interpretation of some reactions in bonds II. Methyl ß-cellobioside. the field in terms of Tappi 52(7): 1321-1325. electron displacement. J. Res. Nat. Bureau Standards 32: 45-59. 3. Brooks, R. D., and Thompson, N. S. 1966. Factors affecting the cleavage of 11. Janson, J., and Lindberg, B. glycosidic bonds in alkali. Tappi 1959. Alkaline hydrolysis of glycosidic 49(8): 362-366. linkages. IV. The action of alkali on some glucopyranosides. Acta Corbett, W. M., and Kenner, J. 4. Chem Scand. 13: 138-143. 1955. The degradation of by alkali. Part IX. Cellobiose, 12. . cellobidose, and cellotetraose, 1960. Alkaline hydrolysis of glucosidic and laminarin, J. Chem. Soc.: linkages. V. The action of alkali 1431-1435. on some methyl furansides. Acta Chem Scand. 14: 2051-2053. 5. Dick, W. E., Jr. 1972. Hydrolysis of intermediate acetox- 13. Kenner, J., and Richards, G. N. onium ions derived from D- 1955. The degradation of carbohydrates glucose, Carbohydr. Res. 21: 255- by alkali. Part XI. 4-0-methyl 268. derivatives of glucose and fruc- tose. J. Chem Soc.: 1810-1812. 6. Dryselius, E., Lindberg, B., and Theander, O. 14. Lindberg, B.

1957. Alkaline hydrolysis of glycosidic 1956. Alkaline hydrolysis of glycosidic linkages II. Investigation of cello- linkages. Svensk Papperstid. 59 bitol, lactitol, and maltitol. Acta (15): 531-534. Chem Scand. 11: 663-667. 15. Machell, G., and Richards, G. N. 7. . 1960. Mechanism of saccharinic a c id 1958. Alkaline hydrolysis of glycosidic formation. Part I. Competing re- linkages III. An investigation of actions in the alkaline degradation some methyl α- and ß-glycopyr- of 4-0-methyl-D -glucose, mal- anosides, Acta Chem. Scand. 12: tose, amylose, and cellulose. J. 340-342. Chem. Soc.: 1924-1931.

8. Gough, T. A., and Simpson, C. F. 16. Minor, J. L., and Kihle, L. E. 1970. Variation in performance of porous 1969. Alkaline stability of , polymer bead columns in gas cellobionic acid, and cellobiitol. chromatography . J . Chromatogr. Tappi 52(11): 2178-2181. 51(2): 129-137.

7 17. Rowell, R. M., Somers, P. J., Barker, S. A., 20. Scott, R. W., Moore, W. E., Effland, M. J., and Stacey, M. and Millett, M. A. 1969. Oxidative alkaline degradation of 1967. Ultraviolet spectrophotometric de- cellobiose. Carbohydr. Res. 11: terminations of hexoses, pen- 17-25. toses, and uronic acids after their reactions with concentrated sul- 18. . furic acid. Anal. Biochem. 21: 1972. Acyl migrations in the synthesis of 68-80. ethyl 4 - 0 - methyl- ß D- gluco- pyranoside. Carbohydr. Res. 23: 21. Whistler, R. L., and Corbett, W. M. 417-424. 1955. Alkaline stability of 2-0-xylapyran- syl-L-arabinose. J. Amer. Chem. 19. , and Green, J. Soc. 77: 3822-3823. 1972. Kinetics of oxidative a 1 k a 1 i n e degradation of end group stab- 22. , and Bemiller, J. N. ilized cellulose models. Tappi 1958. Alkaline degradation of p o 1 y s a c - 55(9): 1326-1327. charides. Adv. Carbohyd. Chem. 13: 302.

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