Esterase-catalysed regioselective 6-deacylation of hexopyranose per-acetates, acid-catalysed rearrangement to the 4-deprotected products and conversions of these into hexose 4- and 6-sulfates

Article abstract of DOI:10.1039/a708596f

The esterase from Rhodosporidium toruloides has been used to catalyse the hydrolysis of a series of per-acetylated α-D-hexopyranoses and α-D-hexopyranosides. Per-acetylated glucose 4, mannose 6, N-acetylgalactosamine 8, galactose 10, methyl α-D-glucoside 12, methyl α-D-mannoside 14 and methyl α-D-galactoside 16 have been selectively cleaved at the C-6 position by the esterase to give the 6-OH derivatives 5, 7, 9, 11, 13, 15 and 17. Acid-catalysed rearrangement of acetates 5, 7, 13, 15, 11, 17 and 9 with 4→6 acetyl migration gives the corresponding 4-deprotected derivatives 22-28, respectively. Hydrolyses of β-D-glucose pentaacetate 20 and α-D-lactose octaacetate 21 have been attempted, but no hydrolyses have been observed. 1,2,3,6-TetraacyIated α-D-hexopyranoses 3 and 22, derivatives of N-acetylglucosamine and glucose respectively, and 2,3,6-triacetylated α-D-hexopyranosides 24 and 25, derivatives of glucose and mannose, respectively, have been hydrolysed by the esterase to the corresponding 4,6-dihydroxy acetates 29, 18, 30 and 31. Acylation of methyl α-D-glucopyranoside 32 catalysed by the esterase provides the C-6 monoacetate 33 and the C-3 monoacetate 34 in 4 and 5% yield, respectively. The sodium salts of N-acetylglucosamine, glucose, N-acetylgalactosamine, galactose and mannose 6-sulfates 38-42, respectively, are prepared in two steps from the 6-deacetylated hexopyranoses 2, 5, 9, 11 and 7, respectively. The sodium salts of N-acetylglucosamine, glucose and mannose 4-sulfates 43-45, respectively, are prepared in two steps from the 4-deacetylated precursors 3, 22 and 26 which are obtained via acid catalysed 4→6 acyl migration of compounds 2, 5 and 7.

Full text of DOI:10.1039/a708596f

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Esterase-catalysed regioselective 6-deacylation of hexopyranose
per-acetates, acid-catalysed rearrangement to the 4-deprotected
products and conversions of these into hexose 4- and 6-sulfates
Tina Horrobin, Chuong Hao Tran and David Crout*
Department of Chemistry, University of Warwick, Coventry, UK CV4 7AL
The esterase from Rhodosporidium toruloides has been used to catalyse the hydrolysis of a series of
per-acetylated á-D-hexopyranoses and á-D-hexopyranosides. Per-acetylated glucose 4, mannose 6,
N-acetylgalactosamine 8, galactose 10, methyl á-D-glucoside 12, methyl á-D-mannoside 14 and methyl
á-D-galactoside 16 have been selectively cleaved at the C-6 position by the esterase to give the 6-OH
derivatives 5, 7, 9, 11, 13, 15 and 17. Acid-catalysed rearrangement of acetates 5, 7, 13, 15, 11, 17 and 9 with
4→6 acetyl migration gives the corresponding 4-deprotected derivatives 22–28, respectively. Hydrolyses of
â-D-glucose pentaacetate 20 and á-D-lactose octaacetate 21 have been attempted, but no hydrolyses have
been observed. 1,2,3,6-Tetraacylated á-D-hexopyranoses 3 and 22, derivatives of N-acetylglucosamine and
glucose respectively, and 2,3,6-triacetylated á-D-hexopyranosides 24 and 25, derivatives of glucose and
mannose, respectively, have been hydrolysed by the esterase to the corresponding 4,6-dihydroxy acetates
29, 18, 30 and 31. Acylation of methyl á-D-glucopyranoside 32 catalysed by the esterase provides the C-6
monoacetate 33 and the C-3 monoacetate 34 in 4 and 5% yield, respectively. The sodium salts of N-
acetylglucosamine, glucose, N-acetylgalactosamine, galactose and mannose 6-sulfates 38–42, respectively,
are prepared in two steps from the 6-deacetylated hexopyranoses 2, 5, 9, 11 and 7, respectively. The sodium
salts of N-acetylglucosamine, glucose and mannose 4-sulfates 43–45, respectively, are prepared in two steps
from the 4-deacetylated precursors 3, 22 and 26 which are obtained via acid catalysed 4→6 acyl migration
of compounds 2, 5 and 7.
series of 4-OH, 6-OH and 4,6-(OH)2 derivatives of acetylated
Introduction
α--hexopyranoses using the crude esterase from R. toruloides,
and acetylation of methyl α--glucopyranoside catalysed by
the enzyme. Also reported is the conversion of the 4- and
6-deprotected species into the corresponding hexose
monosulfates.
A major problem that faces the carbohydrate chemist in the
construction of oligosaccharides is regioselectivity. A regio-
selective synthesis of an oligosaccharide ultimately necessitates
the initial preparation of carbohydrate units with the required
degree of protection. Strategies for the synthesis of partially
protected mono- and oligo-saccharides using traditional chem-
ical techniques have been developed. However, this approach
is hindered by the requirement for multiple protection/
deprotection steps. Alternatives have relied upon methods such
as the partial deacylation of peracylated sugars. However, this
approach is restricted by the position of deacylation and prob-
lems of poor selectivity. In contrast, enzymic techniques have
been used successfully in the selective deacylation of per-
acylated sugars and hence have provided an effective method of
manipulating protecting-group strategies in carbohydrate syn-
thesis. Research involving enzyme-catalysed hydrolysis of per-
acylated sugars has provided an effective method of removing
one or more acyl groups.1 Most of this research has resulted in
the selective cleavage of the anomeric ester in preference to the
cleavage of other primary and secondary esters. However, when
a sugar is derivatized at the anomeric centre as, for example,
with methyl glycosides, cleavage of the primary ester group was
commonly observed. Previously we reported the regioselective
deacetylation of 2-acetamido-2-deoxy-1,3,4,6-tetra-O-acetyl-α-
-glucopyranose 1 to give the 6-OH derivative 2 using the crude
esterase from Rhodosporidium toruloides.2 The 6-OH derivative
2 was subsequently converted to the corresponding 4-OH
derivative 3 via a 4→6 acetyl migration. It was envisaged that the
combined use of chemical and enzymic techniques would pro-
vide a simple general route to the preparation of 6-OH and 4-
OH derivatives of acetylated monosaccharides, which could be
further used in the field of carbohydrate chemistry. Accordingly
we have extended this study to glucose, galactose, N-
acetylgalactosamine and mannose. In this paper is reported
the use of the chemoenzymic method in the preparation of a
Results and discussion
Eight α--peracetylated monosaccharides and derivatives were
subjected to the esterase-catalysed hydrolysis (Scheme 1). The
hydrolyses were conducted by suspending the substrate and the
esterase in citrate buffer (pH 5.0). The mixture was stirred at
30 ЊC and the reaction was monitored by thin-layer chroma-
tography (TLC). Once the starting material was consumed, the
enzyme was removed by filtration. The filtrate was then purified
by conventional solvent/solvent extraction followed by column
chromatography or crystallization. The results are summarized
in Scheme 1b–h and Table 1. Per-acetylated α--glucose 4, α--
mannose 6, N-acetyl α--galactosamine 8 and α--galactose
10, gave the corresponding 6-hydroxy derivatives 5, 7, 9 and 11,
respectively, in good to excellent yield. The α--glycoside ace-
tates 12, 14 and 16 were converted similarly into the 6-hydroxy
derivatives 13, 15 and 17, respectively. The site of the deacetyl-
ation was established by comparing the 1H NMR spectra of the
C-6 deacetylated products with those of the starting materials.
1
In the C-6 deprotected sugars, C-6 protons in the H NMR
spectra are shifted upfield owing to the loss of the deshielding
effect of the acetate groups. Hydrolyses of per-acetylated glu-
cose 4 and N-acetylgalactosamine 8 gave the C-6 deprotected
products 5 and 9 and small amounts of the dihydroxy deriva-
tives 18 and 19 respectively. It was assumed that the dihydroxy
derivatives were formed by a 4→6 acyl migration, followed
by hydrolysis. The hydrolysis of 1,2,3,4,6-penta-O-acetyl-β--
glucopyranose 20 was also investigated. However, no hydrolysis
was observed. Extending the incubation period of the reaction
J. Chem. Soc., Perkin Trans. 1, 1998
1069

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a
Table 1 Rhodosporidium toruloides esterase-catalysed hydrolysis of
(1),(2),3,4,6-(tetra)penta-O-acetyl-α--hexopyranoses
OAc
O
OH
OAc
O
O
AcO
AcO
AcO
HO
AcO
ii
i
Per-acetylated sugar (mmol)
Product (% yield)
AcO
AcNH
AcNH
AcNH
5 (54)a
7 (88)
OAc
OAc
OAc
4 (85.38)
6 (7.69)
1
2
3
8 (0.95)
9 (73)b
11 (67)
13 (77)
15 (70)
17 (85)
10 (5.49)
12 (22.60)
14 (4.12)
16 (5.66)
b
OH
OAc
O
OH
O
HO
AcO
AcO
O
AcO
AcO
i
+
AcO
AcO
AcO
AcO
a 4,6-(OH)2 product 18 was isolated in 1% yield. b 4,6-(OH)2 product
19 was isolated in 6% yield.
OAc
OAc
OAc
OH
4
5
18
Table 2 Conversion of the C-6 deprotected monosaccharides into the
C-4 deprotected monosaccharides via acid-catalysed acyl migration
c
OAc
OAc
OAc
i
O
O
AcO
AcO
AcO
AcO
Substrate (mmol)
Time (t/h)
Product
Yield (%)
OAc
OAc
5 (11.90)
7 (3.59)
9 (39.20)
11 (1.45)
13 (0.99)
15 (0.96)
17 (2.55)
16
44
16
16
16
16
16
22
60
92
7
6
23
28 and 9 (1:1)
d
26
24
25
27
OH
OH
63
49
AcO
AcO
OAc
O
HO
AcO
AcO
AcO
i
O
O
+
AcNH
AcNH
AcNH
OAc
OAc
OAc
8
9
19
in tetrahydrofuran (THF)–acetone (1:1, v/v), was added to
the buffer containing the enzyme. The reaction mixture was
maintained at 30 ЊC and was monitored by TLC. However, the
reaction was still unsuccessful. Upon work-up only starting
material was isolated.
e
AcO
AcO
OAc
OH
AcO
AcO
O
O
i
Within the field of carbohydrate chemistry there is consider-
able interest in the development of simple approaches for the
preparation of C-4 unprotected monosaccharides which could
be useful building blocks for the preparation of higher oligo-
saccharides containing the 1→4 linkage. It is well documented
that acyl groups have the propensity to migrate under both
acidic and basic conditions.4 Albert et al. previously reported
that the use of an O-4-to-O-6 acetyl migration in partially
acetylated hexopyranosides had provided a generally applicable
method for the regiospecific deprotection of hydroxy groups at
C-4.5 Having prepared a series of C-6 deprotected mono-
saccharides, we investigated the possibility of using a 4→6 acyl
migration to convert the C-6 deprotected monosaccharides into
their corresponding C-4 deprotected monosaccharides. The
migration was carried out as follows: the C-6 deprotected sugar
was dissolved in toluene, acetic acid (1%, v/v) was added and
the solution was heated to 80 ЊC for various periods depending
on the substrate. The products were purified by crystallization
or by flash chromatography. The results are illustrated in
Scheme 2 and Table 2. Acetates 5, 7, 13 and 15 (derivatives,
respectively, of α--glucose, α--mannose, methyl α--
glucoside and methyl α--mannoside) were successfully con-
verted into the respective 4-OH derivatives 22, 23, 24 and 25.
AcO
AcO
OAc
OAc
10
11
f
OAc
O
OH
AcO
AcO
O
AcO
AcO
i
i
AcO
AcO
OMe
OMe
12
13
g
OAc
OAc
OH
OAc
O
AcO
AcO
O
AcO
AcO
OMe
OMe
14
15
h
AcO
AcO
OAc
O
OH
AcO
AcO
O
i
AcO
AcO
OMe
OMe
16
17
1
The products were characterized by H and 13C NMR spec-
Scheme 1 Reagents: i, Esterase from R. toruloides; ii, HOAc
1
troscopy. In the above examples the H NMR spectra of the
products showed an upfield shift of the H-4 signal accompanied
by a corresponding downfield shift of the H-6 signal. The
remaining H signals were virtually unaffected. This indicated
OAc
AcO
AcO
OAc
OAc
O
O
1
O
AcO
AcO
O
OAc
OAc
OAc
AcO
that acyl migration from O-4 to O-6 had occurred. Conversion
of the galactosides 11 and 17 into the corresponding 4-OH
derivatives 26 and 27 was achieved. However, the products
could not be purified because further acyl migration occurred
giving complex mixtures of products. Conversion of acetate 9
into the corresponding 4-OH isomer 28 also was problem-
atical. Acetate 9 was found to be insoluble in toluene and hence
no reaction took place. Dimethylformamide (DMF) was used
as a co-solvent to improve the solubility. Thus, compound 9 was
initially dissolved in DMF, toluene and acetic acid were added,
and the reaction mixture was heated to 80 ЊC. After 16 h the
AcO
OAc
20
21
did not result in hydrolysis of the substrate and upon work-up
only starting material was isolated. The result suggested that
the β-isomer was not a suitable substrate for the enzyme.
The enzyme also failed to catalyse hydrolysis of α--lactose
octaacetate 21. One potential problem in this experiment was
the insolubility of α-lactose octaacetate in the citrate phosphate
buffer used. To overcome this problem, the solvent system
described by Khan et al. was used.3 The substrate, dissolved
1070
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a
a
OH
OAc
O
O
HO
AcO
i
HO
AcO
i
i
3
5
7
AcNH
AcO
OAc
OAc
29
22
b
c
b
OH
O
OAc
OAc
HO
AcO
i
i
O
HO
AcO
22
AcO
OAc
OMe
OAc
18
23
c
OH
O
OAc
O
HO
AcO
HO
AcO
i
24
13
15
AcO
AcO
OMe
30
24
d
OAc
OAc
d
OH
i
O
OAc
HO
AcO
O
HO
AcO
i
25
OMe
OMe
25
31
e
OAc
O
HO
AcO
Scheme 3 Reagents: i, Esterase from R. toruloides
i
11
17
9
Table 3 Rhodosporidium toruloides esterase-catalysed hydrolysis of
(1),(2),3,6-(tri)tetra-O-acetyl-α--hexopyranoses
AcO
OAc
26
Substrate (mmol)
Product
Yield (%)
3 (1.44)
22 (1.21)
24 (1.51)
25 (3.19)
29
18
30
31
82
72
35
60
f
OAc
O
HO
AcO
i
AcO
OMe
27
The preferential acylation of primary over secondary
hydroxy groups rarely can be achieved efficiently with free
sugars. Recently, enzymic transformations have been used to
effect specific modification of carbohydrates. In contrast to
chemical techniques the regioselective acylation of sugars by
enzyme-catalysed transesterification using activated esters has
provided an efficient method of preparing monoacylated
sugars.6 Having successfully used the R. toruloides esterase for
the regioselective deacylation of per-acylated monosaccharides,
the possibility was investigated of using this enzyme for the
selective acylation of methyl α--glucopyranoside 32. The
esterase-catalysed acylation was carried out with vinyl acetate
g
OAc
O
HO
AcO
i
9
+
AcNH
OAc
28
Scheme 2 Reagents and conditions: i, HOAc, toluene, 80 ЊC
1H NMR spectrum showed that there was a ~1:1 mixture of
isomers 9 and 28. The mixture was heated at 80 ЊC for 3 days.
The 1H NMR spectrum still showed a 1:1 mixture of the 4-OH
and 6-OH products. Isolation of the required 4-OH species 28
was not achieved.
in
Scheme 4, the reaction yielded two monoacylated glucosides 33
a
THF–triethylamine solvent system.6i As shown in
OH
OAc
OH
Partially protected monosaccharides can be manipulated in a
variety of ways with a view to producing useful building blocks
for the syntheses of higher oligosaccharides. One potential
modification which was of interest was the possibility of
employing the Rhodosporidium esterase for the subsequent
removal of further acyl moieties. Thus, esterase-catalysed
hydrolyses of 4-OH acetates 3, 22, 24 and 25 (derivatives respec-
tively of N-acetylglucosamine, glucose, glucose and mannose)
were examined. The results are shown in Scheme 3 and in
Table 3. In all cases, the esterase hydrolysed the C-6 acetoxy
group of the 4-hydroxy compound to give the corresponding
4,6-dihydroxy derivatives 29, 18, 30 and 31. The position of
cleavage was determined by comparison of the 1H NMR spectra
of the products with those of their respective starting materials.
O
O
O
HO
AcO
HO
HO
HO
HO
+
HO
HO
HO
OMe
OMe
OMe
32
33
4%
34
5%
Scheme 4 Reagents and conditions: R. toruloides, vinyl acetate, THF,
Et3N, 25 ЊC
and 34 in 4 and 5% yield, respectively. Increasing the incubation
period of the reaction did not improve yields. The chemical
preparation of monoacylated glucosides 33 and 34, as well as
requiring multistep procedures, is complicated by the formation
of mixtures of products, requiring complex purification tech-
niques, ultimately lowering the yields of the required products.7
In contrast, the esterase-catalysed acylation of glucosides 33
and 34 was achieved in one step, in low yields, but without
optimization.
1
The H NMR spectrum of each product showed a distinctive
upfield shift of the H-6 signals, confirming that in all cases
cleavage of the primary acetate had been achieved.
J. Chem. Soc., Perkin Trans. 1, 1998
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The ratios of the products of 4→6 acyl migration under the
conditions reported in this study are under thermodynamic
control. The fact that product ratios of 4- and 6-deprotected
species are <100:1 indicated that the free energies of the iso-
mers differ by <3 kcal molϪ1.† Energy minimization of the
isomers using the programme PCMODEL indicated that they
differed by <2 kcal molϪ1. This is the best result that could be
expected at the MMX level and indicates that although it is not
possible to explain the relative stabilities of the isomers in a
qualitative way, the calculations give results that within the
accuracy of the method are consistent with the experimental
observations.
It was surprising that 4→6 acyl migration was not accom-
panied by some 3→4 acyl migration. No evidence of 3-
deprotected species in the product mixture was found. Mini-
mization of the 3-deprotected species in the glucose series
showed that it had a free energy within 2 kcal molϪ1 of the
energies of the 4- and 6-deprotected species. However, the
minimization energies of the oxonium ion intermediates gener-
ated during acyl migration (Scheme 5a,b) are very different.
Fig. 1 Energy-minimized structures of the oxonium ion intermediates
35 and 36 (Scheme 5) in the acid-catalysed rearrangement of 6-
deprotected acetates 5 and 22, respectively
intermediate 36. Accordingly the calculated difference in the
differences in potential energy between substrates 5 and 22 and
corresponding intermediates 35 and 36 for 4→6 and 3→4 acyl
migration, respectively, was 6.2 kcal molϪ1. If the oxonium ion
species 35 and 36 are assumed to be reasonable approximations
to the transition states for the rate-determining steps in the
corresponding acyl migrations (Hammond postulate), the value
of 6.2 kcal molϪ1 can also be taken as a reasonable approxima-
tion to the difference in activation energies for the two
rearrangements. (The free energy of activation is assumed to be
dominated by the enthalpy term; the entropic contribution is
assumed to be approximately the same for the two reactions,
since in both cases the motions of one hydroxy group and one
acetoxy group are frozen in the transition state.) From the rela-
tionship k1/k2 = exp(∆∆G‡/RT), where k1 and k2 are the rate
constants for 1→6 and 1→4 acyl migrations, respectively, and
∆∆G‡ is the difference in activation energies for the two migra-
tions, it follows that at 80 ЊC, the temperature at which the acyl
migrations were carried out, k1/k2 = 2.7 × 104. Although this
figure can only be regarded as approximate, it does indicate that
3→4 acyl migration should be considerably slower than 4→6
migration. It is probable, therefore, that under the conditions of
the rearrangement used in this work, 3→4 acyl migration was
under kinetic control and was too slow to give observable
amounts of 3-deprotected species in the product mixture.
With the 4- and 6-deprotected acetates in hand, attention was
turned to the conversions of these into the corresponding sul-
fates, since this would provide an efficient route to the latter
class of compound. Sulfated carbohydrates are widely distrib-
uted in Nature. They occur in glycoprotein proteins bearing
sulfated asparagine-linked oligosaccharides,8 glycoprotein
hormones,9 brain and nerve tissue,10 glycoproteins of several
enveloped viruses,11 lymphocyte proteoglycans and glycos-
aminoglycans,12 tumour cells13 and selectin-binding counter
receptors such as SLex and ICAM-1.14 They are also known
to possess anti-viral activity,15 inhibit natural cell-mediated
cytotoxicity against K-562 target cells16 and may be involved in
the inhibition of cell migration during cancer metastasis.17
Synthesis of sulfated monosaccharides have attracted the
interest of chemists over the last few decades.18 They have
usually been prepared by reaction of free sugars with sulfating
reagents such as chlorosulfonic acid19 or complexes formed by
sulfur trioxide with pyridine20 or DMF.21 However, these reac-
tions are not regiospecific and present the usual difficulties in
the preparation of single isomers. They usually yield a mixture
of products although these are dominated by the 6-sulfate.
Separation techniques such as column chromatography using
cellulose powder,22 ion-exchange chromatography,23 electro-
phoresis24 and recrystallization of the brucine salt25 have been
used for resolving the isomers obtained by direct sulfation.
However, these techniques are rather laborious and time con-
suming. For the synthesis of a single isomer with the sulfate
group at a predetermined position, selective protection is
required to mask hydroxy functions at all positions except the
one to be sulfated. Chemical synthesis of hexoses with the
a
HO
HO
+O
H+
AcO
AcO
O
O
H
AcO
AcO
AcO
OAc
OAc
5
HO
H
OAc
O
O+
O
HO
AcO
O
AcO
AcO
AcO
OAc
OAc
22
35
b
OAc
O
AcO
H+
HO
AcO
O
HO
O
AcO
AcO
OAc
OAc
O+
22
H
OAc
O
OAc
O
H
O+
AcO
HO
HO
O
AcO
AcO
OAc
OAc
37
36
Scheme 5
The presumed oxonium ion intermediate in 4→6 acyl migration
(35, Scheme 5a) has a six-membered ring which can adopt a
chair conformation. Energy minimization at the MMX level
showed that the epimer shown, with the methyl group attached
to the oxonium carbon atom in the equatorial position, had the
lowest energy. This is shown in the Chem 3D rendering of the
structure minimised using PCMODEL (Fig. 1a). This structure
displays a hydrogen bond between the atoms indicated by
arrows. The minimum-energy conformation found for the cor-
responding intermediate (36, Scheme 5b) for 3→4 acyl migra-
tion contains a more highly strained five-membered ring and
also has an intramolecular hydrogen bond (arrows in Fig. 1b).
The calculated energy difference between the 6-OH and 4-OH
derivatives 5 and 22, was 1.9 kcal molϪ1, the 6-OH compound 5
being the more stable. Similarly the calculated energy for inter-
mediate 35 was 4.3 kcal molϪ1 lower than that calculated for
† 1 cal = 4.184 J.
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Table
4
Sulfation of (1),(2),3,(4)-(tri)tetra-O-acetyl-α--hexo-
required degree of protection is an ardous task. However, prep-
aration of the C-6 and C-4 deprotected monosaccharides
prompted us to investigate their conversion into corresponding
hexose 6- and 4-sulfates.
pyranoses
Starting material (mmol)
Reaction time (h)
Product (% yield)
The sulfur trioxide–pyridine complex was used as the sulfat-
ing reagent. Sulfation of 22 using sulfur trioxide–pyridine com-
plex as the sulfating reagent and DMF as the solvent was
attempted. Surprisingly, no reaction took place after heating at
90 ЊC for two days. When the same reaction was conducted in
pyridine, the reaction was completed in a few hours at room
temp. Accordingly, the sulfations were carried out by treating
sulfur trioxide–pyridine complex with the hexose derivatives 2,
5, 9, 11, 7, 3, 22 and 25 (Scheme 6) in anhydrous pyridine. The
2 (0.91)
5 (1.17)
9 (0.29)
11 (0.58)
7 (2.44)
3 (1.12)
22 (0.37)
23 (0.23)
5
1.5
2
2
2
2
2
7
38 (63)
39 (74)
40 (93)
41 (94)
42 (89)
43 (95)
44 (98)
45 (93)
Table 5 Deacetylation of the protected hexopyranose sulfates
OSO3Na
OSO3Na
Starting material (mmol)
Reaction time (h)
Product (% yield)
i
ii
AcO
AcO
O
2
5
9
HO
HO
O
38 (0.36)
39 (1.7)
6
3
3
2.5
2
8
5
4
46 (78)
47 (92)
48 (61)
49 (83)
50 (94)
51 (82)
52 (80)
53 (88)
OH
OH
OH
AcNH
AcNH
38
OAc
46
40 (0.17)
41 (0.38)
42 (0.67)
43 (0.60)
44 (0.36)
45 (0.90)
OSO3Na
O
OSO3Na
i
AcO
AcO
ii
ii
HO
HO
O
AcO
OAc
OH
39
47
OSO3Na
O
AcO
Deacetylation of acetate sulfates 38–45 was carried out at
room temp. using 2.5 mol equiv. of sodium methoxide. The
products 46–53 (Scheme 6) were purified by anion-exchange
chromatography and gel filtration. Reaction scales and results
are given in Table 5.
OSO3Na
O
HO
i
AcO
HO
AcNH
AcNH
40
OAc
48
AcO
OSO3Na
O
OSO3Na
O
Conclusions
HO
i
Hydrolysis of a series of α--per-acetylated monosaccharides
catalysed by R. toruloides esterase results in cleavage of the
primary acetoxy group with good selectivity. The work
described in this paper provides a useful method for the
regiospecific deacetylation at C-6 in various per-acetylated α--
hexopyranoses and α--hexopyranosides. The advantage of this
procedure is that C-6 deprotected monosaccharides can be isol-
ated in one step. In contrast, the use of traditional chemical
techniques requires the use of complex protection/deprotection
strategies in order to obtain the required degree of protection.
Utilisation of the propensity of acyl groups to migrate under
acidic conditions has ultimately made possible the conversion
of the C-6 partially acylated monosaccharides into the corre-
sponding C-4 deprotected species. This procedure provides a
simple method for regiospecific deprotection of the C-4
position of hexopyranosides. In addition, the present results
demonstrate that the combined use of chemical and enzymic
methods offers a convenient and expeditious method for the
preparation of hexose 4- and 6-sulfates.
ii
ii
ii
11
AcO
HO
OH
AcO
OAc
OSO3Na
OH
41
49
OSO3Na
OAc
O
OH
O
AcO
AcO
i
i
i
7
HO
HO
OH
OAc
42
50
OAc
O
OH
O
NaO3SO
AcO
NaO3SO
HO
3
22
25
OH
AcNH
AcNH
OAc
43
51
OAc
O
OH
O
NaO3SO
AcO
ii
ii
NaO3SO
HO
OH
OH
AcO
OH
OAc
44
52
OAc
OAc
O
OH
OH
O
Experimental
i
NaO3SO
AcO
NaO3SO
HO
The esterase from R. toruloides esterase was a gift from Glaxo
Group Research (Glaxo-Wellcome). 1H NMR spectra were
recorded at 250 MHz using a Bruker ACF 250 spectrometer, or
at 400 MHz using a Bruker ACP 400 spectrometer. 13C NMR
spectra were recorded at 63 MHz using a Bruker ACF 250 spec-
trometer or at 100 MHz using a Bruker ACP 400 spectrometer.
J-Values are quoted in Hz. Mps were determined using a Stuart
Scientific SMP 1 melting point apparatus and are uncorrected.
Optical rotations were recorded on an Optical Activity Ltd
model AA-1000 polarimeter at 589 nm (Na -line) with a path
length of 2 dm. [α]D-Values are given in units of 10Ϫ1 deg cm2
OAc
45
53
Scheme 6 Reagents: i, SO3, pyridine; ii, MeONa
sugar sulfates produced were converted into sodium salts 38–45
(Scheme 6) by anion exchange using an ion-exchange resin. The
reaction scales, times and yields are summarized in Table 4. The
positions of the sulfate groups in 6-sulfates 38–42 were con-
firmed by comparing their 13C NMR spectra with those of the
corresponding 6-OH derivatives 2, 5, 9, 11 and 7. They all
showed a downfield shift of the C-6 signal attributed to the
deshielding effect of the attached sulfate group. 1H NMR spec-
troscopy was used to confirm the structures of the 4-sulfates
g
Ϫ1. Low-resolution mass spectra were recorded on Kratos
MS 80 and VG Analytical Quattro 2 spectrometers. Fast-
atom bombardment (FAB) mass spectra were recorded using
m-nitrobenzyl alcohol (NOBA) or a mixture of glycerol–
thioglycerol (1:1) as matrix. High-resolution mass spectra were
1
43–45. The H NMR spectra of the sugar sulfates 43–45 all
exhibited a downfield shift of the H-4 signals.
J. Chem. Soc., Perkin Trans. 1, 1998
1073

View Article Online
recorded on Kratos MS 80, VG Analytical ZAB-E or Bruker
BioApex 9.4 T FTICR instruments. Software used was
PCMODEL (Serena Software, Bloomington, Indiana) and
Chem 3D (CambridgeSoft Corporation, Cambridge, Mass.).
Potential energies are quoted in kcal molϪ1 where 1 kcal = 4.184
kJ.
(dichloromethane–methanol, 20:1, v/v) to yield compound 7
(2.35 g, 88%) as a syrup. The syrup was redissolved in water (15
cm3), then freeze dried to give compound 7 as a hygroscopic
powder [Found: (M ϩ Na)ϩ, (FAB, Na) 371.0986. C14H20NaO10
requires m/z, 371.095 41]; [α]D27 ϩ61.6 (c 0.34, CHCl3);
νmax(Nujol)/cmϪ1 3475 (OH), 1725 (C᎐O) and 1250 (C᎐O᎐C);
᎐
δH(400 MHz; CDCl3) 1.97 (3 H, s, CH3CO), 2.04 (3 H, s,
CH3CO), 2.12 (3 H, s, CH3CO), 2.12 (3 H, s, CH3CO), 2.46
(1 H, dd, J 5.97 and 8.06, OH), 3.59 (1 H, m, Hb-6), 3.68 (1 H,
m, Ha-6), 3.81 (1 H, ddd, J 2.32, 4.39 and 10, H-5), 5.23 (1 H,
dd, J 1.9 and 3.4, H-2), 5.26 (1 H, app t, J 10 and 10, H-4), 5.35
(1 H, dd, J 3.4 and 10, H-3) and 6.04 (1 H, d, J 1.90, H-1); δC(63
MHz; CDCl3) 20.49 (CH3CO), 20.53 (CH3CO), 20.59
(CH3CO), 20.71 (CH3CO), 60.93 (C-6), 65.62 (C-4), 68.23
General procedure of esterase-catalysed hydrolyses of acetylated
monosaccharides
Per-acetylated hexopyranose was suspended in citrate phos-
phate buffer (pH 5.0, 50 mmol/100 mmol). After addition of
the R. toruloides esterase, the reaction mixture was stirred at
30 ЊC, and was followed by TLC. When complete hydrolysis of
the per-acetylated sugar had occurred, the enzyme was filtered
off and the filtrate was extracted with dichloromethane. The
organic fraction was dried (MgSO4), and concentrated by
reduced-pressure evaporation. The residue was then purified by
flash chromatography or crystallization.
(C-2), 68.44 (C-3), 72.74 (C-5), 90.49 (C-1), 168.15 (C᎐O),
᎐
169.66 (C᎐O), 169.86 (C᎐O) and 170.24 (C᎐O); m/z (rel. ab.)
᎐
᎐
᎐
FAB (NOBA) 371 [(M ϩ Na)ϩ, 30%], 331 (15), 290 (26), 281
(22), 229 (37), 169 (40), 147 (53), 127 (100) and 109 (94).
General procedure for the conversion of C-6 deprotected
monosaccharides into the C-4 deprotected monosaccharides
C-6 Deprotected monosaccharide was added to toluene and the
mixture was heated to 80 ЊC. Acetic acid was added and the
mixture was heated at 80 ЊC for 16 h. The solvent was removed
under reduced pressure to give the C-4 deprotected product
which was purified either by column chromatography or by
crystallization.
2-Acetamido-1,3,4-tri-O-acetyl-2-deoxy-á-D-galactopyranose 9
Acetate 8 (200 mg, 5.14 × 10Ϫ4 mol) was suspended in buffer (9
cm3). Esterase (80 mg) was added and the reaction mixture was
stirred at 30 ЊC for 16 h before being filtered, and the filtrate was
freeze dried. Ethyl acetate (5 cm3) and methanol (2 cm3) were
added to the residue. The mixture was subjected to flash chro-
matography (dichloromethane–methanol, 15:1, v/v) to give
title compound 9 (0.13 g, 73%) as a solid and diol 19 (9.8 mg,
6%) as a syrup; compound 9, mp 189–181 ЊC [Found:
(M ϩ Na)ϩ, (FAB, Na) 371.1079. C14H21NNaO9 requires m/z,
371.111 39]; [α]D23 ϩ150 (c 0.07, CHCl3); νmax(Nujol)/cmϪ1 3600–
1,2,3,4-Tetra-O-acetyl-á-D-glucopyranose 5
Glucose pentaacetate 4 (33.30 g, 85.38 mmol) was suspended in
buffer (350 cm3) at 30 ЊC. Esterase (340 mg) was added and the
mixture was stirred for 16 h. TLC analysis (dichloromethane–
methanol, 15:1, v/v) showed the formation of a major product
(Rf 0.61) and a minor product (Rf 0.19). The solution was evap-
orated under reduced pressure to give a residue, which was
extracted with ethyl acetate (2 × 100 cm3) and ethanol (2 × 100
cm3). The combined organic extracts were filtered, dried
(MgSO4), and evaporated under reduced pressure to yield a
syrup. Purification by flash chromatography (dichloromethane–
methanol, 150:1, v/v) yielded a syrup. Crystallization from
diethyl ether yielded tetraacetate 5 (16.05 g, 54%). A minor
product (Rf 0.19; 407 mg, 1.5%) was isolated, and identified
3300 (OH), 1750 (C᎐O), 1675 (C᎐O) and 1250 (C᎐O᎐C); δ (400
᎐
᎐
H
MHz; CDCl3) 1.92 (3 H, s, CH3CO), 2.02 (3 H, s, CH3CO), 2.14
(3 H, s, CH3CO), 2.17 (3 H, s, CH3CO), 2.58 (1 H, m, OH),
3.45 (1 H, dd, J 6.4 and 11.63, Hb-6), 3.64 (1 H, dd, J 6.4 and
1.63, Ha-6), 4.04 (1 H, app t, J 6.4 and 6.4, H-5), 4.71 (1 H, ddd,
J 3.7, 9.2 and 11.6, H-2), 5.22 (1 H, dd, J 3.2 and 11.6, H-3), 5.37
(1 H, d, J 3, H-4), 5.66 (1 H, d, J 9.2, NH) and 6.17 (1 H, d, J
3.7, H-1); δC(63 MHz; CDCl3) 20.58 (CH3CO), 20.63 (CH3CO),
20.82 (CH3CO), 22.98 (CH3CO), 47.12 (C-2), 60.43 (C-6), 67.51
(C-4), 67.74 (C-3), 71.28 (C-5), 91.15 (C-1), 169.01 (C᎐O),
᎐
170.14 (C᎐O), 171.05 (C᎐O) and 171.12 (C᎐O); m/z (rel. ab.)
᎐
᎐
᎐
FAB (GlythioNa) 370 [(M ϩ Na)ϩ, 28%], 310 (12), 237 (15),
200 (10), 153 (11), 137 (28), 115 (86), 61 (59) and 44 (100);
compound 19, δH(250 MHz; CDCl3) 1.90 (3 H, s, CH3CO), 2.05
(3 H, s, CH3CO), 2.13 (3 H, s, CH3CO), 3.62–3.72 (2 H, m, H2-
6), 4.03 (1 H, app t, J 6.1 and 6.1, H-5), 4.14 (1 H, d, J 2.6, H-4),
4.52 (1 H, dd, J 3.78 and 11.34, H-2), 5.14 (1 H, dd, J 2.6 and
11.34, H-3) and 6.07 (1 H, d, J 3.78, H-1); δC(63 MHz; CDCl3)
21.00 (CH3CO), 21.07 (CH3CO), 22.51 (CH3CO), 47.22 (C-6),
61.61 (C-4), 66.87 (C-3), 71.17 (C-5), 73.65, 92.11 (C-1), 173.46
1
by H NMR as 1,2,3-tri-O-acetyl-α--glucopyranose 18; com-
pound 5, mp 98–100 ЊC (lit.,26 99–101 ЊC) (Found: C, 48.32; H,
5.75. C14H20O10 requires C, 48.26; H, 5.79%); [α]D28 ϩ113 (c 1.26,
CHCl3) {lit.,26 [α]D ϩ119 (c 2.00, CHCl3)}; νmax(Nujol)/cmϪ1
3500 (OH) and 1750 (C᎐O); δ (400 MHz; CDCl ) 1.97 (3 H, s,
᎐
H
3
CH3CO), 1.99 (3 H, s, CH3CO), 2.03 (3 H, s, CH3CO), 2.13 (3
H, s, CH3CO), 2.37 (1 H, m, OH), 3.55 (1 H, m, Hb-6), 3.68 (1
H, m, Ha-6), 3.89 (1 H, ddd, J 2.29, 4.19 and 10, H-5), 5.04 (1 H,
dd, J 3.7 and 10, H-2), 5.07 (1 H, dd, J 9.8 and 10, H-4), 5.48 (1
H, app t, J 10 and 10, H-3) and 6.3 (1 H, d, J 3.7, H-1); δC(63
MHz; CDCl3) 20.48 (CH3CO), 20.58 (CH3CO), 55.28 (OCH3),
60.85 (C-6), 68.75 (C-4), 69.14 (C-2), 69.95 (C-3), 70.86 (C-5),
(C᎐O), 173.83 (C᎐O) and 175.49 (C᎐O).
᎐
᎐
᎐
1,2,3,4-Tetra-O-acetyl-á-D-galactopyranose 11
Galactose pentaacetate 10 (2.5 g, 6.41 mmol) was suspended in
buffer (50 cm3). Esterase (500 mg) was added and the mixture
was stirred at 30 ЊC for 16 h. TLC (dichloromethane–methanol,
15:1, v/v) showed that the reaction was not complete. Further
esterase (300 mg) and buffer (30 cm3) were added. The mixture
was stirred at 30 ЊC for another 7 h. The enzyme was removed
by filtration and the filtrate was freeze dried. The residue was
suspended in dichloromethane (25 cm3). Water (2 cm3) was
added and the resulting mixture was subjected to flash chroma-
tography (dichloromethane–methanol, 40:1, v/v) to give com-
pound 11 as a syrup (1.5 g, 67%) (the syrup could be converted
into a powder by dissolution in water followed by freeze drying)
[Found: (M ϩ Na)ϩ, (FAB, Na) 371.0986. C14H20NaO10
requires m/z, 371.095 41]; [α]D27 ϩ67.1 (c 0.81, CHCl3);
96.62 (C-1) and 169.96, 170.09 and 170.45 (C᎐O); m/z (relative
᎐
abundance) (CI, NH3) 366 [(M ϩ NH4)ϩ, 22%], 331 (6), 306
(14), 289 (72), 229 (34), 187 (39), 169 (26), 127 (10), 115 (49), 98
(28), 83 (43), 60 (34) and 43 (100); compound 18, δH(250 MHz;
D2O), 2.00 (3 H, s, CH3CO), 2.09 (3 H s, CH3CO), 2.15 (3 H, s,
CH3CO), 3.83 (4 H, m, H-4, H-5 and H2-6), 5.07 (1 H, dd, J
3.77 and 10.47, H-2), 5.36 (1 H, m, H-3) and 6.26 (1 H, d, J
3.77, H-1); δC(63 MHz; D2O) 20.41 (CH3CO), 20.77 (CH3CO),
20.80 (CH3CO), 61.21, 68.39, 69.28, 72.42, 73.84, 89.28 (C-1),
169.30 (C᎐O), 169.99 (C᎐O) and 171.38 (C᎐O).
᎐
᎐
᎐
1,2,3,4-Tetra-O-acetyl-á-D-mannopyranose 7
Mannose pentaacetate 6 (3.0 g, 7.69 mmol) was suspended in
buffer (40 cm3). Esterase (90 mg) was added and the mixture
was stirred at 30 ЊC. After 20 h, the reaction mixture was filtered
and the filtrate was freeze dried. The residue was suspended in
ethyl acetate (10 cm3) and subjected to flash chromatography
νmax(Nujol)/cmϪ1 3500 (OH), 1750 (C᎐O) and 1225 (C᎐O᎐C);
᎐
δH(250 MHz; CDCl3) 2.00 (3 H, s, CH3CO), 2.01 (3 H, s,
CH3CO), 2.14 (3 H, s, CH3CO), 2.16 (3 H, s, CH3CO), 3.46
1074
J. Chem. Soc., Perkin Trans. 1, 1998

View Article Online
(1 H, dd, J 6.6 and 11.9, Hb-6), 3.66 (1 H, dd, J 6.6 and 11.9, Ha-
6), 4.05 (1 H, app t, J 6.6 and 6.6, H-5), 5.35 (2 H, m, H-2, -3),
5.48 (1 H, d, J 1.19, H-4) and 6.36 (1 H, d, J 1.69, H-1); δC(63
MHz; CDCl3) 20.42 (CH3CO), 20.52 (CH3CO), 20.77
(CH3CO), 60.62 (C-6), 66.56, 67.33, 68.19 (C-4), 71.41 (C-5),
343.1005]; [α]D26 ϩ149.2 (c 0.12, CHCl3); νmax(Nujol)/cmϪ1 3525
(OH), 1750 (C᎐O) and 1250 (C᎐O᎐C); δ (250 MHz; CDCl )
᎐
H
3
1.99 (3 H, s, CH3CO), 2.08 (3 H, S, CH3CO), 2.15 (3 H, s,
CH3CO), 3.40 (3 H, s, OCH3), 3.49 (1 H, dd, J 6.11 and 11.63,
Hb-6), 3.66 (1 H, dd, J 9.98 and 11.63, Ha-6), 4.04 (1 H, app t,
J 6.6 and 6.6, H-5), 4.98 (1 H, d, J 3.48, H-1), 5.16 (1 H, dd,
J 3.48 and 10.75, H-2), 5.36 (1 H, dd, J 3.49 and 10.75, H-3)
and 5.42 (1 H, dd, J 3.49 and 10.75, H-4); δC(63 MHz; CHCl3)
20.13 (CH3CO), 20.28 (CH3CO), 54.88 (OCH3), 60.33 (C-6),
67.29 (C-3), 67.93 (C-2), 68.31 (C-4), 68.44 (C-5), 96.60 (C-1)
89.56 (C-1), 168.98, 169.80, 169.90 and 170.82 (C᎐O); m/z (rel.
᎐
ab.) FAB (Glythio Na) 371 [(M ϩ Na)ϩ, 100%], 311 (12), 289
(24), 229 (18), 169 (39), 127 (47) and 109 (71).
Methyl 2,3,4-tri-O-acetyl-á-D-glucopyranoside 13
Glucopyranoside 12 (8.16 g, 22.6 mmol) was suspended in buf-
fer (80 cm3) at 30 ЊC. Esterase (240 mg) was added and the
reaction mixture was stirred for 16 h, filtered, and extracted with
dichloromethane (3 × 50 cm3) and ethyl acetate (2 × 50 cm3).
The combined organic extracts were dried (MgSO4), and evap-
orated under reduced pressure to yield a syrup. Crystallization
from diethyl ether yielded compound 13 (5.54 g, 77%); mp 103–
104 ЊC (lit.,27 109.5–110 ЊC; lit.,28 110 ЊC) (Found: C, 48.54; H,
6.24. C13H20O9 requires C, 48.73; H, 6.29%); [α]D28 ϩ125 (c 0.72,
CHCl3) {lit.,27 [α]D ϩ137 (CHCl3); lit.,28 145.5}; νmax(Nujol)/
and 169.56, 170.03 and 170.35 (C᎐O); m/z (rel. ab.) FAB
᎐
(Glythio Na) 343 [(M ϩ Na)ϩ, 100%], 289 (22), 229 (10), 169
(25), 127 (39) and 109 (45).
1,2,3,6-Tetra-O-acetyl-á-D-glucopyranose 22
Glucopyranose derivative 5 (4.08 g, 11.9 mmol) was added to
toluene (76 cm3) and the mixture was heated to 80 ЊC. Acetic
acid (0.76 cm3) was added and the reaction mixture was stirred
at 80 ЊC for 16 h. The solvent was removed under reduced pres-
sure to give a syrup. Crystallization from diethyl ether yielded
tetraacetate 22 (2.44 g, 60%); mp 99–99.5 ЊC (Found: C, 48.38;
H, 5.77. C14H20O10 requires C, 48.26; H, 5.79%); [α]D28 ϩ67.5
(c 0.64, CHCl3) {lit.,26 [α]D24 ϩ56.8 (c 0.88, CHCl3)}; νmax(Nujol)/
cmϪ1 3500 (OH) and 1740 (C᎐O); δ (400 MHz; CDCl ) 1.98 (3
᎐
H
3
H, s, CH3CO), 2.02 (3 H, s, CH3CO), 2.03 (3 H, s, CH3CO), 2.39
(1 H, m, OH), 3.37 (3 H, s, OCH3), 3.55 (1 H, m, Hb-6), 3.68 (1
H, m, Ha-6), 3.75 (1 H, ddd, J 2.3, 4.3 and 10, H-5), 4.88 (1 H,
dd, J 3.6 and 10, H-2), 4.93 (1 H, d, J 3.6, H-1), 4.99 (1 H, dd, J
10 and 9.7, H-4) and 5.49 (1 H, dd, J 9.7 and 10, H-3); δC(63
MHz; CDCl3) 20.48 (CH3CO), 20.58 (CH3CO), (CH3CO),
55.28 (OCH3), 60.85 (C-6), 68.75 (C-4), 69.14 (C-3), 69.95 (C-
cmϪ1 3500 (OH) and 1750 (C᎐O); δ (400 MHz; CDCl ) 1.98 (3
᎐
H
3
H, s, CH3CO), 2.07 (3 H, s, CH3CO), 2.09 (3 H, s, CH3CO), 2.13
(3 H, s, CH3CO), 3.37 (1 H, br s, OH), 3.58 (1 H, app t, J 10 and
10, H-4), 3.92 (1 H, ddd, J 2.3, 3.9 and 10, H-5), 4.22 (1 H, dd,
J 2.3 and 12.4, Hb-6), 4.44 (1 H, dd, J 3.9 and 12.4, Ha-6), 4.99
(1 H, dd, J 3.71 and 10, H-2), 5.29 (1 H, dd, J 10.1 and 10, H-3)
and 6.25 (1 H, d, J 3.71, H-1); δC(63 MHz; CDCl3) 20.35
(CH3CO), 20.67 (CH3CO), 20.73 (CH3CO), 21.29 (CH3CO),
62.4 (C-6), 68.26 (C-4), 69.14 (C-2), 71.92 (C-3), 72.14 (C-5),
5), 70.86 (C-2), 96.62 (C-1), 169.96 (C᎐O), 170.09 (C᎐O) and
᎐
᎐
170.45 (C᎐O); m/z (rel. ab.) FAB (NOBA) 343 [(M ϩ Na)ϩ,
᎐
48%], 331 (7), 321 [(M ϩ H)ϩ, 7], 290 (9), 229 (34), 169 (100),
141 (28), 127 (85) and 109 (82).
89.18 (C-1), 168.97 (C᎐O), 169.87 (C᎐O), 171.09 (C᎐O) and
᎐
᎐
᎐
Methyl 2,3,4-tri-O-acetyl-á-D-mannopyranoside 15
171.57 (C᎐O); m/z (rel. ab.) FAB (NOBA) 371 [(M ϩ Na)ϩ,
᎐
Mannopyranoside 14 (1.49 g, 4.12 mmol) was suspended in
buffer (15 cm3) at 30 ЊC. Esterase (45 mg) was added and the
mixture was stirred for 16 h before being filtered, and extracted
with dichloromethane (2 × 30 cm3). The organic extracts were
dried (MgSO4), filtered, and evaporated under reduced pressure
to yield a syrup. Crystallization from diethyl ether yielded com-
pound 15 as crystals (0.92 g, 70%); mp 95–96 ЊC (lit.,29 97–98 ЊC)
(Found: C, 48.75; H, 6.29. Calc. for C13H20O9: C, 48.73; H,
6.29%); [α]D28 ϩ54.5 (c 0.3, CHCl3) {lit.,29 [α]D25 ϩ54.9 (c 1.1,
CHCl3)}; νmax(Nujol)/cmϪ1 3500 (OH) and 1740 (C᎐O); δ (400
MHz; CDCl3) 1.97 (3 H, s, CH3CO), 2.04 (3 H, s, CH3CO), 2.12
(3 H, s, CH3CO), 2.43 (1 H, s, OH), 3.38 (3 H, s, OCH3), 3.60
(1 H, dd, J 12.62 and 4.25, Hb-6), 3.72 (2 H, m, H-5, Ha-6), 4.69
(1 H, d, J 1.66, H-1), 5.22 (2 H, m, H-2, -4) and 5.35 (1 H, dd,
J 3.44 and 10.16, H-3); δC(63 MHz; CDCl3) 20.54 (CH3CO),
20.58 (CH3CO), 20.72 (CH3CO), 55.12 (OCH3), 61.11 (C-6),
66.29 (C-4), 68.69 (C-3), 69.39 (C-2), 70.33 (C-5), 98.47 (C-1)
29%], 349 [(M ϩ H)ϩ, 5], 331 (9), 311 (5), 290 (10), 229 (100),
187 (24), 169 (21) and 127 (62).
1,2,3,6-Tetra-O-acetyl-á-D-mannopyranose 23
Mannopyranoside derivative 7 (1.25 g, 3.59 mmol) was dis-
solved in toluene (25 cm3). Acetic acid (1 cm3) was added and
the mixture was heated at 80 ЊC. After 44 h, the solvent was
removed by reduced pressure evaporation. The residue was
purified by column chromatography (dichloromethane–
methanol, 40:1, v/v) to yield a syrup. The syrup was dissolved
in water and then freeze dried to give tetraacetate 23 (1.15 g,
92%) as a powder [Found: (M ϩ Na)ϩ, (FAB, Na) 371.0957.
C14H20NaO10 requires m/z, 371.095 41]; [α]D27 ϩ27.3 (c 0.2,
᎐
H
CHCl3); νmax(Nujol)/cmϪ1 3500 (OH), 1750 (C᎐O) and 1250
᎐
(C᎐O᎐C); δH(400 MHz; CDCl3) 2.01 (3 H, s, CH3CO), 2.07
(3 H, s, CH3CO), 2.09 (3 H, s, CH3CO), 2.10 (3 H, s, CH3CO),
3.15 (1 H, d, J 4.49, OH), 3.88–3.79 (2 H, m, H-5, -4), 4.23 (1 H,
dd, J 1.69 and 12.2, Hb-6), 4.43 (1 H, dd, J 4.02 and 12.2, Ha-6),
5.18–5.13 (2 H, m, H-2, -3) and 5.99 (1 H, d, J 1.72, H-1); δC(63
MHz; CDCl3) 20.45 (CH3CO), 20.53 (CH3CO), 20.56
(CH3CO), 20.63 (CH3CO), 62.78 (C-6), 64.70 (C-4), 68.26
and 169.75, 169.95 and 170.69 (C᎐O); m/z (rel. ab.) FAB
᎐
(NOBA) 343 [(M ϩ Na)ϩ, 21%], 331 (14), 321 [(M ϩ H)ϩ, 11],
319 (17), 290 (19), 229 (50), 169 (71), 141 (37), 127 (100) and 109
(93).
(C-2), 70.72 (C-3), 72.69 (C-5), 90.61 (C-1), 168.12 (C᎐O),
᎐
Methyl 2,3,4-tri-O-acetyl-á-D-galactopyranoside 17
169.47 (C᎐O), 170.39 (C᎐O) and 171.49 (C᎐O); m/z (rel. ab.)
᎐ ᎐ ᎐
Galactopyranoside 16 (2.0 g, 5.52 mmol) was suspended in buf-
fer (40 cm3). Esterase (70 mg) was added and the reaction mix-
ture was stirred at 30 ЊC for 20 h. TLC analysis
(dichloromethane–methanol; 9:1, v/v) indicated the reaction
was not complete. Further esterase (100 mg) and buffer (40 cm3)
were added. The reaction mixture was stirred at 30 ЊC for
another 5 h before being filtered and the filtrate was freeze
dried. The residue was suspended in dichloromethane (10 cm3)
and purified by flash chromatography (diochloromethane–
methanol, 40:1, v/v) to yield compound 17 (1.5 g, 85%) as a
syrup (the syrup was converted into a hygroscopic powder by
dissolution in water followed by freeze drying) [Found:
(M ϩ Na)ϩ, (FAB, Na) 343.1030. C13H20NaO9 requires m/z,
FAB (NOBA) 371 [(M ϩ Na)ϩ, 67%], 349 [(M ϩ H)ϩ, 3], 331
(7), 290 (47), 287 (8), 229 (100), 187 (25) and 127 (76).
2-Acetamido-1,3,6-tri-O-acetyl-2-deoxy-á-D-galactopyranose
28
Galactopyranose derivative 9 (45 mg, 39.2 mmol) was added to
toluene (0.79 cm3) and the mixture was heated to 80 ЊC. Acetic
acid (0.01 cm3) was added and the mixture was heated at 80 ЊC
for 16 h. The solvent was removed under reduced pressure to
1
give a solid. The H NMR spectrum showed that the product
was compound 28; no acyl migration had taken place. This was
probably due to the insolubility of product 28 in toluene. To
improve the solubility of compound 28, DMF was used as a
J. Chem. Soc., Perkin Trans. 1, 1998
1075

View Article Online
co-solvent. Glycoside 28 (45 mg, 3.92 × 10Ϫ2 mol) was initially
dissolved in DMF (0.1 cm3), toluene (0.79 cm3) was added, and
the reaction mixture was heated to 80 ЊC. Acetic acid (0.01 cm3)
was added and the mixture was heated at 80 ЊC. After 16 h the
1H NMR spectrum showed that there was a ~1:1 mixture of
compounds 28 and 9. The mixture was heated at 80 ЊC for 3
days. The solvent was removed under reduced pressure to give
(c 0.14, CHCl3); νmax(Nujol)/cmϪ1 3525 (OH), 1750 (C᎐O) and
1250 (C᎐O᎐C); δH(400 MHz; CDCl3) 2.01 (3 H, s, CH3CO),
᎐
2.07 (3 H, s, CH3CO), 2.08 (3 H, s, CH3CO), 2.98 (1 H, d,
J 4.09, OH), 3.35 (3 H, s, OCH3), 3.81–3.74 (2 H, m, H-4, -5),
4.29 (1 H, dd, J 1.16 and 12, Hb-6), 4.42 (1 H, dd, J 4.49 and 12,
Ha-6), 4.63 (1 H, dd, J 1.57, H-1) and 5.17–5.09 (2 H, m, H-2,
-3); δC(63 MHz; CDCl3) 20.61 (3 × CH3CO), 54.91 (OCH3),
63.19 (C-6), 65.42, 69.49 (C-2), 70.47, 71.34 (C-3), 98.43 (C-1),
1
a syrup. The H NMR spectrum showed that there was still a
~1:1 mixture of compounds 28 and 9. The addition of a more
polar co-solvent had altered the position of the equilibrium.
However, isolation of the required 4-deprotected product was
not achieved effectively.
169.83 (C᎐O), 170.59 (C᎐O) and 171.33 (C᎐O); m/z (rel. ab.)
᎐ ᎐ ᎐
FAB (NOBA) 343 [(M ϩ Na)ϩ, 41%], 321 [(M ϩ H)ϩ, 100], 290
(19) and 229 (14).
Methyl 2,3,6-tri-O-acetyl-á-D-galactopyranoside 27
1,2,3,6-Tetra-O-acetyl-á-D-galactopyranose 26
Galactopyranoside 17 (708 mg, 2.55 mmol) was added to tolu-
ene (12.5 cm3) and the mixture was heated to 80 ЊC. Acetic acid
(0.13 cm3) was added to the solution, which was heated at 80 ЊC
for 16 h. The solvent was removed under reduced pressure to
give a syrup ion quantitative yield. Purification by crystalliz-
ation or by column chromatography proved ineffective. The 1H
NMR spectrum showed that compound 27 had been formed as
the major product but that minor amounts of starting material
Galactopyranose derivative 11 (503 mg, 1.45 mmol) was added
to toluene (8.90 cm3) and the mixture was heated to 80 ЊC. Acet-
ic acid (0.09 cm3) was added and the solution was heated at
80 ЊC for 16 h. The solvent was removed under reduced pressure
1
to give a syrup in quantitative yield. The H NMR spectrum
showed that compound 26 had been formed as the major
product but minor amounts of starting material 11 (12.5%
1
as determined from the H NMR integrals) were still present.
1
17 (20% as determined from the H NMR integrals) were still
Purification by crystallization or by column chromatography
proved ineffective; compound 26 [Found: (M ϩ Na)ϩ, (FAB,
Na) 371.0938. C14H20NaO10 requires m/z, 371.095 41]; [α]D27
ϩ136.3 (c 0.16, CHCl3); νmax(Nujol)/cmϪ1 3500 (OH), 1730
present. Purification by crystallization or by column chroma-
tography proved ineffective; compound 27 [Found: (M ϩ Na)ϩ,
(FAB, Na) 343.1029. C13H20NaO9 requires m/z, 343.1005); [α]D27
ϩ69.5 (c 0.79, CHCl3); νmax(Nujol)/cmϪ1 3550 (OH), 1750
(C᎐O) and 1250 (C᎐O᎐C); δ (400 MHz; CDCl ) 1.87 (3 H, s,
᎐
H
3
(C᎐O) and 1225 (C᎐O᎐C); δ (400 MHz; CDCl ) 1.94 (6 H, s,
᎐
H
3
CH3CO), 1.93 (3 H, s, CH3CO), 1.97 (3 H, s, CH3CO), 2.01
(3 H, s, CH3CO), 3.58 (1 H, br s, OH), 4.06 (3 H, m, H-4, -5,
Hb-6), 4.14 (1 H, m, Ha-6), 5.08 (1 H, dd, J 2.80 and 10.8, H-3),
5.28 (1 H, dd, J 3.6 and 10.8, H-2) and 6.18 (1 H, d, J 3.6, H-1);
δC(63 MHz; CDCl3) 20.07 (CH3CO), 20.29 (CH3CO), 20.36
(CH3CO), 20.40 (CH3CO), 62.33 (C-6), 66.09 (C-2), 66.62,
2 × CH3CO), 1.96 (3 H, s, CH3CO), 2.81 (1 H, br s, OH), 3.26
(3 H, s, OCH3), 3.91 (1 H, m, H-5), 3.99 (1 H, m, H-4), 4.14
(2 H, m, H2-6), 4.82 (1 H, d, J 2.97, H-1) and 5.09 (2 H, m, H-2,
-3); δC(63 MHz; CDCl3) 2.304 (CH3CO), 20.37 (CH3CO), 20.41
(CH3CO), 54.78 (OCH3), 62.77 (C-6), 67.15, 67.18, 67.72 (C-2),
69.76 (C-3), 96.69 (C-1), 169.92 (C᎐O), 170.18 (C᎐O) and
᎐
᎐
170.62 (C᎐O); m/z (rel. ab.) FAB (GlythioNa) 343 [(M ϩ Na)ϩ,
69.47 (C-3), 69.90, 89.45 (C-1), 168.87 (C᎐O), 169.79 (C᎐O),
᎐
᎐
᎐
170.08 (C᎐O) and 170.69 (C᎐O); m/z (rel. ab.) FAB (GlythioNa)
᎐
᎐
100%], 289 (52), 229 (18), 169 (40), 141 (20), 127 (56) and 103
(67).
371 [(M ϩ Na)ϩ, 100%], 311 (12), 289 (19), 229 (10), 169 (12),
127 (28) and 109 (20).
2-Acetamido-1,3-di-O-acetyl-2-deoxy-á-D-glucopyranose 29
Glucopyranoside 3 (500 mg, 1.44 mmol) was suspended in buf-
fer (6.25 cm3) at 30 ЊC. Esterase (18 mg) was added and the
reaction mixture was stirred at 30 ЊC for 16 h. The mixture was
concentrated on the freeze drier, extracted with ethanol (3 × 5
cm3), filtered and evaporated under reduced pressure to yield a
solid. Recrystallization from acetone yielded compound 29
(0.36 g, 82%) as crystals; mp 166–167 ЊC (decomp.) [(Found:
(M ϩ Na)ϩ, (FAB Na) 328.0978. C12H19NNaO8 requires m/z,
328.100 83]; [α]D27 ϩ62.5 (c 0.08, MeOH); νmax(Nujol)/cmϪ1 3700–
Methyl 2,3,6-tri-O-acetyl-á-D-glucopyranoside 24
Glucopyranoside derivative 13 (317 mg, 9.91 × 10Ϫ4 mol) was
added to toluene (5.6 cm3) and the mixture was heated to 80 ЊC.
Acetic acid (0.056 cm3) was added to the solution, which was
heated at 80 ЊC for 16 h. The solvent was removed under
reduced pressure to give a syrup. Purification by flash chroma-
tography (toluene–ethyl acetate, 5:1, v/v) yielded compound 24
(Rf 0.53; 199 mg, 63%) as a syrup [Found: (M ϩ Na)ϩ, (FAB,
Na) 343.0999. C13H20NaO9 requires m/z, 343.1005]; [α]D28 ϩ120
(c 0.27, CHCl3) (lit.,30 [α]D20 ϩ100.8); νmax(Nujol)/cmϪ1 3550
3200 (OH), 1735 (C᎐O) and 1650 (C᎐O, NHAc); δ (400 MHz;
᎐
᎐
H
(OH), 1750 (C᎐O) and 1250 (C᎐O᎐C); δ (400 MHz; CDCl )
᎐
H
3
D2O) 1.93 (3 H, s, CH3CO), 2.09 (3 H, s, CH3CO), 2.19 (3 H, s,
CH3CO), 3.79 (4 H, m, H-4, -5, H2-6), 4.28 (1 H, dd, J 3.68 and
10.89, H-2), 5.20 (1 H, dd, J 8.92 and 10.89, H-3) and 6.05 (1 H,
d, J 3.68, H-1); δC(63 MHz; D2O) 20.86 (CH3CO), 20.92
(CH3CO), 22.29 (CH3CO), 51.19 (C-2), 60.62 (C-6), 67.85
2.06 (3 H, s, CH3CO), 2.07 (3 H, s, CH3CO), 2.10 (3 H, s,
CH3CO), 3.05 (1 H, d, J 5.4, OH), 3.38 (3 H, s, OCH3), 3.54 (1
H, m, H-4), 3.80 (1 H, ddd, J 2.29, 4.4 and 10, H-5), 4.28 (1 H,
dd, J 2.29 and 12.21, Hb-6), 4.44 (1 H, dd, J 4.4 and 12.21,
Ha-6), 4.83 (1 H, dd, J 3.68 and 9.8, H-2), 4.88 (1 H, d, J 3.68,
H-1) and 5.28 (1 H, app t, J 9.8 and 9.8, H-3); δC(63 MHz;
CDCl3) 20.62 (CH3CO), 20.68 (CH3CO), 20.73 (CH3CO),
55.15 (OCH3), 62.72 (C-6), 69.17 (C-4), 69.55 (C-5), 70.47
(C-4), 73.61 (C-3), 74.45 (C-5), 91.74 (C-1), 173.20 (C᎐O),
᎐
174.21 (C᎐O) and 175.18 (C᎐O); m/z (rel. ab.) FAB (NOBA)
᎐
᎐
328 [(M ϩ Na)ϩ, 55%], 306 [(M ϩ H)ϩ, 15], 246 (100), 186 (18),
156 (9) and 144 (14).
(C-2), 72.74 (C-3), 96.73 (C-1), 170.17 (C᎐O), 171.36 (C᎐O)
᎐
᎐
and 171.46 (C᎐O); m/z (rel. ab.) FAB (NOBA) 343 [(M ϩ Na)ϩ,
᎐
43%], 331 (9), 321 [(M ϩ H)ϩ, 100], 303 (6), 290 (32), 229 (77),
1,2,3-Tri-O-acetyl-á-D-glucopyranoside 18
Glucopyranoside 22 (420 mg, 1.21 mmol) was suspended in
buffer (5 cm3) at 30 ЊC. Esterase (15 mg) was added and the
reaction mixture was stirred at 30 ЊC. After 16 h, the reaction
mixture was filtered, and then extracted with dichloromethane
(2 × 50 cm3) and ethyl acetate (2 × 10 cm3). The combined
organic extracts were dried (MgSO4), and evaporated under
reduced pressure to yield compound 18 (266 mg, 72%) as a
syrup [Found: (M ϩ Na)ϩ, (FAB, Na) 329.0849. C12H18NaO9
requires m/z, 329.084 85]; [α]D27 ϩ89.3 (c 0.14, MeOH);
169 (51), 127 (65), 115 (31) and 109 (51).
Methyl 2,3,6-tri-O-acetyl-á-D-mannopyranoside 25
Mannopyranoside derivative 15 (336 mg, 9.64 × 10Ϫ4 mol) was
added to toluene (5.9 cm3) and the mixture was heated to 80 ЊC.
Acetic acid (0.06 cm3) was added and the reaction mixture was
stirred at 80 ЊC for 16 h. The solvent was removed under
reduced pressure to give a syrup. Purification by chroma-
tography (dichloromethane–methanol, 75:1, v/v) yielded com-
pound 25 (164 mg, 49%) as a syrup [Found: (M ϩ Na)ϩ, (FAB,
Na) 343.1017. C13H20NaO9 requires m/z, 343.1005]; [α]D27 ϩ30
νmax(Nujol)/cmϪ1 3450 (OH), 1725 (C᎐O) and 1250 (C᎐O᎐C);
᎐
δH(400 MHz; D2O) 2.07 (3 H, s, CH3CO), 2.15 (3 H, s, CH3CO),
1076
J. Chem. Soc., Perkin Trans. 1, 1998

View Article Online
2.24 (3 H, s, CH3CO), 3.86 (4 H, m, H-4, -5, H2-6), 5.07 (1 H,
dd, J 3.68 and 10, H-2), 5.36 (1 H, app t, J 10 and 10, H-3) and
6.28 (1 H, d, J 3.68, H-1); δC(63 MHz; D2O), 20.69 (CH3CO),
20.97 (CH3CO), 21.01 (CH3CO), 60.59 (C-6), 67.49 (C-4), 70.54
(C-2), 73.29 (C-3), 74.63 (C-5), 90.33 (C-1), 173.03 (C᎐O),
3100 (OH) and 1750 (C᎐O); δ (400 MHz; D O) 2.10 (3 H, s,
᎐
H 2
CH3CO), 3.39 (3 H, s, OCH3), 3.42 (1 H, dd, J 9.2 and 10, H-4),
3.54 (1 H, dd, J 3.7 and 9.6, H-2), 3.64 (1 H, dd, J 9.6 and 9.2,
H-3), 3.82 (1 H, ddd, J 2.3, 5.2 and 10, H-5), 4.24 (1 H, dd, J 5.2
and 12.2, Hb-6), 4.37 (1 H, dd, J 2.30 and 12.20, Ha-6) and 4.77
(1 H, d, J 3.77, H-1); δC(63 MHz; D2O) 20.83 (CH3CO), 55.79
(OCH3), 64.04 (C-6), 69.89 (C-5), 70.19 (C-4), 71.79 (C-2),
173.29 (C᎐O) and 174.24 (C᎐O); m/z (rel. ab.) FAB (NOBA)
᎐
᎐
329 [(M ϩ Na)ϩ, 45%], 307 [(M ϩ H)ϩ, 15], 289 (17), 247 (100)
and 187 (23).
73.59 (C-3), 100.01 (C-1) and 174.79 (C᎐O); m/z (FAB, Thi-
᎐
oglyNa) 259 [(M ϩ Na)ϩ, 100%]; compound 34; mp 133–134 ЊC
[Found: (M ϩ Na)ϩ, (FAB, Na) 259.0798. C9H16NaO7 requires
m/z, 259.079 37]; [α]D20 ϩ162.5 (c 0.12, MeOH); νmax(Nujol)/
Methyl 2,3-di-O-acetyl-á-D-glucopyranoside 30
Glucopyranoside 24 (484 mg, 1.51 mmol) was suspended in
buffer (6.25 cm3) at 30 ЊC. Esterase (15 mg) was added and the
reaction mixture was stirred at 30 ЊC. After 16 h, the reaction
mixture was filtered, and concentrated on the freeze drier. The
residue was extracted with ethyl acetate (2 × 10 cm3), and the
combined organic extracts were dried (MgSO4), and evaporated
under reduced pressure to give compound 30 (146 mg, 35%) as
a syrup [(Found: (M ϩ Na)ϩ, (FAB, Na) 301.0928. C11H18NaO8
requires m/z, 301.089 93]; [α]D27 ϩ114.5 (c 0.1, MeOH);
cmϪ1 3600–3100 (OH) and 1740 (C᎐O); δ (250 MHz; D O) 2.14
᎐
H
2
(3 H, s, CH3CO), 3.42 (3 H, s, OCH3), 3.56 (1 H, dd, J 9.5 and
9.75, H-4), 3.77–3.68 (3 H, m, H-2, -5, Hb-6), 3.85 (1 H, dd, J
2.13 and 12.13, Ha-6), 4.83 (1 H, d, J 3.77, H-1) and 5.10 (1 H,
app t, J 9.5 and 9.5, H-3); δC(63 MHz; D2O) 21.19 (CH3CO),
55.79 (OCH3), 60.97 (C-6), 68.36 (C-4), 70.15 (C-5), 72.04
(C-2), 76.18 (C-3), 99.79 (C-1) and 174.64 (C᎐O); m/z (FAB,
᎐
ThioglyNa) 259 [(M ϩ Na)ϩ, 10%], 237 [(M ϩ H)ϩ, 19], 205
νmax(Nujol)/cmϪ1 3500 (OH), 1750 (C᎐O) and 1250 (C᎐O᎐C);
(14), 145 (18), 75 (35) and 56 (50).
᎐
δH(400 MHz; D2O) 2.07 (3 H, s, CH3CO), 2.10 (3 H, s,
CH3CO), 3.40 (3 H, s, OCH3), 3.79–3.70 (3 H, m, H-4, -5,
Hb-6), 3.86 (1 H, m, Ha-6), 4.92 (1 H, dd, J 3.7 and 10.13, H-2),
4.97 (1 H, d, J 3.7, H-1) and 5.24 (1 H, dd, J 8.96 and 10.13,
H-3); δC(63 MHz; D2O) 20.76 (CH3CO), 20.97 (CH3CO), 55.52
(OCH3), 60.85 (C-6), 68.17 (C-4), 71.59 (C-2), 72.02 (C-5),
General procedure for the preparation of hexose acetate sulfates
38–45
Sulfur trioxide–pyridine complex was added to the 4- or 6-
deprotected α-hexopyranose acetate in anhydrous pyridine. The
mixture was stirred at room temp. and the reaction was fol-
lowed by TLC (dichloromethane–methanol, 10:1, v/v). On
completion of the reaction, water was added to destroy the
excess of sulfur trioxide. Pyridine was removed by co-
evaporation with water under reduced pressure (<35 ЊC). The
residue was redissolved in water and passed through an anion-
exchange column (Dowex 1X2-400, ClϪ form). The column was
washed with water and eluted with aq. NaCl. Compound 45
was eluted with 0.2  NaCl, compounds 39, 43 and 44 were
eluted with 0.5  NaCl, compounds 38, 40 and 42 were eluted
with 1.0  NaCl and compound 41 was eluted with 1.5  NaCl.
The eluate was concentrated and subjected to gel filtration with
water as eluent (Sephadex G25, column dimensions 2.5 cm × 90
cm) to remove NaCl. Fractions containing the sulfated sugar
were combined, concentrated and freeze dried to give the
product.
73.67 (C-3), 97.15 (C-1), 173.51 (C᎐O) and 174.26 (C᎐O); m/z
᎐
᎐
(rel. ab.) FAB (NOBA) 579 [2(M ϩ Na)ϩ, 5%], 557
[2(M ϩ H)ϩ, 5], 301 [(M ϩ Na)ϩ, 32], 279 [(M ϩ H)ϩ, 66], 247
(100), 217 (7), 187 (28), 145 (8) and 127 (22).
Methyl 2,3-di-O-acetyl-á-D-mannopyranoside 31
Mannopyranoside 25 (1.02 mg, 3.19 mmol) was suspended in
buffer (12.5 cm3) at 30 ЊC. Esterase (36 mg) was added and the
reaction mixture was stirred at 30 ЊC for 16 h before being fil-
tered, and concentrated on the freeze drier. The residue was
extracted with ethyl acetate (2 × 30 cm3), and the combined
organic extracts were dried (MgSO4), and evaporated under
reduced pressure to yield a solid. Recrystallization from acetone
yielded compound 31 (531 mg, 60%), mp 136–137 ЊC [Found:
(M ϩ Na)ϩ, (FAB, Na) 301.0882. C11H18NaO8 requires m/z,
301.089 93]; [α]D27 ϩ45.7 (c 0.7, MeOH); νmax(Nujol)/cmϪ1 3475
(OH), 1750 (C᎐O) and (C᎐O᎐C); δ (400 MHz; D O) 2.08 (3 H,
General procedure for the deacetylation
᎐
H
2
s, CH3CO), 2.17 (3 H, s, CH3CO), 3.44 (3 H, s, OCH3), 3.83–
3.75 (2 H, m, H-5, Hb-6), 3.94–3.88 (2 H, m, H-4, Ha-6), 4.82
(1 H, d, J 1.7, H-1), 5.08 (1 H, dd, J 3.39 and 9.85, H-3) and
5.25 (1 H, dd, J 1.7 and 3.39, H-2); δC(63 MHz; D2O) 20.85
(CH3CO), 20.95 (CH3CO), 55.66 (OCH3), 61.19 (C-6), 65.04
(C-4), 70.22 (C-2), 72.76 (C-3), 73.19 (C-5), 98.86 (C-1), 173.63
(C᎐O) and 173.93 (C᎐O); m/z (rel. ab.) FAB (NOBA) 579
Acetate and sodium methoxide (2.5 mol equiv.) were suspended
in anhydrous methanol. The mixture was stirred at room temp.
and the reaction was followed by TLC (propan-1-ol–
nitromethane–water, 10:9:2, v/v). When deacetylation was
complete, the reaction was quenched by the addition of water.
The solution was neutralized by passage through a cation-
exchange column (Dowex, 50W-X8, Hϩ form). The neutralized
solution was then passed through an anion-exchange column
(Dowex 1X2-400, ClϪ form). The column was washed with
water and eluted with aq. NaCl. Compounds 47, 49, 50, 51 and
53 were eluted with 0.2  NaCl, compounds 46 and 52 were
eluted with 0.5  NaCl and compound 48 was eluted with 1.0 
NaCl. The eluate was evaporated under reduced pressure
(<35 ЊC) and the residue was subjected to gel filtration with
water as eluent (Sephadex G25; column dimensions 2.5 cm × 90
cm). Carbohydrate-containing fractions were combined, con-
centrated and freeze dried to give the sulfated hexose.
᎐
᎐
[2(M ϩ Na)ϩ, 4%], 557 [2(M ϩ H)ϩ, 8], 301 [(M ϩ Na)ϩ, 30],
279 [(M ϩ H)ϩ, 100], 278 (6), 277 (6), 259 (7), 247 (94) and 127
(25).
Methyl 6-O-acetyl-á-D-glucopyranoside 33 and methyl 3-O-
acetyl-á-D-glucopyranoside 34
Triethylamine (2.00 cm3, 7.17 × 10Ϫ3 mol), vinyl acetate (13.00
cm3, 7.05 × 10Ϫ2 mol) and R. toruloides esterase (10 g) were
added to a solution of glucopyranoside 32 (3.25 g, 1.68 × 10Ϫ2
mol) in THF (50 cm3). After stirring of the mixture at 25 ЊC for
1.5 h, TLC analysis showed the formation of two products (Rf
0.42, 0.3; ethyl acetate–ethanol, 9:1, v/v). The suspension was
filtered through Celite, the Celite was washed with ethyl acetate
(3 × 30 cm3), and the filtrate was evaporated under reduced
pressure to give an orange syrup. Purification by flash chroma-
tography (ethyl acetate–ethanol, 20:1, v/v) yielded products 33
(150 mg, 4%) and 34 as syrups. Crystallization from acetone
yielded 3-acetate 34 (199 mg, 5%); 6-acetate 33 [Found:
(M ϩ Na)ϩ, (FAB, Na) 259.080 00. C9H16NaO7 requires m/z,
259.079 37]; [α]D22 ϩ32.6 (c 0.46, MeOH); νmax(Nujol)/cmϪ1 3600–
Sodium 2-acetamido-1,3,4-tri-O-acetyl-2-deoxy-á-D-gluco-
pyranose 6-sulfate 38
A mixture of the N-acetylglucosamine derivative 22 (317 mg,
0.91 mmol), anhydrous pyridine (10 cm3) and sulfur trioxide–
pyridine complex (360 mg, 2.26 mmol) was stirred at room
temp. for 5 h. Water (30 cm3) was added and the product was
purified as described in the general procedure to give sulfate 38
(260 mg, 63%) as a powder (decomp. 186 ЊC) [Found: (M ϩ
Na)ϩ, 472.0519. C14H20NNa2O12S requires m/z, 472.0502];
J. Chem. Soc., Perkin Trans. 1, 1998
1077

View Article Online
[α]D20 ϩ62.7 (c 0.5, water); δH(400 MHz; D2O) 1.96 (3 H, s,
CH3CO), 2.06 (3 H, s, CH3CO), 2.10 (3 H, s, CH3CO),
2.23 (3 H, s, CH3CO), 4.11 (1 H, dd, J 2.3 and 11.6, Ha-6), 4.16
(1 H, dd, J 4 and 11.6, Hb-6), 4.34 (1 H, ddd, J 2.3, 4 and 10,
H-5), 4.49 (1 H, dd, J 3.65 and 11, H-2), 5.17 (1 H, app t, J 10
and 10, H-4), 5.37 (1 H, dd, J 10 and 11, H-3) and 6.12 (1 H,
d, J 3.65, H-1); δC(100 MHz; D2O) 20.73 (CH3CO), 20.84
(CH3CO), 20.92 (CH3CO), 22.36 (CH3CO), 50.84 (C-2), 66.48
(C᎐O), 173.29 (C᎐O), 173.46 (C᎐O) and 173.86 (C᎐O); m/z (rel.
᎐ ᎐ ᎐ ᎐
ab.) 473 [(M ϩ Na)ϩ, 100%], 451 (50), 446 (50), 369 (60) and
311 (80).
Sodium 1,2,3,4-tetra-O-acetyl-á-D-mannopyranose 6-sulfate 42
A mixture of mannopyranoside 7 (850 mg, 2.44 mmol),
anhydrous pyridine (15 cm3) and sulfur trioxide–pyridine com-
plex (1.0 g, 6.28 mmol) was stirred at room temp. for 2 h. Water
(30 cm3) was added and the mixture was purified as described in
the general procedure to give sulfate 42 (980 mg, 89%) as a
powder (decomp. 166 ЊC) [Found: (M ϩ Na)ϩ, 473.0333.
C14H19Na2O13S requires m/z, 473.0342]; [α]D21 ϩ58.2 (c 0.5,
water); δH(400 MHz; D2O) 2.04 (3 H, s, CH3CO), 2.12 (3 H, s,
CH3CO), 2.21 (3 H, s, CH3CO), 2.23 (3 H, s, CH3CO), 4.13
(1 H, dd, J 2.3 and 11.6, Ha-6), 4.20 (1 H, dd, J 3.65 and 11.6,
Hb-6), 4.34–4.38 (1 H, m, H-5), 5.37–5.45 (3 H, m, H-2, -3, -4)
and 6.09 (1 H, d, J 2, H-1); δC(100 MHz; D2O) 20.81
(2 × CH3CO), 20.87 (2 × CH3CO), 66.04 (C-6), 66.45, 69.21,
(C-6), 69.02, 70.09, 71.64, 91.26 (C-1), 172.87 (C᎐O), 173.22
᎐
(C᎐O), 173.90 (C᎐O) and 175.15 (C᎐O); m/z (rel. ab.) 472
᎐
᎐
᎐
[(M ϩ Na)ϩ, 60%], 368 (35), 310 (35), 119 (40), 87 (60) and 82
(100).
Sodium 1,2,3,4-tetra-O-acetyl-á-D-glucopyranose 6-sulfate 39
The glucose derivative 5 (406.2 mg, 1.17 mmol) was dissolved in
anhydrous pyridine (10 cm3). Sulfur trioxide–pyridine complex
(470 mg, 2.95 mmol) was added. The mixture was stirred at
room temp. for 1.5 h. Water (30 cm3) was added and the product
was purified as described in the general procedure to give com-
pound 39 (390 mg, 74%) as a powder (decomp. 190 ЊC) [Found:
(M ϩ Na)ϩ 473.0329. C14H19Na2O13S requires m/z, 473.0342];
[α]D23 ϩ61.6 (c 0.25, water); δH(400 MHz; D2O) 2.05 (3 H, s,
CH3CO), 2.06 (3 H, s, CH3CO), 2.09 (3 H, s, CH3CO), 2.22 (3 H,
s, CH3CO), 4.11 (1 H, dd, J 2.5 and 11.8, Ha-6), 4.15 (1 H, dd,
J 3.8 and 11.8, Hb-6), 4.38 (1 H, ddd, J 2.5, 3.8 and 10.3, H-5),
5.21 (1 H, dd, J 9.8 and 10.3, H-4), 5.23 (1 H, dd, J 3.7 and 9.8,
H-2), 5.50 (1 H, app t, J 9.8 and 9.8, H-3) and 6.30 (1 H, d,
J 3.7, H-1); δC(100 MHz; D2O) 20.63 (CH3CO), 20.83
(2 × CH3CO), 20.91 (CH3CO), 66.36, 68.41, 69.88, 70.15,
70.34, 70.86, 91.32 (C-1), 172.08 (C᎐O), 173.35 (C᎐O), 173.39
᎐
᎐
(C᎐O) and 173.51 (C᎐O); m/z (rel. ab.) 473 [(M ϩ Na)ϩ, 100%],
᎐
᎐
451 (40), 311 (55) and 301 (30).
Sodium 2-acetamido-1,3,6-tri-O-acetyl-2-deoxy-á-D-gluco-
pyranose 4-sulfate 43
The N-acetylglucosamine derivative 3 (390 mg, 1.12 mmol)
was dissolved in anhydrous pyridine (13 cm3). Sulfur trioxide–
pyridine complex (340 mg, 2.50 mmol) was added. The mix-
ture was stirred at room temp. for 2 h. Water (10 cm3) was
added and the mixture was then freeze dried. The residue was
purified as described in the general procedure to give sulfate
43 (483 mg, 95%) as a powder (decomp. 177 ЊC) [Found:
(M ϩ Na)ϩ, 472.0510. C14H20NNa2O12S requires m/z,
472.0502]; [α]D19 ϩ46.8 (c 0.25, water); δH(250 MHz; D2O) 1.91
(3 H, s, CH3CO), 2.04 (3 H, s, CH3CO), 2.06 (3 H, s, CH3CO),
2.16 (3 H, s, CH3CO), 4.13–4.19 (1 H, m, H-5), 4.23 (1 H, dd,
J 2.6 and 12.5, Ha-6), 4.31 (1 H, dd, J 3.8 and 12.5, Hb-6),
4.40 (1 H, dd, J 3.8 and 11, H-2), 4.51 (1 H, app t, J 9 and 9, H-
4), 5.34 (1 H, dd, J 9 and 11, H-3) and 6.04 (1 H, d, J 3.78, H-1);
δC(100 MHz; D2O) 20.81 (2 × CH3CO), 21.00 (CH3CO),
22.23 (CH3CO), 50.81 (C-2), 63.07 (C-6), 70.33, 71.08, 74.24,
71.34, 89.89 (C-1), 172.78 (C᎐O), 173.15 (2 × C᎐O) and 173.98
᎐
᎐
(C᎐O); m/z (rel. ab.) 473 [(M ϩ Na)ϩ, 80%], 371 (40) and 149
᎐
(100).
Sodium 2-acetamido-1,3,4-tri-O-acetyl-2-deoxy-á-D-galacto-
pyranose 6-sulfate 40
The N-acetylgalactosamine derivative 9 (100 mg, 0.29 mmol)
was dissolved in anhydrous pyridine (5 cm3). Sulfur trioxide–
pyridine complex (120 mg, 0.75 mmol) was added. The mix-
ture was stirred at room temp. for 2 h. Water (10 cm3) was
added and the mixture was freeze dried. The residue was puri-
fied as described in the general procedure to give sulfate 40
(125 mg, 93%) as a powder (decomp. 177 ЊC) [Found:
(M ϩ Na)ϩ 472.0502. C14H20NNa2O12S requires m/z,
472.0502]; [α]D22 ϩ70 (c 0.25, water); δH(400 MHz; D2O) 1.93
(3 H, s, CH3CO), 1.99 (3 H, s, CH3CO), 2.17 (3 H, s, CH3CO),
2.18 (3 H, s, CH3CO), 4.00–4.08 (2 H, m, H2-6), 4.51 (1 H, t,
J 6, H-5), 4.58 (1 H, dd, J 3.65 and 11.6, H-2), 5.28 (1 H, dd, J 3
and 11.6, H-3), 5.52 (1 H, d, J 3, H-4) and 6.15 (1 H, d, J 3.65,
H-1); δC(100 MHz; D2O) 20.64 (2 × CH3CO), 20.84 (CH3CO),
22.32 (CH3CO), 47.07 (C-2), 66.31 (C-6), 68.05, 68.80, 69.26,
91.67 (C-1), 172.98 (C᎐O), 173.46 (C᎐O), 173.95 (C᎐O) and
91.10 (C-1), 172.87 (C᎐O), 173.92 (C᎐O), 174.57 (C᎐O) and
᎐
᎐
᎐
175.21 (C᎐O); m/z (rel. ab.) 472 [(M ϩ Na)ϩ, 100%], 370 (50)
᎐
and 149 (60).
Sodium 1,2,3,6-tetra-O-acetyl-á-D-glucopyranose 4-sulfate 44
A mixture of the glucose derivative 22 (130 mg, 0.37 mmol),
anhydrous pyridine (10 cm3) and sulfur trioxide–pyridine com-
plex (140 mg, 0.88 mmol) was stirred at room temp. After 2 h,
water (15 cm3) was added and the mixture was freeze dried. The
residue was purified as described in the general procedure to
give sulfate 44 (165 mg, 98%) as a powder [Found: (M ϩ Na)ϩ,
473.0342. C14H19Na2O13S requires m/z, 473.0342]; [α]D20 ϩ56.6
(c 0.5, water); δH(250 MHz; D2O) 1.88 (3 H, s, CH3CO), 2.05
(3 H, s, CH3CO), 2.08 (3 H, s, CH3CO), 2.20 (3 H, s, CH3CO),
4.19–4.32 (3 H, m, H-5, H2-6), 4.58 (1 H, app t, J 9.3 and 9.3, H-
4), 5.17 (1 H, dd, J 3.78 and 10.46, H-2), 5.51 (1 H, dd, J 9.3 and
10.46, H-3) and 6.25 (1 H, d, J 3.78, H-1); δC(63 MHz; D2O)
20.79 (CH3CO), 21.02 (2 × CH3CO), 21.30 (CH3CO), 63.14 (C-
᎐
᎐
᎐
175.31 (C᎐O); m/z (rel. ab.) 472 [(M ϩ Na)ϩ, 100%], 368 (40)
᎐
and 310 (60).
Sodium 1,2,3,4-tetra-O-acetyl-á-D-galactopyranose 6-sulfate 41
A mixture of the galactose derivative 11 (200 mg, 0.58 mmol),
anhydrous pyridine (10 cm3) and sulfur trioxide–pyridine com-
plex (250 mg, 1.57 mmol) was stirred at room temp. for 2 h.
Water (30 cm3) was added and the mixture was freeze dried.
The residue was purified as described in the general procedure
to give sulfate 41 (245 mg, 94%) as a powder [Found:
(M ϩ Na)ϩ, 473.0337. C14H19Na2O13S requires m/z, 473.0342];
[α]D22 ϩ82.4 (c 0.25, water); δH(400 MHz; D2O) 2.03 (3 H, s,
CH3CO), 2.07 (3 H, s, CH3CO), 2.209 (3 H, s, CH3CO), 2.211
(3 H, CH3CO), 4.07 (1 H, dd, J 6.30 and 10.95, Ha-6), 4.10 (1
H, dd, J 6 and 10.95, Hb-6), 4.59 (1 H, app t, J 6 and 6.3, H-5),
5.39 (1 H, dd, J 3.7 and 10.6, H-2), 5.47 (1 H, dd, J 3 and 10.6,
H-3), 5.60 (1 H, d, J 3, H-4) and 6.36 (1 H, d, J 3.7, H-1);
δC(100 MHz; D2O) 20.70 (2 × CH3CO), 20.76 (CH3CO), 20.89
(CH3CO), 66.24, 67.40, 68.77, 68.86, 69.41, 90.38 (C-1), 172.93
6), 69.98, 70.62, 71.02, 73.97, 89.88 (C-1), 172.97 (C᎐O), 173.35
᎐
(C᎐O), 174.29 (C᎐O) and 174.72 (C᎐O); m/z (rel. ab.) 473
᎐
᎐
᎐
[(M ϩ Na)ϩ, 100%] and 371 (60).
Sodium 1,2,3,6-tetra-O-acetyl-á-D-mannopyranose 4-sulfate 45
A mixture of the mannose derivative 23 (80 mg, 0.23 mmol),
anhydrous pyridine (5 cm3) and sulfur trioxide–pyridine com-
plex (90 mg, 0.57 mmol) was stirred at room temp. (15 ЊC). After
7 h, water (15 cm3) was added and the mixture was purified as
described in the general procedure to give sulfate 45 (97 mg,
93%) as a powder (decomp. 149 ЊC) [Found: (M ϩ Na)ϩ,
473.0334. C14O19Na2O13S requires m/z, 473.0342]; [α]D22 ϩ51.2
1078
J. Chem. Soc., Perkin Trans. 1, 1998

View Article Online
(c 0.25, water); δH(250 MHz; D2O) 2.03 (3 H, s, CH3CO), 2.08
(3 H, s, CH3CO), 2.14 (3 H, s, CH3CO), 2.17 (3 H, s, CH3CO),
4.17–4.26 (2 H, m, H-5, Ha-6), 4.36 (1 H, dd, J 3.77 and 12.49,
Hb-6), 4.70 (1 H, app t, J 9.9 and 9.9, H-4), 5.29 (1 H, dd, J 2
and 3.5, H-2), 5.44 (1 H, dd, J 3.5 and 9.9, H-3) and 6.00 (1 H,
d, J 2, H-1); δC(100 MHz; D2O) 20.87 (2 × CH3CO), 21.05
(2 × CH3CO), 63.14 (C-6), 69.60, 69.68, 71.10, 71.83, 91.08
and 10.46, Ha-6), 3.84 (1 H, dd, J 3.20 and 10.46, Hb-6), 3.88–
4.29 (4 H, m, 4 × HC-O), 4.57 (0.6 H, d, J 7.85, H-1β) and 5.23
(0.4 H, d, J 3.78, H-1α); δC(63 MHz; D2O) 68.15, 68.53, 69.06,
69.17, 69.36, 69.76, 69.94, 72.57, 73.39, 73.57, 93.21 and 87.28;
m/z (rel. ab.) 259 [(M Ϫ Na)Ϫ, 100%] and 97 (30).
Sodium D-mannopyranose 6-sulfate 50
(C-1), 172.08 (C᎐O), 173.28 (C᎐O), 173.60 (C᎐O) and 174.57
᎐
᎐
᎐
The sulfate 42 (303 mg, 0.67 mmol) and sodium methoxide (110
mg, 2.04 mmol) were suspended in anhydrous methanol (20
cm3). The mixture was stirred at room temp. for 2 h. Water (20
cm3) was added and the mixture was purified as described in
the general procedure to give sulfate 50 (180 mg, 94%) as a
syrup [Found: (M ϩ Na)ϩ, 304.9916. C6H11NNa2O9S requires
m/z, 304.9914]; [α]D23 ϩ17.8 (c 0.5, water); δH(250 MHz; D2O)
3.55–3.72 (2 H, m, 2 × HC-O), 3.83 (0.57 H, dd, J 3.19 and
9.59, HC-O), 3.90–3.93 (1 H, m, HC-O), 3.95–4.02 (0.57 H, m,
HC-O), 4.14–4.33 (2 H, m, H2-6), 4.90 (0.43 H, d, J 0.87, H-1β)
and 5.15 (0.57 H, d, J 1.45, H-1α); δC(63 MHz; D2O) 67.04,
67.29, 68.26, 70.95, 71.23, 71.40, 73.01, 74.80, 94.63 and 95.00;
m/z (rel. ab.) 305 [(M ϩ Na)ϩ, 100%].
(C᎐O); m/z (rel. ab.) 473 [(M ϩ Na)ϩ, 100%], 371 (90) and 289
᎐
(40).
Sodium 2-acetamido-2-deoxy-D-glucopyranose 6-sulfate 46
The sulfate 38 (160 mg, 0.36 mmol) and sodium methoxide
(50 mg, 0.93 mmol) were suspended in anhydrous methanol
(25 cm3). The mixture was stirred at room temp. for 6 h. Water
(25 cm3) was added and the mixture was purified as described in
the general procedure to give the sulfate 46 (90 mg, 78%) as a
powder (decomp. 164 ЊC) [Found: (M ϩ Na)ϩ, 346.0182.
C8H14NNa2O9S requires m/z, 346.0179]; [α]D21 ϩ24.2 (c 0.5,
water); δH(400 MHz; D2O) 1.95 (3 H, s, CH3CO), 3.42–3.98
(4 H, m, 4 × HC᎐O), 4.11–4.26 (2 H, m, H2-6), 4.64 (0.4 H, d,
J 8.63, H-1β) and 5.11 (0.6 H, d, J 3.32, H-1α); δC(100 MHz;
D2O) 22.60 (CH3CO), 22.87 (CH3CO), 54.60, 57.21, 67.82,
70.17, 70.28, 70.41, 71.29, 74.37, 91.59 (C-1α), 95.68 (C-1β),
175.19 and 175.47; m/z (rel. ab.) 346 [(M ϩ Na)ϩ, 25%], 244
(15) and 79 (100).
Sodium 2-acetamido-2-deoxy-D-glucopyranose 4-sulfate 51
The sulfate 43 (270 mg, 0.60 mmol) and sodium methoxide (70
mg, 0.13 mmol) were suspended in anhydrous methanol (45
cm3). The mixture was stirred at room temp. for 8 h. The reac-
tion was quenched by addition of water (40 cm3) and the mix-
ture was purified as described in the general procedure to give
sulfate 51 (160 mg, 82%) as a powder (decomp. 157 ЊC) [Found:
(M ϩ Na)ϩ, 346.0179. C8H14NNa2O9S requires m/z, 346.0179];
[α]D22 ϩ19.0 (c 0.5, water); δH(250 MHz; D2O) 2.00 (3 H, s,
CH3CO), 3.52–4.23 (6 H, m, 6 × HC-O), 4.69 (0.4 H, d, J 8.48,
H-1β) and 5.17 (0.6 H, d, J 2.62, H-1α); δC(63 MHz; D2O) 22.76
(CH3CO), 23.04 (CH3CO), 54.74, 57.28, 61.20, 61.37, 70.06,
Sodium D-glucopyranose 6-sulfate 47
The sulfate 39 (766 mg, 1.70 mmol) and sodium methoxide (280
mg, 5.18 mmol) were suspended in anhydrous methanol (50
cm3). The mixture was stirred at room temp. for 3 h. The reac-
tion was quenched by addition of water (50 cm3). The mixture
was purified as described in the general procedure to give
sulfate 47 (440 mg, 92%) as a powder (decomp. 170 ЊC) [Found:
(M ϩ Na)ϩ, 304.9915. C6H11NNa2O9S requires m/z, 304.9914];
[α]D14 ϩ23.3 (c 0.5, water); δH(250 MHz; D2O) 3.16–3.24 (0.6 H,
m, HC-O), 3.37–3.70 (3 H, m, 3 × HC-O), 3.93–4.00 (0.4 H, m,
HC-O), 4.10–4.29 (2 H, m, H2-6), 4.61 (0.6 H, d, J 7.85, H-1β)
and 5.17 (0.4 H, d, J 5.17, H-1α); δC(63 MHz; D2O) 67.94,
70.05, 70.09, 70.30, 72.16, 73.44, 74.58, 74.79, 76.37, 92.99
(C-1α) and 96.81 (C-1β); m/z (rel. ab.) 305 [(M ϩ Na)ϩ, 100%]
and 99 (75).
70.66, 73.19, 75.19, 77.77, 78.14, 91.28, 95.68, 175.40 (C᎐O)
᎐
and 175.68 (C᎐O); m/z (rel. ab.) 346 [(M ϩ Na)ϩ, 12%] and 79
᎐
(100).
Sodium D-glucopyranose 4-sulfate 52
The sulfate 44 (160 mg, 0.36 mmol) and sodium methoxide (60
mg, 1.11 mmol) were suspended in anhydrous methanol (25
cm3). The mixture was stirred at room temp. After 5 h, the
reaction was quenched by addition of water (25 cm3). The mix-
ture was purified as described in the general procedure to give
sulfate 52 (80 mg, 80%) as a hygroscopic powder [Found:
(M ϩ Na)ϩ, 304.9916. C6H11NNa2O9S requires m/z, 304.9914];
[α]D14 ϩ34.8 (c 0.5, water); δH(400 MHz; D2O) 3.30 (0.6 H, dd, J 8
and 9.5, β anomer), 3.55–3.94 (5 H, m, 5 × HC-O), 4.12 (1 H,
app t, J 9.4, H-4β), 4.13 (1 H, app t, J 9.6, H-4α), 4.64 (0.6 H, d,
J 7.96, H-1β) and 5.21 (0.4 H, d, J 3.65, H-1α); δC(100 MHz;
D2O) 61.22, 61.39, 70.52, 72.04, 72.13, 74.74, 75.09, 75.18,
77.59, 77.82, 92.51 and 96.61; m/z (rel. ab.) 305 [(M ϩ Na)ϩ,
100%], 203 (30) and 99 (15).
Sodium 2-acetamido-2-deoxy-D-galactopyranose 6-sulfate 48
The sulfate 46 (78 mg, 0.17 mmol) and sodium methoxide (28
mg, 0.52 mmol) were suspended in anhydrous methanol (10
cm3). The mixture was stirred at room temp. After 3 h, the
reaction was quenched by addition of water (10 cm3) and the
mixture was purified as described in the general procedure to
give sulfate 48 (34 mg, 61%) as a hygroscopic powder [Found:
(M ϩ Na)ϩ, 346.0176. C8H14NNa2O9S requires m/z, 346.0179];
[α]D22 ϩ38.6 (c 0.5, water); δH(400 MHz; D2O) 2.002 (3 H, s,
CH3CO), 2.004 (3 H, s, CH3CO), 3.69 (0.4 H, dd, J 3.32 and
10.62, HC-O), 3.82–4.30 (6.6 H, m, HC-O), 4.62 (0.4 H, d, J
8.29, H-1β) and 5.19 (0.6 H, d, J 3.65, H-1α); δC(63 MHz; D2O)
22.78 (CH3CO), 23.03 (CH3CO), 50.95, 54.30, 67.96, 68.09,
68.39, 68.48, 69.12, 69.20, 71.72, 73.57, 175.52 (C-1α) and
175.80 (C-1β); m/z (rel. ab.) 300 [(M Ϫ Na)Ϫ, 100%], 199 (20)
and 45 (55).
Sodium D-mannopyranose 4-sulfate 53
The sulfate 45 (400 mg, 0.90 mmol) and sodium methoxide
(170 mg, 3.15 mmol) were suspended in anhydrous methanol
(40 cm3). The mixture was stirred at room temp. After 4 h,
the reaction was quenched by addition of water (30 cm3).
The mixture was purified as described in the general procedure
to give sulfate 53 (220 mg, 88%) as a powder (decomp. 171 ЊC)
[Found: (M ϩ Na)ϩ, 304.9911. C6H11NNa2O9S requires
m/z, 304.9914]; [α]D14 ϩ13.6 (c 0.5, water); δH(250 MHz; D2O)
3.46 (0.33 H, ddd, J 2.33, 6.10 and 9.60, H-5α), 3.65–4.00
(4.67 H, m, 4.67 × HC-O), 4.25 (0.33 H, app t, J 9.6, H-4β),
4.32 (0.67 H, app t, J 9.6, H-4α), 4.86 (0.33 H, d, J 1.16,
H-1β) and 5.12 (0.37 H, d, J 2.04, H-1α); δC(63 MHz; D2O)
61.50, 61.56, 69.89, 71.56, 72.00, 72.60, 75.27, 75.85, 76.17,
94.40 and 94.45; m/z (rel. ab.) 259 [(M Ϫ Na)Ϫ, 100%] and 97
(35).
Sodium D-galactopyranose 6-sulfate 49
The sulfate 41 (173 mg, 0.38 mmol) and sodium methoxide
(60 mg, 1.11 mmol) were suspended in anhydrous methanol
(10 cm3). The mixture was stirred at room temp. for 2.5 h. The
reaction was quenched by the addition of water (10 cm3) and
the mixture was purified as described in the general procedure
to give sulfate 49 (90 mg, 83%) as a powder (decomp. 150 ЊC)
[Found: (M ϩ Na)ϩ, 304.9913. C6H11NNa2O9S requires
m/z, 304.9914]; [α]D14 ϩ46.8 (c 0.5, water) {lit.,31 [α]D14 ϩ47};
δH(250 MHz; D2O) 3.45 (0.6 H, dd, J 7.85 and 9.88, HC-O),
3.63 (0.6 H, dd, J 3.48 and 9.88, HC-O), 3.76 (0.4 H, dd, J 3.48
J. Chem. Soc., Perkin Trans. 1, 1998
1079

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3431; (x) G. Carrea, S. Riva, F. Secundo and B. Danieli, J. Chem.
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Paper 7/08596F
Received 26th November 1997
Accepted 26th November 1997
1080
J. Chem. Soc., Perkin Trans. 1, 1998