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 1
Forest Products Laboratory
Forest Service U.S. Department of Agriculture INTRODUCTION
Of the chemical pulp produced in the United
States, 80 percent is produced by an alkaline
kraft process; thus, determining the reactions
taking place when cellulose is subjected to aqueous
alkali has been of great importance. Much of the
work has dealt with the degradation caused by the
endwise depolymerization (peeling) reaction that
2
gives rise to isosaccharinic acid (4, 13, 15, 22) .
Not only has the mechanism of this reaction been
studied, but also a mechanism to modify the reducing D-glucose end unit to stop or at least to
slow down this endwise degradation (16, 19).
Of equal interest has been the study of the degradation of cellulose caused by the splitting of
internal glycosidic linkages. Cleavage of these
bonds between internal D -glucose units gives rise
to new end units that then undergo the endwise
depolymerization reaction. Increases in cellulose
yield during alkaline pulping ultimately depend on
retention of glycosidic bonds,
In the research on the alkaline degradation of
cellulose, many environmental factors have been
considered. The variables of temperature, type of
alkali, alkali concentration, addition of oxygen, and
various stabilizing chemicals have all been examined, and their individual and combined effects
have been determined. Much has been written on
these environmental effects; however, little atten-
tion has been directed to the effects of reactive
hydroxyl groups adjacent to the glucosidic linkages. These
ß-dihydroxyl groups contribute
significantly in degrading cellulose under alkaline
conditions.
linked hemiIt has long been recognized that
celluloses have much greater stability to dilute
alkali than any of the polysaccharides (1, 21).
This results from the inability of the hydroxyl
group on carbon atom 2 -hydroxyl) to form a
carbonyl group necessary for the degradation to
proceed at room temperature to saccharinic
acids (22, p. 298).
Several workers have studied the mechanism
of alkaline glycosidic cleavage (2, 3, 6, 7, 11, 12, and
14), and the most widely accepted mechanism is
shown in figure 1. In the alkaline solution, the
hydroxyl oxygens will be ionized. As shown, the
C-2 oxygen anion is trans to the $-glycosidic
linkage, and can easily displace the C-1 oxygen
bond to form an epoxide B between carbons 1 and
2. This epoxide is then hydrolyzed to a glucosyl
anion, which degrades further.
It can be seen from A in figure 1 that other
trans arrangements are in the molecule. In fact,
in the ß-D-glucopyranoside structure, all hydroxyl
groups are trans to each other. Thus, as easily
as C-2 hydroxyl can assist in the elimination of
1 Maintained a t Madison, Wis., in cooperation w i t h t h e U n i v e r s i t y o f Wisconsin.
2
Underlined numbers i n parentheses r e f e r t o L i t e r a t u r e C i t e d a t t h e end of t h i s report.
FPL 188
Figure
I.--Mechanism of
alkaline glycosidic cleavage.
Figure
2.--Alkaline degradation
of
2-2-substituted
2
(M
glycosides.
140
232)
(M
140
231) the C-1 hydroxyl, so C-2 can eliminate C-3; C-3,
C-2; C-3, C-4; and even the C-6 hydroxyl is in
close proximity to eliminate C-1 or C-4 hydroxyls.
Lindberg and coworkers (6) studied the influence
of cis-trans hydroxyl groups in model glycosides,
and found that in all cases a trans hydroxyl in a
position alpha to the glycosidic bond hydrolyzed
faster under alkaline conditions than did the cis
configuration. They also found that by blocking
the a-hydroxyl group, the rate of hydrolysis was
slowed (11), but not stopped as might have been
expected. These degradations were at 170° C. in
10 percent sodium hydroxide; under these conditions, blocking the C-2 hydroxyl did not offer very
much protection. This not only results from the
rather severe conditions but, as stated earlier,
can result from the trans hydroxyl on C-3 aiding
in eliminating the C-2 blocking group that in turn,
aided in eliminating the glycosidic linkage (fig. 2).
Since both the glycoside and the C-2 hydroxyl in
these experiments were substituted with methyl
groups, it was impossible to determine the origin
of the methyl alcohol detected after the degradation. For this reason, ethyl 2-0-methyl-ßD
-glucopyranoside triacetate was prepared here-to
differentiate between the points of elimination. Degrading this model determines whether the blocking group is eliminated simultaneously with glycoside hydrolysis or whether a different mechanism
is in effect. Of equal interest was the complete
removal of the a-hydroxyl group. For this purpose,
ethyl 2-deoxy-D-glucopyranoside w a s synthesized.
To closely simulate a cellulose molecule, a
model compound, ethyl 4-0-methyl-ßD
- -glucopyranoside, has been synthesized. Degrading this
derivative will determine the mode of degradation
in the internal linkages of cellulose.
modification of the one originally given by Janson
and Lindberg (11). Pyridine (25 ml.) and acetic
anhydride (10 ml.) were added to 2-?-methylD-glucose (5 g.) (5, 9). The mixture was allowedto
stand overnight at room temperature, then concentrated at 60° C. to a thick oil. The syrup was
dissolved in acetic acid (20 ml,): then acetyl
bromide (25 ml.) was added. After cooling the
solution to 10° C., water (0.5 ml.) was added
dropwise over 15 minutes. The solution was
warmed to room temperature, and allowed to
stand for 2 hours. The mixture was then extracted
with chloroform (2 x 100 ml.), and the combined
chloroform extracts were washed successively
with sodium bicarbonate-ice solution and ice
water. The chloroformwas then dried over sodium
sulfate, and concentrated at 40°C. to a thick lightyellow syrup. The syrup was dissolved in dry
benzene (7 ml.), and ethanol (30 ml.) was added.
To this calcium sulfate (5 g.) and silver carbonate
(6 g.) were added, and the mixture stirred overnight at roomtemperature. After filtration through
a Celite pad, the filtrate was concentrated to a
syrup that showed one major product of R Solf
vent A, 0.60. The syrup was dissolved in a small
volume of chloroform, and applied to the top of
a chromatographic column (2.5 x 50 cm.) packed
with Mallinckrodt SilicAR CC-4 (100-200 mesh).
Elution with solvent A followed by combination
and concentration of the pure fractions gave 1.7
grams of I. Crystallization from hot ethanol gave
m.p. 96-97° C.,
-22.7°C. (c22 chloroform).
Anal, calc. for C
H O:
C, 51.72; H, 6.94.
15 24 9
Found: C, 51.78; H, 6.72. (II).--2-
Et h y 1 2-deoxy-α-D-glucopyranoside
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,
and the resin removed by filtration. Air evaporation of the ethanol gave a small yield of crystalline product, and on further evaporation gave a
thick syrup, Redissolution of the syrup in hot
ethanol followed by air evaporation gave more
crystalline product. Repeating this procedure four
times, a total crystalline yield of 3.2 grams was
achieved. Recrystallization from hot ethanol gave
EXPERIMENTAL
Purity of crystalline products was determined
by thin-layer chromatography on silica g e 1
H-coated glass plates irrigated with either ethyl
acetate-hexane (1:1, v/v) or chloroform-ethanol
(4: 1, v/v). Components were located by spraying
with sulfuric acid (10 pct.) in ethanol, then charring until permanent spots became visible.
Ethyl 3, 4, 6 - tri -0-acetyl-2-0-methyl-ß-D-
m.p. 124° C.
R solvent B 0.43.
f
glucopyranoside (I).--The following procedure is a
3
+120° C. (c, 3.09, water).
Anal. calc. for C H O : C, 50.00; H, 8.33.
8 16 5
Found: C, 49.94; H, 8.16. Ethyl 4-0-methyl-ß-D-glucopyranoside
(III).--
Table I.--Rate of a l k a l i n e degradation of model glycoside
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 sugar
addition with a stream of oxygen-free nitrogen,
The vessels were sealed with threaded caps, and
placed in an oil bath at 170° ±0.2° C. At various
intervals, samples were collected by cooling and
opening the containers. Aliquots of these solutions
were treated with sulfuric acid, and analyzed
spectrophotometrically as described by Scott
et al. (20). The optical density was read at
322 nanometers for compounds I and III and at
306 nanometers for compound II. The method determines the hydroxy methyl furfural produced
when the unreacted glucosyl moiety is dehydrated
in sulfuric acid. Solutions of known concentrations
of the three compounds were used for preparing
standard reference curves.
The amounts of ethanol and methanol produced
by the degradation were determined by gas
chromatography (8) on a Porapak Q-S 80-100 mesh
(Waters Assn., Inc., Framingham, Mass.) column
(1/8 in. x 6 ft.); carrier gas, N ;flow rate, 20 ml./
1 See Literature Cited (11).
this reason, the stainless steel reactors were
used.
Because of the possible leakage of gas, no quantitative significance was given to the ethanol and
methanol production. However, assuming the rate
and extent of evaporation, if any, was the same
for the two alcohols, the ratio of ethyl alcohol to
methyl alcohol is given a s an indication of the
extent of elimination of the two groups from the
derivatives,
In the ethyl 2-0-methyl-ß-D-glucopyranoside,
the rate of degradation is just half that of the 2-2hydroxy compound. There is little protection of the
glucoside from the methyl group at 170° C. From
samples at 1, 12, 24, 48, and 136 hours, the ratio
of methyl to ethyl alcohol was constant at 1.4.
If all the glycosidic bonds were broken by assistance from the 2-hydroxyl once the methyl group
was eliminated, the ratio would be 1. Since it is
1.4, about 30 percent of the glucosidic bonds a r e
broken without the participation of the C-2
hydroxyl. This shows that more than one mechanism is involved in the alkaline hydrolysis of
2-2-substituted glucosides.
In ethyl 2-deoxy-α- D-glucopyranoside, the rate
of degradation is 8 times slower than that in the
2-hydroxy derivative. This shows quite dramatically the effect of the 2-position in the alkaline
hydrolysis. The fact that this is ana-glycoside
has no significance on the degradation, since the
2-hydroxyl is absent. In this situation, there may
be some assistance from C-6 hydroxy a s in the
reaction-forming levo-glucosan.
In the degradation of ethyl 4-2-methyl-6-Eglucopyranoside, two possible mechanisms might
be considered. In one, the ethyl glycoside is
2
min.; isothermal temperature, 130° C.
The amount of D-glucoisosaccharinic acid produced from III was determined as described (17).
RESULTS AND DISCUSSION
The first order rate constants determined from
the spectrophotometry data are given in table 1.
It was found that in the runs when both methanol
and ethanol were produced the stainless steel
tubes were not able to contain the pressure developed, and part of the sample evaporated. In one
experiment at 150° C., sealed Pyrex glass tubing
was substituted, but the glass reacted with the
sodium hydroxide and caused a drop in alkalinity
as follows: Time 0, N = 2.5; 22 h, N, 1.6; 48 h,
N, 1.25; 120 h, N, 1.0; and 170 h, N, 0.82. For
FPL 188
4
eliminated as just described giving rise to
4-2-methyl glucose. This, in turn, is degraded
by a ß-alkoxycarbonyl mechanism (10) that eliminates the 4-2-methyl group, and is followed by a
benzilic acid type rearrangement (17) to give Dglucoisosaccharinic acid. In a second possible
mechanism, the 4-2-methyl group is eliminated
independently from the glycoside with assistance
from the trans C-3 hydroxyl or C-6 hydroxyl.
The rate of degradation of III is not significantly
slower than that of the C-4 hydroxy derivative,
which indicates that the rate-controlling step is
still the eliminating of the glycoside. The ratio of
EtOH/MeOH was 1.6; this means that 40 percent
more glucosidic bonds are breaking than are the
4-?-methyl ether bonds. The only major degradation product, as determined by ion exchange
chromatography, was D-glucoisosaccharinic acid;
however, the ratio of III/isosaccharinic acid was
1.4, which showed that 30 percent of III goes into
reaction pathways other than saccharinic acid
formation. At this high-alkali concentration and
temperature, enough energy is in the system to
cause the epoxide intermediates to degrade by
any number of pathways. On the basis of these
results, it is impossible to assign any one mechanism of degradation. From earlier experiments,
isosaccharinic acid formation seems favored at
lower temperatures.
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
4
1
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., Northern Region Research Laboratory, U.S. Department of Agriculture, Peoria, Illinois for furnishing part of 2-0-methyl-D-glucose.
FPL 188
6
LITERATURE CITED 1.
2.
Best, E. V., and Green, J. W.
1969. Alkaline cleavage of glycosidic
bonds II. Methyl ß-cellobioside.
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3.
Brooks, R. D., and Thompson, N. S.
1966. Factors affecting the cleavage of
glycosidic bonds in alkali. Tappi
49(8): 362-366.
4.
5.
6.
7. 8.
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1952. N-(3,4,6-t r i a c e t y l - D - g l u c o -
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1963. Alkaline stability of 2-0-(4-0m e t h y l -a - D - g l u c o p y r a n o syluronic
acid)-D-xylopyranose.
Acta Chem. Scand. 17(2): 545-546.
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paring 2-substituted glucose derivatives. J. Amer. Chem. Soc.
7 4 1498-1500.
10. Ibell, H. S.
1944. Interpretation of some reactions in
the carbohydrate field in terms of
electron displacement. J. Res.
Nat. Bureau Standards 32: 45-59.
11.
Corbett, W. M., and Kenner, J.
1955. The degradation of carbohydrates
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cellobidose,
and cellotetraose,
and laminarin, J. Chem. Soc.:
1431-1435.
12.
Dick, W. E., Jr.
1972. Hydrolysis of intermediate acetoxonium ions derived from Dglucose, Carbohydr. Res. 21: 255268.
Dryselius,
E.,
Lindberg,
B.,
and
Theander, O.
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1958.
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7
.
1960. Alkaline hydrolysis of glucosidic
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13.
Kenner, J., and Richards, G. N.
1955. The degradation of carbohydrates
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14.
Lindberg, B.
1956. Alkaline hydrolysis of glycosidic
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(15): 531-534.
15.
Machell, G., and Richards, G. N.
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Minor, J. L., and Kihle, L. E.
1969. Alkaline stability of gluconic acid,
cellobionic acid, and cellobiitol.
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Alkaline hydrolysis of glycosidic
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some methyl α- and ß-glycopyranosides, Acta Chem. Scand. 12:
340-342.
Gough, T. A., and Simpson, C. F.
1970. Variation in performance of porous
polymer bead columns in gas
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51(2): 129-137.
Janson, J., and Lindberg, B.
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18. 19.
20. Scott, R. W., Moore, W. E., Effland, M. J.,
and Millett, M. A.
1967. Ultraviolet spectrophotometric determinations of hexoses, pentoses, and uronic acids after their
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68-80.
Rowell, R. M., Somers, P. J., Barker, S. A.,
and Stacey, M.
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cellobiose. Carbohydr. Res. 11:
17-25.
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.
Acyl migrations in the synthesis of
ethyl
4 - 0 - m e t h y l - ß D-glucopyranoside. Carbohydr. Res. 23:
417-424.
, and Green, J.
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55(9): 1326-1327.
21.
Whistler, R. L., and Corbett, W. M.
1955. Alkaline stability of 2-0-xylapyransyl-L-arabinose. J. Amer. Chem.
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