SYNTHESIS AND KINETICS OF HYDROLYSIS OF
SOME GLYCOSIDE-GLYCOSIDASE MODELS

Thai: for the Degree of Ph. D.
MICHIGAN STATE UNIVERSITY
WiIIard Loye Macison

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This is to certify that the

thesis entitled
Synthesis and Kinetics of Hydrolysis of Some

Gl ycos ide-Gl ycos idase Model 3

presented by

Willard Loye Madson

has been accepted towards fulfillment
of the requirements for

Ph. D. degree in Biochemistry

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ABSTRACT

SYNTHESIS AND KINETICS OF HYDROLYSIS OF SOME GLYCOSIDE-
GLYCOSIDASE MODELS

by Willard L. Madson

A series of acetal and ketal models for the glycoside-glycosidase
system were prepared. These included 1,2-O-isopropy1idene-QPD-glucose,
6-O-methyl-l,2—O-isopropylidene-a—D—glucose, 6—deoxy-6-methanethiol—l,2-
O-isopropylidene-a-D-glucose, methyl-(6-O-methyl)-a~D~ga1actoside, methyl-
(6—deoxy—6-methanethiol)-a-D—ga1actoside, and u(S)-(y—oxobutyl)-imidazole
diethyl ketal. The rates of the acid-catalyzed hydrolysis of these compounds
were determined by means of direct spectrophotometric, polarimetric, and ‘
colorimetric methods. The kinetics of these hydrolysis reactions indicate
that anchimeric assistance by nucleophiles does occur in some of these cases.
Participation by both the sulfur of the -SCH3 group and by the imidazole
nitrogen is indicated; increased hydrolysis rates of acetal and ketal
derivatives containing these groups were observed in several instances.
These results are compatible with the well-established mechanism of acetal
and ketal hydrolysis involving an intermediate carbonium ion. A properly
oriented nucleophile can stabilize this transition state and thus increase
the rate of hydrolysis. It seems that enzymatic hydrolysis by glycosidases
might well proceed in a similar fashion with the enzyme positioning a
nucleophile in such a way that it can participate in stabilization of the
glycosyl carbonium ion. This type of "onium" ion may also be involved in
the transosylase activity of these enzymes; the transferring species may be

an immonium or sulfonium ion.

SYNTHESIS AND KINETICS OF HYDROLYSIS OF SOME GLYCOSIDE-

GLYCOSIDASE MODELS

By

Willard Loye Madson

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Biochemistry

196a

\}

ACKNOWLEDGMENT

The author wishes to express his thanks to Dr. John C. Speck, Jr.
for his interest, suggestions, cooperation, and sense of humor during the
course of this investigation.

Most grateful acknowledgment is due to my wife, Cari, whose faith,
understanding, and encouragement helped make all of this possible.

Acknowledgment is also due to the Department of Biochemistry and

the National Institutes of Health for financial support of this work.

ii

I.

II.

TABLE OF CONTENTS

INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . . .

Historical Background . . . . . . . . . . . . . . . . . . . .

A. Acetal Hydrolysis.

B. Glycosidases .

EXPERIMENTAL. . . . . . . . . . . . . . . . . . . . . . . . . . .

Reagents. . . . . . . . . . . . . . . . . . . . . . . . .
Preparation of 1,2-O—Isopropylidene-ohD-Glucose . . . . .

Preparation of 1,2-O-Isopropylidene-6-O—(p-Toluene-
SUlfony1)_Q—D—GIUCOSB o o o o o a o o o o o o o o o o o 0

Preparation of 1,2-O-Isopropylidene-fi-Deoxy-fi-Methane-
thi01ߣ1ucose O I O O O O O O O O O O O O O O O O O 0

Preparation of 1,2-O-Isopropylidene-6-O-Methy1-a—
D-Glucose . . . . . . . . . . . . . . . . . . . . . . . .

Preparation of l,2:3,h-Di-O-IsopropylidenenafiD-
Galactose . . . . . . . . . . . . . . . . . . . . . . . .

Preparation of l,2:3,QFDi-O-Isopropylidene-6-O-(p-
Toluenesulfonyl)-orD~Galactose. . .'. . . . . . . . . . .

Preparation of l, 2: 3, AFDi-O-Isopropylidene-6-Deoxy—6-
Methanethiol-a-D-Galactose. . . . . . . . . . . . .

Preparation of Methyl- (6-Deoxy—6-Methanethiol)-orD—
Galactoside . . . . . . . . . . . . . . . . . . . . .
Preparation of l,2:3,u-Di—O-Isopropylidene—6-O—Methyl-

orD-Galactose . . . . . . . . . . . . . . . . . . . . . .
PrepAration of Methyl-(6-O-Methy1)-o~D-Ga1actoside. . . .
Preparation of h(5)-Hydroxymethyl Imidazole Picrate . . .
Preparation of 4(5)-Chloromethyl Imidazole Hydrochloride.

Preparation of h(5)-(y-Oxobutyl)-Imidazole. . . . . . . .

iii

Page

11
11

12

1.2

1h

15

16

17

18

19

20
22
22
23

23

TABLE OF CONTENTS (Cont.)
Page

0. The Preparation of 4(5)-(y—0xobutyl)-Imidazole
Diethyl Ketal . . . . . . . . . . . . . . . . . . . . . . 24

P. Methods Used to Determine Rates of Hydrolysis . . . . . . 26

1. Direct Spectrophotometric Method. . . . . . . . . . . 26

2. Polarimetric Method . . . . . . . . . . . . . . . . . 26

3. Colorimetric Determination of Reducing Sugars . . . . 27

Q. Preparation of Buffers. . . . . . . . . . . . . . . . . . 28

III. RESULTS AND DISCUSSION. . . . . . . . . . . . . . . . . . . . . . 29
A. Derivatives of l,2-O-Isopropylidene-orD-Glucose . . . . . 29

B. Rates of Hydrolysis of Methy1-(6-Deoxy-6-Methanethiol) and
Methyl-(6-O~Methy1)-a~D-Galactosides. . . . . . . . . . . 31

C. Rate of Hydrolysis of 4(5)-(y—Oxobutyl)-Imidazole Diethyl
Ke tal O O O O O O O O O O O O O O O 0 0 O O I O O O O O O 33

D. General Comments. . . . . . . . . . . . . . . . . . . . . 35
IV. SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

V. REFERENCES. . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

iv

TABLE

II.

III.

LIST OF TABLES

Rates of Acid-Catalyzed Hydrolysis of Some Derivatives of
1,2—O—Isopropylidene-ahD-Glucose. . . . .

Rates of Acid-Catalyzed Hydrolysis of Methyl-(6-O-Methyl)—
and Methyl-(6—Deoxyh6-Methanethiol)-orD-Galactosides.

Rate of Acid-Catalyzed Hydrolysis of 4(5)- (y-Oxobutyl)-
Imidazole Diethyl Ketal . . . . . . . . . . . .

Page

30

32

34

SYNTHESIS AND KINETICS OF HYDROLYSIS OF SOME GLYCOSIDE-
GLYCOSIDASE MODELS

I. INTRODUCTION

Glycosidases and transosylases have in common the ability to catalyze
cleavage of the carbon-oxygen bond of glycosides as shown in the following
expression involving a pyranoside. Glycosidases without transosylase
activity catalyze only hydrolysis, whereas glycosidases having transosy-
lase activity catalyze transfer of the glycosyl moiety to substances other

0 l O

*fl-r x +ROH

than water. There is some evidence to indicate that this class of enzymes
may include those which catalyze hydrolysis or formation of glycosides
having nitrogen or other heteroatomsattached to the glycosyl carbon.
_Since the substrates of these enzymes (glycosides) may be considered
as cyclic acetals, it has appeared that a better understanding of the
factors influencing acetal hydrolysis might give some insight into the
mechanism of action of these enzymes. The mechanism of hydrolysis of
acetals, as shown in the general expression below, has been well
established (1). Polar, resonance, and steric effects on the reactivity
of acetals have been determined and correlated (1, 2). It is interesting
to note, however, that no evidence of neighboring group participation has
been shown previously, although several of the substituents studied are

0“

known to be effective in this manner.

 

 

 

 

R1 R1
+
\Cu-“OR' + H20 3945-» \czzo + R'OH

/

R2 R2

If the above mechanism is accurate, the transition state must have
considerable carbonium ion character (as evidenced by hyperconjugative and
steric effects). It is reasonable, then, to expect that neighboring
groups on the acetal (i.e., nucleophiles) might stabilize this transition
state and give rise to increased hydrolysis rates. If this is shown to
be true, then enzymatic catalysis might proceed in a similar manner with
an appropriate nucleophile positioned so as to participate in transition
state stabilization.

Initial work in this laboratory indicated that there is an effect by
neighboring nucleophiles. When the rates of hydrolysis of methoxy and
methanethiol acetal were investigated, a thousand-fold difference was
observed even though the inductive effects of methoxy and methanethiol
are very similar (3).

The possible participation of imidazole nitrogen as a nucleophile

is important to the investigation because of the obvious implication to

histidine as a participant in enzyme catalysis. No direct evidence
has been obtained previously for the involvement of histidine in enzyme
catalysis. Thus, it has seemed that investigation of the rates of
hydrolysis of acetals containing nitrogen at positions suitable for
intramolecular participation would be most valuable. In addition,
it appeared that the acid-catalyzed hydrolysis of glycosides containing
nucleophiles. especially at the 6 position of the glycosyl portion, might
show any neighboring group effect more directly.

This thesis will present the work done to prepare some of these
model compounds, the kinetic data that was collected, and some of the
possible implications to the mechanism of action of glycosidases -- and

perhaps to transosylases in general.

O
Historical Background

 

A. Acetal Hydrolysis

The rates of hydrolysis of acetals and the factors influencing these
rates have been under investigation for over fifty years. As early as
1908, Fitzgerald and Lapworth (u) looked at the hydrolysis of acetals and
were able to make estimates of their speeds. The first systematic study
of acetal hydrolysis was made in the early 1920's by Skrabal and workers (5).
They investigated the hydrolysis of alkyl and acyl (substituted at the‘
alcohol) acetals under both alkaline and acidic conditions. A step-wise

mechanism was postulated for these hydrolyses:

k
RCH<OX)2 + H20 —$+ RCH(OX)(OH) + XOH <1)

I k2
R-CHO + xou (2)

Based on the observation that the rate constants for acid hydrolysis of the
alkyl acetals were constant, Skrabal suggested that the measured constants
represented the velocity at which the half acetals were formed and that

the decomposition to the aldehydes occurred at a much higher velocity.
Later work by Skrabal (6) in which he varied the aldehyde rather than the
alcohol, showed a rapid change in the hydrolysis rates going from the
pentaerythritol acetals of formaldehyde, acetaldehyde, propionaldehyde,

to acetone. Still later, in 1939, Skrabal (7) showed that the effects of
alkyl substituents (on the carbonyl portion) on the rates of hydrolysis

of acetals were additive.

In 192A, Hermans (8), looking at the reaction of acetone with glycols,
said that only the C-0 bonds of the carbon atom of the carbonyl, and
never those of the alcohol, were broken since no change in configuration
was observed. This view was supported by Palomaa and Aalto (9) in 1933
in their work to determine the influence of oxygen on the aldehyde component.
More recent work by Alexander, 23 31. (10), O'Gorman and Lucas (11), and
Garner and Lucas (12) on the hydrolysis of acetals derived from optically
active alcohols [(a) D(+)-2-butanol, (b) D(+)-2-octanol, and (c)
D(-)-2,3butanediol] substantiated these conclusions; in each instance, the
alcohol recovered from the hydrolysis of the acetal had not undergone
racemization.

Looking at some cyclic acetals, Leutner (13) in the early 1930's was
able to show that the speed of hydrolysis is increased by replacing

hydrogen in the alcohol portion by methyl. He also showed a six-fold

increase in stability as the ring size went from five to six and a
similar increase between the six-membered and seven-membered rings.

The presently accepted mechanism was first suggested by O'Gorman
and Lucas (11) after examining rate studies by earlier workers [Skrabal (7)
and Hammett (14)] and their own observations on the hydrolysis of acetals
of optically active alcohols. They again noted that the effect of change
in structure operates on the aldehyde carbon rather than on the alkyl
carbon atom since changes in the nature of the aldehyde part of the
molecule exerted more profound effects on the hydrolysis rate than
changes in the alcohol portion. They stated that the hydrolysis may be
considered to be a typical SNl reaction in which the aldehyde carbon atom,

not the alkyl carbon atom, forms the carbonium ion:
H

R\\C//OR fl:+ R\\C//OR a. R\\C+
H//’\\OR H// \\OR H// \\OR

+ ROH

McIntyre and Long (15),observing the acid-catalyzed hydrolysis of
methylal,showed a direct relationship between the logs of the first-order
rate constants and the Hammett acidity functions. This supports the. idea
that the activated complex behaves like the conjugate acid of methylal
(i.e., there are no water molecules firmly bound in the transition state).
Hammett and Zucker (16) showed this relationship to be valid in earlier
work on acid-catalyzed hydrolysis reactions.

The most recent work on the mechanism of acetal hydrolysis and the
effects of substituents on the hydrolysis was carried out by Taft and

Kreevoy (1, 2, 17). They correlated the inductive, resonance, and steric

effects on the reactivity of the acetals and further supported the

concept that the transition state has considerable carbonium ion character
at the carbonyl carbon atom. Taft and Kreevoy found that the second-order
rate constant for the acid-catalyzed hydrolysis of acetal in fifty percent
aqueous dioxane showed a three-fold increase when run in deuterium oxide.
Orr and Butler (18) observed the same phenomenon, earlier, in water.
Kilpatrick (19) obtained similar results with dimethyl and ethylene acetals
in deuterium chloride-deuterium oxide solutions. The greater hydrolysis
rates in deuterium oxide indicate that a prototropic equilibrium precedes

the rate determining step.

B. Glycosidases

Koshland, in 1954, proposed three main schemes to account for the facts
that were known about enzymatic transfer (20). Single displacement, in
which the substrate and reactant are fixed on the enzyme and the attacking
group enters on the side opposite the leaving group,was the first. This
implies inversion at the glycosidic bond. This has been proposed for the
maltose phosphorylase reaction (21). The second was a double displacement
in which the preliminary attack is on the substrate by a nucleophile on the
enzyme surface, followed by splitting of the glycosidic bond and the forma-
tion of a covalent bond between the enzyme and the residue to be transferred;
the enzyme-substrate intermediate then is attacked by the entering group
resulting in a double inversion and retention of configuration. This
mechanism is supported by the exchange of phosphorus by sucrose phosphory-
lase (22). Third, a frontal displacement mechanism was suggested in which

the substrate is fixed on the enzyme's surface in such a way that the

attacking group can come in on the side on which the leaving group is
attached, again with retention of configuration.

This third possibility of Koshland's has been supported by
Jermyn (23) who has considered fixation of the acceptor molecule on the
binding site as necessary before reaction occurs, and by Stringer and
Tsuchiya (24) in their work on the activation of dextran sucrase by
methyl-ahD-glucoside.

Mayer and Larner (25), in examining the a-amylases from hog pancrease
and B. subtillus and the B-amylase from sweet potatoes, suggested formation
of a carbonium ion at carbon 1 of the glucose moiety (after orientation of
the enzyme surface) followed by a stereospecific hydration. Thoma. and‘
Koshland (26) found a quantitative inversion in sweet potato B—amylase and
stated that no carbonium ion can be formed and that the mechanism probably
involves a single displacement (a displacement of the -OR group) in which
the approach of water is guided by the enzyme.

Thoma and Koshland (27), looking at the inhibition of B-amylase by
cycloamyloses, have suggested an induced fit theory in which (a) a change
is induced in the protein's geometry by the substrate when it becomes
attached to the binding site, (b) a precise orientation of the catalytic
groups is required, and (c) the substrate induces this orientation by the
modification it produces in the protein geometry. This is in opposition
to the theories which suggest some kind of preformed fit between enzyme
and substrate which contributes to the specificity and proper orientation

of the catalytic centers.

Cohn (28) was the first to show that the transferred moiety in
transosylase catalyzed reactions was glycosyl. Using 018 labeled
inorganic phosphate, glycogen and muscle phosphorylase, she showed that
all of the O18 was in the inorganic phosphate and glucose phosphate at
the end of the reaction. No loss to water occurred as would have been
the case if the phosphorus-oxygen bond had broken. Koshland and Stein (29),
in examining yeast invertase in the presence of H2018, found the glucose
formed did not contain any 018; the fructose, therefore, is liberated as
fructosyl. Similar results with H2018 were obtained with hog pancrease and
B. subtillus a-amylase and sweet potato B-amylase by Mayer and Larner
(25, 30). Using glycogen as a substrate, they found that 90 percent of the
018 used was contained in the 1 position of the saccharides that were formed.
Maltose, used as a control, contained only a trace of 018. They concluded
that both of the amylases hydrolytically rupture the 01-0 bond of the 1,u
linkages. Bunton and workers (31, 32), examining the acid-catalyzed
hydrolysis of a number of glycosides in H2018, stated that the results are
best interpreted in terms of a mechanism involving pyranosyl carbonium ions.
They concluded that the hydrolysis proceeds by a unimolecular decomposition
of the conjugate acid and involves breaking of the hexose-oxygen bonds
only. Their results, however, do not distinguish between cyclic and acylic
pyranosyl carbonium ions. The one exception noted by Bunton was that of the
t-butyl—B-D-glycopyranoside which involves a bond breaking between the
t-butyl group and the glycoside oxygen atom. The formation of such an alkyl
carbonium ion will presumably occur in any glycoside for which the stability

of such an ion is greater than that of the glycopyranosyl carbonium ion.

 

 

The question of a cyclic versus an acylic carbonium ion in the
transition state is still an open one. There does appear to be more
evidence for the acylic form in acid-catalyzed hydrolysis. Some of this
evidence comes from the interconversion of furanosides and pyranosides. In
the formation of glycosides, the aldoses, normally present as pyranoses,
give a mixture of the pyranosides and furanosides (33). The proportion of
the less stable furanosides initially present gradually decreases as the
reaction reaches equilibrium, suggesting cleavage of the oxygen ring. The
hydrolysis of the glycosides may proceed by a similar mechanism. Additional
evidence in support of the acyclic mechanism is found in the acetolysis of
methyl tri-O-acetyl-B-D-arabinopyranoside which, when catalyzed with zinc
chloride or sulfuric acid gives a mixture of the two isomeric penta-O-
acetyl-D-arabinose methyl hemicaetals in good yield (3h).

Many suggestions have been made concerning the groups present at the
active sites of transosylases. Involvement of the sulfhydryl group has
been proposed for a number of these enzymes (35). Competitive protection
by the substrate against inhibition by sulfhydryl reagents has been shown
(36). A quantitative relationship between the binding of sulfhydryl groups
by p-mercuribenzoate and the decrease of activity for a B-galactosidase has
been observed (37). Reversible inactivation of B-amylase using p-chloro-
mercuribenzoate is known to involve at least six sulfhydryl groups (38).
None of these results, however, give any direct evidence for the involve-
ment of sulfhydryl at the active site; these groups may be necessary for

the configurational requirements of the enzymes.

10

Wallenfels found (39), by using the pH activity curve for a B-galac—
tosidase from E. coli, indication of two groups at pK 5.8 to 6.7 and
7.8 to 8.6. The first is supposed to be an imidazole group, and the latter
a sulfhydryl group. Thoma and Koshland (40) explained the Vfi versus pH
plot of sweet potato B-amylase on the basis of two active groups, one having
pK h.3 and the other pK 7.1, and suggested that they are a carboxyl and
an imidazole group, respectively. They considered that a sulfhydryl group
is involved also.

Neely (41) found two pK values for dextran sucrase at 9.9 to 5.1 and
5.5 to 5.7. Neely proposed that a carboxyl group (pK 4.9-5.1) acts as a
nucleophile and an imidazole group (pK 5.5-5.7) as an electrophile. Neely
also exposed solutions of the enzyme and methylene blue to intense light and
observed inactivation of the enzyme and concommitant destruction of histidine;
substrate protected the activity and suppressed the destruction of histidine.
These results suggest that histidine may participate in the enzymatic

catalysis.

II. EXPERIMENTAL

A. Reagents

 

1. General
All inorganic salts, concentrated acids, general organic reagents,
etc. were reagent grade.

2. Solvents

 

a. Acetone, anhydrous
Reagent grade acetone was dried over anhydrous calcium sulfate
(Drierite) and filtered through paper before use.

b. Benzene, anhydrous
Reagent grade benzene was dried by azeotropic distillation..

c. Chloroform, anhydrous
Reagent grade chloroform was washed with concentrated sulfuric
acid, then water, and dried over anhydrous sodium sulfate
before being distilled.

d. Dioxane-l,u, anhydrous
Reagent grade dioxane (2 l) was refluxed with 27 m1 of
concentrated hydrochloric acid and 200 ml of water under
nitrogen for 12 hours. Solid potassium hydroxide was added
until it no longer dissolved and a layer separated. The.
dioxane was decanted, fresh potassium hydroxide added, decanted
again into a clean flask, and refluxed with sodium for 12 hours.
The dioxane was then distilled under nitrogen. This purified
dioxane was redistilled from sodium just before use.

e. Ethyl alcohol, anhydrous
'Gold Shield (Commercial Solvents Corporation) ZOO-proof ethyl

alcohol was refluxed with and distilled from barium oxide.

11

12

f. Methyl alcohol, anhydrous
Reagent grade methyl alcohol was refluxed with magnesium
shavings and distilled.
g. Pyridine, anhydrous
Reagent grade pyridine was distilled from calcium hydride.
3. Organics
a. Fructose (Levulose)
Nutritional Biochemicals Corporation 1
b. Galactose, anhydrous
Pfanstiehl
c. Glucose (Dextrose), anhydrous
Baker reagent grade or Pfanstiehl
d. p-Toluenesulfonyl chloride
Eastman Kodak p-toluenesulfonyl chloride was sublimed before
using.
4. Others
2,4-Dinitrophenylhydrazine solution
0.4 g of 2,4-dinitrophenylhydrazine was dissolved in 10 m1

of 72 percent perchloric acid and 20 ml of water.

B. Preparation of 1,2-O-Isopropylidene-a—D-Glucose
The method used is that of Mehltretter, et a1. (42), as described by
Schmidt (43). I
C. Preparation of lJ2-O-Isopropylidene-6—O—(p-Toluenesulfonyl)-o~D-G1ucose

The procedure is a modification of the method described by

Tipson (44).

13

A solution of 33 g (0.15 mole) of l,2-O-Isopropy1idene-orD-glucose
and 150 ml of dry pyridine was cooled to 5° in a SOC-m1 two-neck round-
bottom flask equipped with a drying tube and an addition funnel. A
solution of 30 g (0.154 mole) of p-toluenesulfonyl chloride in 150 m1 of
dry chloroform was added slowly, with swirling, over a period of 1 hour
while cooling was continued. The temperature of the mixture was kept at
0° for 7 hours and then the temperature was allowed to rise gradually to
room temperature. Ten hours after the flask was removed from the cold bath,
it was cooled again to 0° in ice-salt. A few pieces of ice were added to
the mixture and it was transferred to a Teflon-stoppered separatory funnel
and washed with the following:

(1) five 100-m1 portions of ice-cold 20 percent potassium bisulfate,

(2) two lOO-ml portions of ice-cold water,

(3) five loo-ml portions of ice-cold 20 percent potassium bisulfate,

(4) two IOO-ml portions of ice-cold water,

(5) two lOO-ml portions of ice-cold saturated sodium bicarbonate, and

(6) one 100-m1 portion of ice-cold water.
The last three washings (4, 5, and 6) were done by decantation in a beaker
since the mixture had become too thick to handle in the funnel. The
resulting solid was filtered and dried over silica gel in a vacuum
desiccator in the cold room (1°). The filtrate was placed in a separatory
funnel and the chloroform layer separated, dried over anhydrous sodium
sulfate, and then evaporated to dryness under reduced pressure (water pump).

The solid that formed was added to that obtained above.

14

The crude product was recrystallized by dissolving it in ether (about
100 ml for each 35 g of crude product), filtering, clouding the filtrate with
ligroin, and placing it in the refrigerator. The crystals that formed were
filtered and dried over silica gel in the cold room (1°). The yield was
about 30 g. The melting point was 98 - 100°; the literature value (45)

is 101 - 108°.

D. Preparation of 1,2-0-Isopropy1idene-fi-Deoxy-fi-Methanethiol-a—D-Glucose
The method used is a modification of the procedure described by
Raymond (46 ) .
A lOO-ml round-bottom flask was equipped with a gas inlet tube, reflux
condenser (for which a gas exit tube was arranged leading to an ethyl
alcohol bubbler), and an electric heating mantle. Fifty ml of absolute
methyl alcohol and 2.07 g (0.09 mole) of freshly cut sodium were placed in
the flask. After all of the sodium had reacted, methyl mercaptan was bubbled
into the solution until it was saturated (8 g added). The system was
flushed with nitrogen and 2.18 g (0.06 mole) of 1,2-O-isopropylidene—6—
(p-toluenesulfonyl)-o-D-g1ucose was added. The nitrogen flushing was
continued. After about 5 minutes, the mixture became thick due to the
precipitation of sodium tosylate. The mixture was allowed to stand at room
temperature for 4 hours. It then was poured into a flask and 240 ml of ether
added to precipitate the salts. The mixture was filtered and an additional
30 ml of ether used to rinse the flask. The filtrate was refiltered into a
500-ml round-bottom flask and an additional 30 ml of ether was used to
rinse the original flask (total volume of ether used was 300 ml). An

alternate and preferred method used to remove the salts was to filter the

15

original mixture containing the sodium tosylate and then add about

120 ml of absolute methyl alcohol. One ml of water was added to the

solution and carbon dioxide was bubbled through the mixture to precipitate
the sodium ion as sodium carbonate. The precipitate was removed by
centrifugation. The precipitate was washed with a little dry methyl

alcohol and the combined washing and supernatant solutions evaporated under
reduced pressure (water pump). The resulting syrup—precipitate was extracted
with six 50-m1 portions of dry ether and the filtered extracts evaporated
(water pump). The product then was distilled as indicated below.

The ether-methanol solution was evaporated under reduced pressure
(water pump). The dark syrup was dissolved in 25 m1 of dry acetone and-
treated with acetone-washed Amberlite MB-l until the PH was about 7. The
mixture was much lighter in color after this treatment. The mixture was
filtered, the flask and resin rinsed well with dry acetone, and the
solution stored overnight at room temperature.

The solution was transferred portion-wise to a small distillation
apparatus, the solvent removed under reduced pressure (water pump), and
the syrup distilled under a nitrogen atmosphere at reduced pressure. The
bath temperature was 150°, the distillation temperature was 110 - 120°,
and the pressure about 0.1 mm. The yield was 7 g.[a]§$8 -7.6° in ethyl
alcohol (c. 11.9); the literature values (46) are [0133 -3.5° and —7.6°

in ethyl alcohol (C- 2.3).

E. Preparation of 1,2-0—Isoprgpy1idene-G-O-Methyl-ohD-Glucose

.This was prepared from 1,2-O-isopropy1idene-6-O-(p-toluenesulfonyl)-
a~D—g1ucose according to the method of Levene and Raymond (47).
[a]§$8 -6.9 in chloroform (c. 17.3); literature value (47) is [a]3° -6.0

in chloroform.

16

F. Preparation of l,2:3,4FDi-O-Isopropylidene-a-D-Galactose

This product was prepared using a modification of the procedure
described by Schmidt (48).

A three-neck 5-1 round-bottom flask was equipped with a thermometer,
stirring motor (or Teflon-coated magnetic stirring bar), and an addition
funnel. Two-hundred g of powdered anhydrous galactose (powdered in a
Waring Blender) and 4 l of acetone were placed in the flask and mixture
cooled while stirring to 10°. Concentrated sulfuric acid (160 ml) then
was added with stirring in 20-ml portions at 15 minute intervals. During
addition of the acid, the temperature of the mixture was kept at 4 to 10°.
Stirring, then, was continued after all of the acid had been added and-the
mixture allowed to warm gradually to room temperature over a period of
about 5 hours. At the end of this reaction time, the mixture was cooled
again to approximately 10° and a solution of sodium hydroxide (245 g in
300 m1 of water) was added slowly so that the temperature did not rise
above 15° (an alternate and preferred method of neutralization is to use
dry ammonia gas). After the mixture reached pH 9, solid sodium bicarbonate
(20 g) and sodium carbonate (5 g) were added to keep the mixture near
neutrality.

After standing overnight, the pH was near 5 so an additional 10 g
of sodium bicarbonate and 10 g of sodium carbonate were added to bring the
pH to 7. The mixture was filtered and the filtrate evaporated under
reduced pressure (water pump). After all of the acetone had been removed,
about 1 lof water was added and the evaporation continued to remove

acetone condensation products. Anhydrous ether (150 ml) was added to the

17

resulting salt-syrup mixture and the mixture filtered. The filtrate was

evaporated under reduced pressure (water pump). One-hundred and fifteen g
of product was distilled (this was about one-half of the product obtained)
under reduced pressure at a bath temperature of 130 - 140°C and a pressure

of 0.5 - 0.3 mm.

G. Preparation of l,2:3,4-Di—O—Isgpr9pylidene—6—Oegp-Toluenesulfonyl)—a—
—D-Ga1actose

 

This compound was prepared using a modification of the method described
by Tipson (44).

TWenty-six g (0.1 mole) of 1,2:3,4Pdi—O-isopropylidene-orD—galactose
was poured into a 250-ml flask equipped with a Teflon-coated magnetic
stirring bar. Anhydrous acetone (27.5 ml) and anhydrous pyridine (17.5 ml)
were added and the flask stoppered. The mixture was stirred until the
isopropylidene derivative dissolved. The solution was cooled in cold water
and the flask closed with a stopper fitted with a thermometer. While the
solution was stirred, 22.8 g (0.12 mole) of p-toluenesulfonyl chloride was
added in small portions over a 1 hour period so that the temperature was
kept below 30°. A precipitate formed after most of the tosyl chloride had
been added. The mixture was kept at room temperature overnight (12 hours).
After the mixture had been cooled to 0° in an ice-salt bath, 10 ml of water
was added slowly so that the temperature did not rise above 5°. The
mixture was poured into 250 m1 of cold water. The syrup that separated
became thick and white but did not crystallize. The mixture then was
extracted with three 100-ml portions of chloroform. The chlorofonn

solution was washed in a separatory funnel with the following:-:

18

(1) five 100-m1 portions of ice-cold 20 percent potassium bisulfate,

(2) two lOO-ml portions of ice-cold water,

(3) two lOO-ml portions of saturated sodium bicarbonate, and

(4) one 100-m1 portion of water.

The chloroform portion then was dried over anhydrous sodium sulfate. The
chloroform solution was filtered and the sodium sulfate rinsed several
times with dry chloroform. The combined filtrate and rinses were
evaporated under reduced pressure (water pump). The resulting thick

syrup was stored under vacuum over silica gel in the cold room (1°). After
about 10 days, the syrup crystallized.

The crude product (42 g) was recrystallized in three batches in the~
following manner. The crude product was dissolved in a volume of hot
isopropyl alcohol equal to the weight of product. The solution was cooled
to room temperature and then nucleated with a small seed of the crude
product. As soon as crystallization began, a volume of n-hexane equal to
the volume of isopropyl alcohol was added, the mixture stirred for a few
minutes, and then filtered. The crystals were washed with a little dry
n-hexane. After a few minutes of standing, another crop of crystals appeared
in the filtrate and these were recovered also. The combined crystalline
material was dried under vacuum over phosphorous pentoxide. The yield was
31.2 g. The melting point of the product was 88 - 89°; the literature

value (49) is 89 - 91°.

H. Preparation of 1,2:3,4~Di-O-Isopropylidene-6-Deoxy—6-Methanethiol-a-
-D-Galactose

A 1-1 three-neck round-bottom flask was equipped with reflux condenser,

19

gas inlet tube, and drying tube. Two-hundred ml of dry n-propyl alcohol
was placed in the flask and the system flushed with dry nitrogen. Freshly
cut sodium (3.45 g, 0.15 g-atom) was added. After all of the sodium had
reacted, the solution was saturated with methyl mercaptan (8.2 g added).
The system was again flushed with dry nitrogen and 37.9 g (0.094 mole)
of l,2:3,4-di-O-isopropylidene-6-O-(p-toluenesulfonyl)-a-D-galactose was
added. The mixture was heated under reflux for 3.5 hours. A precipitate
formed. The mixture was cooled, filtered, and the precipitate washed well
with dry n-propyl alcohol. Two ml of water were added to the solution and
then carbon dioxide bubbled through the solution to precipitate the excess
base as sodium carbonate. The mixture was centrifuged, the precipitate
washed well with dry n-propyl alcohol, and the combined washing and super-
natant solutions evaporated under reduced pressure (water pump). The
residue (containing a small amount of precipitate) was taken up in 100 ml
of anhydrous acetone, filtered, and the filtrate evaporated to a light
yellow syrup. Yield was 20.6 g. The syrup can be distilled under reduced
pressure at a bath temperature of 128°, a distillation temperature of 102 -
104°, and a pressure of 0.3 - 0.5 mm to yield a water-white product.
[a]§;8 -72.u° in methyl alcohol (c- 6.8).
Analysis:

Calculated: % C 53.77 % H 7.64 % S 11.04

Found: 53.87 7.66 11.11

1. Preparation of Methyl-(6-Deoxy-6-Methanethiol)-o~D-Galactoside
A solution of 11.6 g (0.04 mole) of 1,2:3,4-di-O-isopropylidene-6-

-deoxy—6-methanethiol-o—D-galactose in 12 ml of absolute methyl alcohol

20

was placed in a glass tube (l-inch gauge glass sealed at one end).
Twelve ml of a solution of dry hydrogen chloride gas in absolute methyl
alcohol (3.5 percent in hydrogen chloride) was added to the tube. An
additional 12 ml of absolute methyl alcohol was used to rinse the original
flask and this was added to the tube. The tube was cooled in an ice-salt
bath and sealed. It then was heated in a water bath for 50 hours at 100°
(after about 5 to 19 minutes, the contents became very dark but became
lighter about 2 hours later). The sealed tube was cooled, opened, and
the contents evaporated under reduced pressure (water pump). The product
precipitated as soon as the alcohol was removed. The solid was taken up in
a minimum of absolute methyl alcohol, filtered, washed with dry ethyl ether,
and dried over phosphorous pentoxide. Yield was about 5 g. It was
recrystallized from hot ethyl alcohol. The melting point of twice-recry—
stallized product was 167 - l67.5°; [a :38 +156° in methyl alcohol (c. 1.1).
Analysis:

Calculated: %c 42.84 7.11 7.19 %s 14.30.

Found: 42.95 7.07 14.16
J. Preparation of l,2:3,4-Di-O—Isgpropy1idene-6-O-Methyl-o-D-Galactose

This derivative was prepared by the method of Goldstein, Hamilton,
and Smith (50).

A 2-1 three-neck roundebottom flask was equipped with a Teflon-coated
magnetic stirrer, reflux condenser, two addition funnels, a thermometer, and
a pH electrode. A solution of 50 g (0.19 mole) of l,2:3,4~di-O—isopropyl-
idene-—o~D—galactose in 95 m1 of acetone was placed in the flask. A 30

percent aqueous solution (405 ml) of sodium hydroxide was placed in one of

21

the addition funnels. Dimethyl sulfate (162 ml) was placed in the other
funnel (CAUTION - dimethyl sulfate is very toxic. Ammonia is a specific
antidote). Some of the sodium hydroxide solution was added to the flask
to make the solution in the flask basic (pH 11). Dimethyl sulfate then
was added, with stirring, in small portions. The pH was kept between 10
and 11 by additions of the sodium hydroxide solution. During the addition
of the first 20 to 30 m1 of dimethyl sulfate, the temperature was kept at
about 15° by using an ice bath. The bath then was removed and the addition
of the sodium hydroxide solution and dimethyl sulfate controlled so that
the temperature remained below 50°. The addition of the dimethyl sulfate
required about 5 hours. At the end of this time, the remainder of the
sodium hydroxide solution was added dropwise so that the temperature did
not rise above 50°C. The mixture then was stirred overnight (12 hours).

The resulting mixture was extracted with chloroform. During the
first extraction (100 ml) the chloroform layer was on top, while in the
following two extractions (each with 50 ml), the chloroform layer was on
the bottom. The combined chloroform extracts were dried over anhydrous
sodium sulfate overnight.

The filtered chlorofonm solution was evaporated under reduced pressure
(water pump). The resulting light yellow syrup was distilled under reduced
pressure. The bath temperature was 110° and the pressure 0.05 - 0.1 mm.

The almost water-white syrup was redistilled ona"Bantamware" set-up
using a small fractionating column. The product distilled at a bath
temperature of 110 - 120°C, a distillation temperature of 86 - 92°, and a
pressure of 0.10 - 0.20 mm. The product was water-white. Yield was about

35 g.

22

K. Preparation of MethylfL6-O-Methy1)-a-D-Ga1actoside

A solution of 7.0 g (0.025 mole) of l,2:3,4Fdi-O-isopropylidene-6-O-
methyl-chD-galactose in 10 ml of absolute methyl alcohol was placed in a
1-inch gauge-glass tube sealed at one end. The original flask was rinsed
with an additional 10 ml of dry methyl alcohol and this added to the tube.
Three m1 of a solution of dry hydrogen chloride in absolute methyl alcohol
(12 percent in hydrogen chloride) then was added to the tube followed by
another 7 ml of absolute methyl alcohol. The tube was cooled in an ice-
salt bath and sealed. The contents of the tube were mixed and the tube
heated in a water bath for 26.5 hours at 100°. The contents of the tube
became almost black after 30 minutes of heating but became lighter in color
as heating was continued. The tube was cooled to room temperature, opened,
and the contents transferred to a small round-bottom flask (the tube was
rinsed with a little dry methyl alcohol). The solution was evaporated
under reduced pressure (water pump). The syrupy residue did not
crystallize and was dissolved in dry methyl alcohol, Norite added to the
solution, and this mixture filtered and evaporated again under reduced
pressure (water pump). After standing at room temperature for a week,
the syrup crystallized.

The crystalline mass was recrystallized once from methyl alcohol
and twice from absolute ethyl alcohol. The yield was about 2 g. The
rotation of this thrice crystallized product was [o]§$8 = +173° in water

(c. 2.9); the literature value (so) is [ajg°= +165° in water (c. 1).

L. Preparation of 4(5)-Hydrgxymethyl Imidazole Picrate
This product was prepared according to the procedure of Totter and

Darby (51).

23

M. Preparation of 4(5)-Chloromethyl Imidazole Hydrochloride

This product was prepared from 4(5)-hydroxymethyl imidazole picrate
using the procedure of Mehler, Tabor, and Bauer (52).

N. Preparation of 4(5)+(y-Oxobutyl)-Imidazole

This procedure combines the method described by Marvel, Johnson,
and Hager(53,54) for the preparation of ketones via acetoacetic ester and a
modification of the procedure described by Pyman (55) for the preparation
of 4(5)-(y—oxobuty1)-imidazole.

A 500-ml round-bottom flask was equipped with a Teflon-coated magnetic
stirring bar, addition funnel, and reflux condenser. Two-hundred ml of
absolute ethyl alcohol (distilled from barium oxide) and 9.2 g (0.4 mole)
of freshly cut sodium were placed in the flask. After all of the sodium had
reacted, 26 g (0.2 mole) of freshly distilled acetoacetic ester was slowly
dripped into the stirred solution. After all of the ester had been added,
the solution was cooled in an ice bath and 30 g (0.2 mole) of 4(5)-chloro-
methyl imidazole hydrochloride was added. A precipitate formed immediately.
The mixture was stirred at room temperature for 1 hour and then refluxed
(bath temperature about 85°) for 3 hours. The mixture was cooled to room
temperature and the salt removed by centrifugation. The salt was washed
three times with absolute alcohol. After each washing, the salt was
separated by centrifugation. The combined supernatant solutions were
evaporated under reduced pressure (water pump).

The residual syrup from this operation was poured, with stirring, into
200 ml of 5 percent aqueous sodium hydroxide in a 500-ml round-bottom

flask. The mixture was stirred for 4 hours. At the end of this time,

24

the unsaponified material was removed by an ether extraction (about 50 m1
of ether used). The aqueous layer was placed in a 500-ml flask and 20 ml
of 50 percent sulfuric acid added dropwise with stirring. Stirring was
continued until the evolution of carbon dioxide stopped (about 3 hours).
The mixture then was treated with solid potassium carbonate until the pH
was about 7. The mixture was stored overnight at room temperature. VThe
dark solution then was placed in a 2-1 round-bottom flask and evaporated
under reduced pressure (water pump). When all but about 50 to 75 ml of
water had been removed, the mixture was extracted with chloroform. The
aqueous layer must be slightly basic during the extractions. Three extrac-
tions were made, each with about 75 m1 of chloroform. The combined chloroé
form extracts were dried over anhydrous sodium sulfate and evaporated under
reduced pressure (water pump). The dark syrup was then put in a vacuum
desiccator over barium oxide at 0.1 to 0.5 mm pressure until the syrup
crystallized. The crude product was then purified by two sublimations at

a bath temperature of 80 - 90° and a pressure of 0.1 - 0.2 mm. The yield
was 8 g. The melting point of the twice sublimed product was 79 - 80°;

the literature value (55) is 80 - 81°.

0. The Preparation of 4(5)-(y-Oxobutyl)-Imidazole Diethyl Ketal
A mixture of 5.7 g(ogumole) of 4(5)-(y-oxobutyl)-imidazole, 14 m1 of
absolute ethyl alcohol (distilled from barium oxide), and 26 ml of freshly
distilled triethylorthoformate were placed in a 100-ml round-bottom flask
equipped with a drying tube. The ketone was dissolved by swirling the
contents of the flask. Concentrated sulfuric acid was added; enough to

redissolve the white precipitate that formed during the initial addition

25

of the acid plus about a three-drop excess past this point (total volume

of acid added was about 1.6 ml). The solution was allowed to stand over-
night (12 hours) at room temperature. The solution was transferred to a
300-ml flask and a solution of sodium ethoxide was added until the pH was

i 11. The flask was equipped with a reflux condenser, drying tube, and
eletric heating mantle. The mixture was refluxed one hour. After standing
at room temperature for 2 days, the mixture was evaporated under reduced
pressure (water pump). The salt-syrup residue was extracted with about

50 m1 of dry benzene. The salts were removed by centrifugation. The salts
were washed twice with benzene; in each case the salts were removed again
by centrifugation. The combined supernatant solutions were evaporated under
reduced pressure (water pump). After the benzene was removed, the thin
syrup was transferred to a small distillation setup and distilled under
reduced pressure. The product distilled at a bath temperature of 115 -
123°, a distillation temperature of 75 - 77°, and a pressure of 0.05 -

0.01 mm. The yield was 2.2 ml. Assuming an 6270 of 30 for the ketone
(obtained from the absorbance at 270-mu using weighed samples of the pure
ketone), absorbance measurements of the product indicated that it contained
from 15 - 25 percent ketone. All attempts to purify this product have
failed. Gas chromatography with a number of columns operated under a wide
range of conditions did not give a pure sample (as indicated by the
absorbance at 270-mu). Treatment of a solution of the product with basic
alumina (Brockman Activity 1) did not remove an appreciable amount of the
ketone impurity. An attempt to prepare a larger amount of product and then

to fractionally distillthis product failed.

26

If the impurity is the parent ketone, this should not affect the
rate of hydrolysis and, thus, some preliminary rate studies were made of

'this impure product (see Results and Discussion).

P. Methods Used to Determine Rates of Hydrolysis
1. Direct Spectrophotometric Method

A Beckman Model DU Spectrophotometer equipped with a water-jacketed
temperature—controlled cell compartment was used. A circulating water bath
(Precision Scientific Company) was connected to the cell compartment and
the water temperature adjusted so that the temperature within the cells was
maintained at 25 i 0.05°.

In a typical run, a solution of the acetal or ketal and a solution of
the appropriate buffer or acid were equilibrated in a water bath at 25° for
about 3 minutes. The solutions then were mixed thoroughly, a portion of
the resulting solution placed in a 1.0 cm silica cell and the absorbance
at 270-mu of this solution determined at 2-minute intervals. The reaction
was followed well past half-life, usually to 70 - 80 percent of completion.

The logarithms of increments in 270-mu absorbance of these reaction
mixtures (usually over 6 - 8 minutes) were plotted versus time, and the
k1 values then were determined from the slopes of these plots.

2. Polarimetric Method

The Carl Zeiss Photoelectric Precision Polarimeter was used for
polarimetric measurements. The hydrolysis reactions were carried out in
a water-jacketed l-decimeter polarimeter cell. Using a circulating water
bath (Precision Scientific Company), the temperature in the cell was main-

tained at 60 i 0.1° (corrected).

27

Before each run, the cell was equilibrated at 60° for about 15 minutes.
A sample of the glycoside was weighed in a beaker and 8 m1 of 1.003N
hydrochloric acid was added. After thorough mixing, the solution was
placed in the polarimeter cell. The cell was kept outside of the polari-
meter during most of the run because of the obvious danger of damage to the
instrument in case of a leak. At regular intervals, usually 4 hours, the
electronics of the instrument were allowed to warm up, the zero point was
checked, and the optical rotation of the reaction solution determined. The
reaction was followed to 80 - 90 percent completion.

The change in optical rotation was plotted versus time and the k1
values determined from the slopes of these plots.

3. Colorimetric Determination of Reducing Sugars

This method is based on the amount of color produced by the reaction
of a reducing sugar (one of the hydrolysis products) with 3,5—dinitrosali—
cylic acid (56).

The reagent was prepared by moistening 10 g of 3,5-dinitrosalicylic
acid with a few drops of water, adding this to 200 ml of 2N aqueous sodium
hydroxide and bringing the solution to 500 ml with water. Three-hundred g
of potassium sodium tartrate were added and the mixture brought to l l with
water.

The hydrolysis reactions were run in glass-stoppered flasks in a
temperature-controlled circulating oil bath maintained at 75 (or 85) i 0.1°.
The samples were weighed into a flask and the appropriate amount of 0.46N
perchloric acid (preequilibrated in the oil bath for 15 minutes) added. At

regular intervals, aliquots were withdrawn from the flask and placed in

28

SO-ml volumetric flasks. An amount of 0.5N aqueous sodium hydroxide equal
to the aliquot was added to each volumetric flask to stop the reaction.
After a number of samples had been obtained in this manner, the color was

developed by adding 4 ml of the 3,5-dinitrosalicylic acid reagent to each

flask, heating the flasks (unstoppered) in a boiling water bath for 6
minutes, removing the flasks, and then diluting the contents to the SO-ml
mark with distilled water. After thorough mixing, the absorbances at
540 mu were determined on a Beckman Model DU Spectrophotometer in 1.0 cm
Corex cells.

Plots were made of the change in absorbance versus time and the k1

values calculated from the slopes of these plots.

Q. Preparation of Buffers

 

Buffer No. l

 

A mixture of 1.7 g acetic acid and 9.51 ml of 2.584N aqueous sodium
hydroxide was diluted to 100 ml with distilled water. The pH of the
resulting solution was found to be 5.37. The concentration of acetic acid

is 0.2833M; the concentration of acetate ion is 0.345nn.

Buffer No. 2

 

A mixture of 45 g of acetic acid and 150 m1 of 1.638N aqueous sodium
hydroxide was diluted to l l with distilled water. The pH of the resulting
solution was found to be 4.10. The concentration of acetic acid‘iB 0.750M;

the concentration of acetate ionvas 0.2457M.

III. RESULTS AND DISCUSSION

A. Derivatives of lyg—O-Isopropylideneeg:Q:Glucose

The rates of hydrolysis of three isopropylidene derivatives of QED-
glucose are shown in Table I. The substitution at position 6 of the glucose
moiety was varied from -OH to -OCH3 to -SCH3 to determine whether or not a
nucleophile could operate from this position by anchimeric assistance to
give increased hydrolysis rates. The observed rates indicate that there is
no effect in changing -OH to -OCH3, even though the latter is a somewhat
better nucleophile. There is, however, a significant change in rate with the
-SCH3 substituted derivative suggesting that this group may participate in a
transition state stabilization (as shown below) and thus result in an;

increased hydrolysis rate. One must remember in examining this effect that

Hz-S-CH3 H2 -.-CH3
HO -H 0 Ho—c-‘H
o
H OH H H+
H a
l -e- II H
3
3

there are three possible bond cleavages in these hydrolysis reactions and

  

 

the nucleophile can influence the rate of hydrolysis of only one of these

(i.e., at bond 1). Thus, the overall effect on the hydrolysis rate will

sz-S-CHs
HO -H H

H H

be decreased.

30

.Aowom cagednowomv ZHNH.H mmz mesa mmmnu mo Ham :fl coHumuucoocoo owom 05H *

.msnuwucwo~ .mwwwum mo canon cw commoumxo
mum mosam> one .oonuos may we mflwmuoo one you newuoom Houcoswwomxo onu can "use“ msmuo>
coccQLOmnc cw owcoao can no muofim one no moaofim ecu scum oocwauouoo mums monam> moose +

 

am.a

 

am.H
30.0 H oa.~ 30.0 H Hm.H
Hm.H omoosfiulo
JoIHo«:uoecnquIoI%xooQ
AN.H lolocoowazmoum0mHIOIN.H
aa.~
mo.H
mo.o H oo.~ mo.o H N~.H
AH.H
omOUDHUImloramnuoz
2 . H lonelaaafl aeoaaoflloum . H
AH.H
HH.H
30.0 H mo.H 30.0 H o~.~ oN.H
omoosHUIQIo
m~.H locoowazaouQOmHIOIm.~
AHI.omm HIOHOS.H N «IOHV AHI.00m «Iodv Aal.oom ¢IOHV
Hmosac> «x mosfio> ax owcuo>< +mosflo> ax ocsomaoo

 

omoosaolalolocoowHmmoum0mHIOlN.H mo mo>wum>wuon meow mo mwmzaouozm omnzgcuoolowo< mo moumm .H mqm<9

31

B. Rates of Hydrolysis of Methyl—(6—Deoxy-6Hnethanethiol) and Methyl-
(6-O~Methyl)-o~D—Galactosides

 

 

The rates of hydrolysis of these two glycosides were determined by
both polarimetric and colorimetric methods (see experimental section for
details of methods). Table 11 lists the various k1 and k2 values that
were calculated from these runs. Some difficulty was experienced in
obtaining good linear plots with the colorimetric method; this was
probably due to slight variations in the color development procedure since
it was impossible to develop the color in all of the samples at the same
time. The polarimetric method was much better since it involved the direct
measurement of a physical property of the reaction solution.

Comparison of the k2 values for the acid-catalyzed hydrolysis of
these two galactosides shows that the -SCH3 substituted (at the 6
position of the galactosyl) hydrolyzes at a rate greater than the -OCH3
substituted derivative. This would support the results observed with
the 1,2-O-isopropy1idene glucose derivatives and again suggest the

anchimeric participation of the -SCH3 group as shown below.

CH -—S-CH
CHz-S-CH3 2 + 3
O H + 11 O
H H + HOCH
OH —‘*' OH 3

O O

32

.menHHchoH Housuos mo msuou :H commoumxo mum mosHm> omoeH +

 

. L oonopoonOIQlo

I com o OB. . com I . oHH oaHHo o

H H- H H x e-oH x_:a m H- x e oH x co m .oe . H . H H IHHHeHmz n eVIHHaHm:
oonouomHmw

H-.eaa H-aHos.H x e-oH x we.e H-.uea x e-oH x Aw.e aoe oHuHmaHneHom leis-HHoHeHmaenHmz

telaxomcleleHnHaz

. monOHomHmUIQLc
.Umm 0 CE. . .Umm l Oak GSHHO O .
H- H- H H x e-oH x mm H H- x m OH x o“ m .mm . H . H o IHHHeHaz-o-eolHanHez

oonouomHmo
H-.omm H-mHOE.H x eloH x ::.H H-.omw x nuoH X Ho.o amw oHuumEHHoHoo .IQJOIAHoanocmnwmz
lonesome-eVIHHeHez

 

Has HHH .aame vogue: ecsoaaoo

 

moowmouomamwlololAHoHnuocmnuoz
lolhxooQIonHxnuoz ram AHmaquIOIoVIHmnHoE mo mHmzHouozm oonzamumolowo< mo moumm .HH mqm<H

33

C. Rate of Hydrolysis of 4(5)-(yj0xobuty1)flmidazole Diethyl Ketal

The direct spectrophotometric method (see experimental section) was
used to determine the rate of hydrolysis of this ketal. The ketal was
dissolved in peroxide-free dioxane and an equal amount of the appropriate
buffer (preparation of these buffers was described in the experimental
section) used for the acid catalyst. The hydrogen ion concentration was
calculated using the ionization constant for acetic acid in 50 percent
aqueous dioxane at 25° interpolated from the data of Harned and Owen (59).
Harned and Owen reported ionization constants for acetic acid as
4.93 X 10'7 in 45 percent dioxane at 25° and 4.78 X 10'9 in 70 percent
dioxane at 25°. Interpolation gives a value of 2.63 x 10'7 in 50 percent
dioxane at 25°.

The observed second order constants for the hydrolysis of 4(5)-

(y~oxobutyl)-imidazole diethyl ketal lie within the allowable experimental

error and are approximately 100 times as great as the second order constants
for the diethyl ketal of methyl ethyl ketone (1). This data strongly
indicates anchimeric assistance in this hydrolysis reaction by nitrogen of
the imidazole ring, and it demonstrates_the possibility of nucleophilic
assistance by imidazole (of histidine residues) in glycosidase—catalyzed

hydrolysis reactions.

-C2H5

-CH2-CH2 -CH3 If... 2 —CH3
HC//$ -CaH5 H2 i'x
\\c/ _H

\H

34

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“coupon an :H oHoo oHuooo mo acoumaoo :oHuoanoH ozu.wcwoo moawauouov on»: ocoHuowusoocoo_:oH +m any .0

.aaauwuoon Hogans: no canon :H commounwo one mosHo> soapy .o
.occHqucm.coHuooou HocHu on» :H meoHuoHucoocco any one omega .n

.coHuooo Hauaoawuomxo onu :H oonHuouoo ow enemas: omocu mo :oHuouonoum use .c,

 

 

H-.aaa a-oHoa.H e-oH x ~a.n a-.eua a-oH x_:H.m z_aN~H.o z_omam.o N .oz Haaeu
HanaeHc «HoaaeHaH
v.59. a....eHe_a.H 12 x 3.... a-daa e-oH x 3H 2 $26 2 33.0 H 62 123330-313:
m H :o o o oo HO 0H 00. no : unease
e x a 3 pH H a a <8 aHe. < .H <8 a mu m e o

 

Hausa Hanuown oHowooHaHIAHmuonOXOLrvIAmv: mo onHHouohm oouhHouooloHo< mo mama .HHH mqm<H

35

D. General Comments

 

Although the evidence for anchimeric assistance by potential
nucleophiles in the hydrolysis of acetals, ketals, and glycosides is not
conclusive, it strongly suggests such a possibility. Both the sulfur of
-SCH3 and the imidazole nitrogen appear to participate in this manner. Of
these nucleophiles, the nitrogen of the imidazole ring appears to have the
greatest potential as a participant in enzymatic hydrolysis by glycosidases.
The imidazole immonium ion may also be involved in the transfer reactions
of these enzymes; the transferred moiety being an imidazole immonium ion or,
possibly, a sulfonium ion.

One aspect of acetal hydrolysis that has not been examined is the
problem of electron-withdrawing groups located near the carbonyl carbon
atom. In the glycosides, the -OH at carbon 2 of the glycosyl moiety results
in a greatly increased stability. For example, the hydrolysis of 2-
methoxytetrahydropyran proceeds rapidly with dilute acids at room
temperature (60) while the pentopyranosides require elevated temperatures.
During enzymatic catalysis, the enzyme must be able to overcome the effect
of such electron-withdrawing groups on carbon 2 of the glycosyl moiety;

possibly by means of hydrogen bonding.

IV. SUMMARY

1. A number of acetal and ketal models for the glycoside-glycosidase
system were prepared. These included 1,2-O-isopropy1idene-a—D-glucose,
6-O-methyl—l,2-O-isopropylidene-o~D-glucose, 6—deoxy—6-methanethiol-l,2-0-
isopropylidene-orD-glucose, methyl-(6-O-methyl)-a-D-galactoside, methyl-
(6-deoxy—6-methanethiol)—o—D—galactoside, and 4(5)—(y—oxobutyl)-imidazole
diethyl ketal.

2. The acid-catalyzed rates of hydrolysis of the above compounds were
determined using a number of methods including direct spectrophotometric,
polarimetric, and colorimetric methods.

3. The kinetic data obtained suggest that some form of anchimeric
assistance by nucleophiles does take place in these acid-catalyzed
hydrolysis reactions. Both the sulfur of the -SCH3 group and the imidazole
nitrogen appear to participate in this manner; acetal and ketal derivatives

containing these groups have increased hydrolysis rates.
4. It seems that enzymatic hydrolysis of glycosides may involve a

similar participation of a nucleophile; positioned by the enzyme so as

to stabilize the intermediate carbonium ion and thus increase the rate of
hydrolysis. This "onium" ion may be the species involved in the enzymatic
transfer reactions of these enzymes; the transferred moiety being an

immonium or sulfonium ion.

36

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REFERENCES

M. M. Kreevoy and R. W. Taft, J. Am. Chem. Soc. 11, 3146 (1955); J. Am.
Chem. Soc. 11, 5590-5 (1955).

M. M. Kreevoy, C. R. Morgan, and R. W. Taft, J. Am. Chem. Soc. Z6,
4236-9 (1956).

J. C. Speck, Jr., personal communication.
E. Fitzgerald and A. Lapworth, Proc. Chem. Soc. 24, 153 (1908).

(a)' A. Skrabal and A. Schiffier, Z. physik. Chem. 22, 290-313 (1921),
(b) A. Skrabal and K. H. Mirth, Z. physik. Chem. 111, 98-108 (1924),
(c) A. Skrabal and I. Sawivk, Z. physik. Chem. 122, 357-70 (1926).

A. Skrabal and M. Slatewa, Z. physik. Chem. 119, 305-18 (1926).

A. Skrabal, Ber. 72B, 446 (1939).

P. H. Hermans, Ber. 57B, 824-7 (1924).

M. H. Palomaa and V. Aalto, Ber. 66B, 468-72 (1933).

E. R. Alexander, H. M. Bush, and G. L. Webster, J. Am. Chem. Soc. 15,
3173 (1952).

J. M. O‘Gorman and H. J. Lucas, J. Am. Chem. Soc. 12, 5489 (1950).
H. K. Garner and H. J. Lucas, J. Am. Chem. Soc. 12, 5497 (1950).
R. Leutner, Monatsh. 66, 317-52 (1932); Monatsh. 66, 222-48 (1935).

L. P. Hammett, ”Physical Organic Chemistry," McGraw-Hill Book
Company, New York, N. Y., 1940, p. 304.

D. McIntyre and F. A. Long, J. Am. Chem. Soc. 16, 3240 (1954);
F. A. Long and D. McIntyre, J. Am. Chem. Soc. Z6, 3243 (1954).

L. P. Hammett and L. Zucker, J. Am. Chem. Soc. 61, 2791 (1939).
M. M. Kreevoy, J. Am. Chem. Soc. Z6, 4236-9 (1956).

W. J. C. Orr and J. A. V. Butler, J. Chem. Soc., 330 (1937).

M. Kilpatrick, J. Am. Chem. Soc. 66, 1036-8 (1963).

D. E. Koshland, Jr., in "The Mechanism of Enzyme Action,” W. D.

McElroy and G. Blass, eds., Johns Hopkins Press, Baltimore, 1954,
p. 608.

37

(21)

(22)

(23)
(24)
(25)
(26)

(27)

(28)
(29)
(30)

(31)

(32)

(33)

(34)

(35)
(36)

(37)

(38)

(39)

(40)

38

R. A. Dedonder, Ann. Rev. Biochem. 66, 370 (1961).

M. Doudoroff, S. A. Barker, and W. Z. Hassid, J. Biol. Chem. 168,
725 (1947).

M. A, Jermyn, Science 126, 12 (1957).

C. S. Stringer and H. M. Tsuchiya, J. Am. Chem. Soc. 66, 6620 (1958).
F. C. Mayer and J. Larner, J. Am. Chem. Soc. 61, 188-93 (1959).

J. A. Thoma and D. E. Koshland, Jr., J. Biol. Chem. 262, 2511 (1960).
D. E. Koshland, Jr., in "The Enzymes," P. D. Boyer, H. Lardy, and
ngMyrback, eds., Academic Press, New York, N. Y., 2nd ed., V61. 1,
p; 305; J. Am. Chem. Soc. 62, 3329 (1960).

‘M. J. Cohn, J. Biol. Chem. 266, 771 (1949).

D. E. Koshland, Jr., and S. S. Stein, J. Biol. Chem. 266, 139 (1954).

F. C. Mayer and J. Larner, Biochim at Biophys. Acta 22, 465 (1958).

C. Armour, C. A. Bunton, S. Patai, L. H. Selmand, and C. A. Vernon,
J. Chem. Soc., 412 (1961).

C. A. Bunton, T. A. Lewis, D. R. Llewellyn, and C. A. Vernon, J.
Chem. Soc., 4419 (1955).

P. A. Levene, A. L. Raymond, and R. T. Dillon, J. Biol. Chem. 22,
699 (1932); D. F. Mowery,.Jr., J. Am. Chem. Soc. 11, 1667 (1955).

E. M. Montgomery, R. M. Hann, and C. S. Hudson, J. Am. Chem. Soc. 62,
1124 (1937).

R. A. Dedonder, Ann. Rev. Biochem. 66, 370 (1961).
J. D. Duerkson and J. Halvorson, J. Biol. Chem. 233, 1113 (1958).

K. Wallenfels, "Colloque international sur la biochimie des glucida
(Gif-sur-YVette, 1960)," Bull. soc. chim. bio. 62, 1715 (1960).

J. P. Riehm, Doctoral Thesis, Michigan State University (1961).
K. wallenfels and 0. P. Malhotra, in "The Enzymes," P. D. Boyer,
H. Lardy, and K. Myrback, eds., Academic Press, New'YOrk, N. Y.,
2nd ed., Vol. 4, p. 409.

J. A. Thoma and D. E. Koshland, Federation Proc. 12, 46 (1960).

(41)

(42)

(43)

(44)

(45)
(46)
(47)

(48)

(49)

(50)

(51)
(52)

(53)

(54)

(55)

(56)

(57)

39

W. B. Neely, Arch. Biochem. Biophys.._z2, 297 (1958); J. Am. Chem.
Soc. 66, 2010 (1958).

C. L. Mehltretter, B. H. Alexander, R. L. Mellies, and C. E. Rist,
J. Am. Chan. Soc. 16, 2424 (1951).

O. T. Schmidt, "Methods in Carbohydrate Chemistry," R. L. Whistler
and M; L. Wolfrom, eds., Academic Press, New York, N. Y., 1963,
Vol. II,.p. 322.

R. S. Tipson, "Methods in Carbohydrate Chemistry," R. L. Whistler
and M. L. Welfrom, eds., Acadanic Press, New York, N. Y., 1963,
Vol. II, p. 248.

H. Ohle and K. Spencker, Ber. 62, 1836 (1926).

A. L. Raymond,-J. Biol. Chem. 107, 85-96 (1934).

P. A. Levene and A. L. Raymond, J. Biol. Chem. 21, 751 (1932).

o. T. Schmidt, "Methods in Carbohydrate Chemistry," R. L. Whistler
and M. L. Walfrom, eds., Academic Press, New York, N. Y., 1963,
Vol. II, p. 324.

A. B. Foster, W. G. Overend, M. Stacey, and L. F. Wiggins, J. Chem.
Soc., 2542 (1949).

I. J. Goldstein, J. K. Hamilton, and F. Smith, J. Am. Chem. Soc. 22,
1190 (1957).

J. R. Totter and W. J. Darby, Org. Syn. 26, 64 (1944).

A. H. Mehler, H. Tabor, and H. Bauer, J. Biol. Chem. 197, 475 (1952).

C. 8. Marvel and F. D. Hagar, in "Organic Syntheses," A. H. Blatt, ed.,
John Wiley and Sons, Inc., New York, N. Y., 1941, 2nd ed., Coll. Vol. I,
p. 248.

J. R. Johnson and F. D. Hagar, in "Organic Syntheses," A. H. Blatt, ed.,
John Wiley and Sons, Inc., New York, N. Y., 1941, 2nd ed., Coll. Vol.

I, p. 351.

F. L. Pyman, J. Chem. Soc. 22, 2176 (1911).

Rene Evard, Doctoral Thesis, Michigan State University (1959)..

E. A. Moelwyn-Hughes, Trans. Faraday Soc. 22, 503 (1929); Trans.
Faraday Sec. g, 81 (1929).

(58) H. S. Isbell and H. L. Frush, J. Research Natl. Bur. Standards 26,
125 (1940).

(59) H. S. Harned and B. B. Owen, "The Physical Chemistry of Electrolytic
Solutions," Reinhold Publishing Corp., New York, N. Y., 1950, p. 581.

(60) R. Paul, Compt. rend. 197, 1652 (1933).

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