Regioselective enzymatic hydrolysis of acetylated pyranoses and pyranosides using immobilised lipases. An easy chemoenzymatic synthesis of α- and β-D-glucopyranose acetates bearing a free secondary C-4 hydroxyl group

Article abstract of DOI:10.1016/S0008-6215(02)00113-1

Protected sugars with only one free hydroxyl group are useful building blocks for the synthesis of a large number of glycoderivatives. In order to avoid the problems of the classical chemical synthesis, we studied the regioselective enzymatic hydrolysis of different fully acetylated glycopyranoses and glycopyranosides. The main challenge was to obtain the hydrolysis of the substrates in only one position, with high regioselectivity, while avoiding any further hydrolysis towards partially acetylated sugars. Candida rugosa (CRL) and Pseudomonas fluorescens (PFL) lipases (EC 3.1.1.3) immobilised on octyl agarose afforded regioselective hydrolysis only in the 6- and 1-positions, respectively. Furthermore, a new one-pot chemoenzymatic approach has been developed in order to obtain α- and β-protected glucopyranoses bearing a free secondary C-4 hydroxyl group. For instance, 1,2,3,6-tetra-O-acetyl-α-D-glucopyranose was easily synthesised in good overall yield (70%) starting from 1,2,3,4,6-penta-O-acetyl-α-D-glucopyranose by regioselective enzymatic hydrolysis in the 6-position, catalysed by CRL, followed by a temperature- and pH-controlled acyl migration.

Full text of DOI:10.1016/S0008-6215(02)00113-1

Carbohydrate Research 337 (2002) 1615–1621
www.elsevier.com/locate/carres
Regioselective enzymatic hydrolysis of acetylated pyranoses and
pyranosides using immobilised lipases. An easy chemoenzymatic
synthesis of a- and b-
D
-glucopyranose acetates bearing a free
secondary C-4 hydroxyl group
a,
a
a
b
Marco Terreni, * Raul Salvetti, Laura Linati, Roberto Fernandez-Lafuente,
b
b,
b
Gloria Fern a´ ndez-Lorente, Agatha Bastida, * Jos e´ M. Guisan
a
Pharmaceutical Biocatalysis Laboratories, Department of Pharmaceutical Chemistry, Via Taramelli 12, Uni6ersity of Pa6ia, Pa6ia, Italy
Laboratorio de Tecnolog ´ı a Enzim a´ tica, Departamento de Biocat a´ lisis, Instituto de Cat a´ lisis, CSIC, Cantoblanco, 28049 Madrid, Spain
b
Received 15 October 2001; accepted 29 April 2002
Abstract
Protected sugars with only one free hydroxyl group are useful building blocks for the synthesis of a large number of
glycoderivatives. In order to avoid the problems of the classical chemical synthesis, we studied the regioselective enzymatic
hydrolysis of different fully acetylated glycopyranoses and glycopyranosides. The main challenge was to obtain the hydrolysis of
the substrates in only one position, with high regioselectivity, while avoiding any further hydrolysis towards partially acetylated
sugars. Candida rugosa (CRL) and Pseudomonas fluorescens (PFL) lipases (EC 3.1.1.3) immobilised on octyl agarose afforded
regioselective hydrolysis only in the 6- and 1-positions, respectively. Furthermore, a new one-pot chemoenzymatic approach has
been developed in order to obtain a- and b-protected glucopyranoses bearing a free secondary C-4 hydroxyl group. For instance,
1
,2,3,6-tetra-O-acetyl-a-
D-glucopyranose was easily synthesised in good overall yield (70%) starting from 1,2,3,4,6-penta-O-acetyl-
a- -glucopyranose by regioselective enzymatic hydrolysis in the 6-position, catalysed by CRL, followed by a temperature- and
pH-controlled acyl migration. © 2002 Elsevier Science Ltd. All rights reserved.
D
Keywords: Regioselective hydrolysis; Acetylated pyranosides; Lipase; Acetyl group migration
1. Introduction
For example, 1,2,3,6-tetra-O-acetyl-a-D-glucopyran-
ose and related compounds, may be of particular inter-
est due to the presence of (1“4)-glycosidic bonds in
natural oligosaccharides and in those of pharmaceutical
interest. However, only few papers describing suitable
Pure regioisomers of O-acetyl pyranosides bearing
only one free hydroxyl group (AP) may be used as key
intermediates for the synthesis of a large number of
glycoderivatives (oligosaccharides, sugar esters, gly-
3
,4
1
,2
chemical syntheses of APs have been published. The
,2,3,4,6-penta-O-acetyl-a- -pyranose (1) could eco-
copeptides, etc.). AP intermediates can be readily and
selectively modified at their free hydroxyl group and are
soluble in most organic solvents. Moreover, the pro-
tected final products can be easily deacetylated by very
mild chemical or enzymatic processes. However, the
preparation of APs with only one free hydroxyl group
requires by classical chemical synthesis multistep reac-
tions that may pose environmental problems for large-
scale production, due to the use of organic solvents and
toxic reagents.
1
D
nomically be used in regioselective deacetylation for
large-scale preparation of APs. Although a large num-
ber of procedures has been reported, suitable processes
for the chemical hydrolysis of 1 only afford the
5
deacetylation at the anomeric position. An alternative
approach for obtaining protected sugars with only one
free secondary hydroxyl group, including the 4-posi-
tion, employs dispiroketals for regioselective protection
6
of vicinal diols.
*
Corresponding authors. Tel.: +39-382-507-361; fax: +
The complexity of the classical chemical approaches
3
9-382-422-975
E-mail address: markt@pbl.unipv.it (M. Terreni).
for regioselective protection/deprotection of sugars
0
008-6215/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(02)00113-1

1
616
M. Terreni et al. / Carbohydrate Research 337 (2002) 1615–1621
makes the use of enzymatic catalysts such as lipases or
esterases an attractive and important tool for AP
preparation. However, enzymatic deacylations of fully
acylated pyranosides described in literature have some
drawbacks. In most cases the reactions are very slow
or proceed with poor selectivity and yield. In addition
these reactions often afford complex mixtures of tetra-,
tri-, di-, and monoacetates, along with free monosac-
charides such as glucose.
In the present work we have extended the previous
studies in order to obtain regioisomers of APs bearing
only one free hydroxyl group in different position. We
report the results obtained studying the regioselective
enzymatic hydrolysis of different acetylated pyran-
osides, catalysed by CRL and PFL immobilised on
octyl agarose. In particular, biocatalytic processes suit-
able for obtaining different regioisomers of AP in high
yields that avoid any further hydrolyses towards par-
tially acetylated or free sugars are described. Further-
more, a new, efficient chemoenzymatic process for the
2
,7
Some lipase-catalysed hydrolyses of fully acylated
pyranosides have been reported to afford deacetylation
8
,9
at the anomeric or C-6 positions
in good yields.
easy preparation of protected a- and b-D-glucopyran-
oses bearing a free secondary C-4 hydroxyl group is
proposed.
Regioselective hydrolysis at the primary position can be
routinely obtained by protection of the C-1 hydroxyl
group as the alkyl glycoside. The hydrolysis of sugar
esters in positions other than the anomeric or the C-6
position with good regioselectivity and yield has never
been reported. Furthermore, fully acetylated sugars are
usually poor substrates when soluble lipases are used,
whereas pentanoyl or longer chain esters result in im-
2. Results and discussion
Enzymatic hydrolysis of 1,2,3,4,6-penta-O-acetyl-h-D-
glucopyranose (1).—In the hydrolysis of 1 catalysed by
lipases isolated from CRL and PFL, at acid pH, the
regioselective hydrolysis only in the 6- and 1-positions,
respectively, is obtained affording (Scheme 1) the corre-
sponding tetra acetyl glucopyranoses (AP): 1,2,3,4-
8
proved activity.
10
In a previous communication we have reported that
Candida rugosa (CRL) and Pseudomonas fluorescens
(
PFL) afford APs in good yield by hydrolysis of 1.
The main problem was to design a very active catalyst
able to perform the selective hydrolysis of acetylated
pyranoses in only one position. New lipase derivatives
prepared by hydrophobic absorption on octyl agarose
tetra-O-acetyl-a-
D
-glucopyranose (1a) and 2,3,4,6-
-glucopyranose (1c).
tetra-O-acetyl-a,b-
D
In order to clarify the mechanism of the enzymatic
hydrolysis of these substrates, we have considered the
effect of different reaction conditions on the regioselec-
tivity and yields. The influence of pH has been found to
play a pivotal role in hydrolysis selectivity. In fact, as
reported in Table 1, using CRL, as the pH increases,
1
1
gel have been used as catalysts. These immobilised
lipases display good activity and regioselectivity in the
hydrolyses performed in aqueous homogeneous
1
0
media.
Scheme 1.

M. Terreni et al. / Carbohydrate Research 337 (2002) 1615–1621
1617
Table 1
Hydrolysis of a-
a
D
-glucose pentaacetate catalysed by CRL and PFL at different pH
Lipase
pH
TAG % yield (t, h)
1a
1b
1c
Other TAG
CRL
CRL
CRL
CRL
CRL
CRL
PFL
PFL
4.00
5.00
6.00
7.00
8.00
9.00
5.00
7.00
90 (2 h)
75 (1.5 h)
60 (1.5 h)
57 (1 h)
46 (1 h)
34 (4 h)
100
98.3
–
–
–
–
–
–
–
–
–
2.3
2
17.3
32
–
1.7
2.8
70
61
28
–
94.9
28
21.7
40
–
86 (4.5 h)
\98 (3 h)
100
100
–
–
–
a
Reaction conditions: phosphate buffer 25 mM, 20% acetonitrile; substrate concentration=10 mM; 160 U of immobilised
lipase in 20 mL of reaction mixture.
R=OAc; R =H; R =OAc; R =OAc; R =H; R =
R,R =H,OH; R =OAc; R =OAc; R =H; R =OAc (1c)
1 2 3 4 5
1
2
3
4
5
OAc (2)
R=H; R =OMe; R =OAc; R =OAc; R =H; R =
R=H; R =OMe; R =OAc; R =OAc; R =H; R =OH
1 2 3 4 5
1
2
3
4
5
OAc (3)
(3a)
R=OMe; R =H; R =OAc; R =OAc; R =H; R =OH
R=OMe; R =H; R =OAc; R =OAc; R =H; R =
1
2
3
4
5
1
2
3
4
5
OAc (4)
(4a)
R=OBu; R =H; R =OAc; R =OAc; R =H; R =OAc R=OBu; R =H; R =OAc; R =OAc; R =H; R =OH (5a)
1
2
3
4
5
1
2
3
4
5
(
5)
R=H; R =OAc; R =NHAc; R =OAc; R =H; R =
R=H; R =OAc; R =NHAc; R =OAc; R =H; R =OH
1 2 3 4 5
(6a)
1
2
3
4
5
OAc (6)
R=OAc; R =H; R =OAc; R =H; R =OAc; R =OAc R=OAc; R =H; R =OAc; R =H; R =OAc; R =OH (7a)
1
2
3
4
5
1
2
3
4
5
(
7)
R=H; R =OAc; R =OAc; R =H; R =OAc; R =OAc R=H; R =OAc; R =OAc; R =H; R =OAc; R =OH (8a)
1
2
3
4
5
1
2
3
4
5
(
8)
Scheme 2.
regioselectivity and yield of APs markedly decrease.
Performing the hydrolysis at pH 4, a 90% yield and
complete regioselectivity toward the 6-position are ob-
tained, whereas a low yield and a mixture of com-
Enzymatic hydrolysis of different acetylated pyran-
osides.—We have studied the hydrolysis of different
acetylated pyranosides (substrates 2–8) catalysed by
CRL and PFL (Scheme 2). As shown in Table 2,
pounds 1a and 1,2,3,6-tetra-O-acetyl-a-
D
-gluco-
1,2,3,4,6-penta-O-acetyl-b- -glucopyranose (2) was
D
pyranose (1b) are observed under basic conditions. On
the other hand PFL gives a/b 1c (a 70:30 mixture of a
and b anomers) in quantitative yields at all pH values
tested.
preferentially hydrolysed at the anomeric position with
PFL to give a/b 1c in good yield. CRL was poorly
active towards this substrate and, surprisingly, afforded
also product 1c, although in very low yields (13%).
In order to also obtain the b anomers of APs bearing
only one free hydroxyl group at the 6-position, the
study of the regioselectivity of CRL and PFL was
enlarged to include substrates in which the anomeric
position was protected with an alkyl group such as
methyl or butyl. The results obtained in the hydrolysis
of the a and b anomers of methyl 2,3,4,6-tetra-O-acet-
The decrease of yield and the change in regioselectiv-
ity observed with CRL at basic pH may be assessed as
a probable consequence of an acetyl group migration
from the 4-position to the 6-position, which occurs in
neutral or basic medium. The enzyme first hydrolyses
the substrate in the 6-position, but because of acyl
group migration, further hydrolysis can occur with a
consequent reduction of the global AP’s yield. At pH 7,
for example, 1b can be obtained as a major product
yl- -glucopyranoside (compounds 3 and 4) are re-
D
ported in Table 2. Using CRL as catalyst, the best
activity was again obtained towards the a anomer 3;
however, the b anomer 4 was also hydrolysed at a
positive rate (complete hydrolysis achieved in ꢀ20 h).
(
70:30 ratio with compound 1a), but the global yield of
AP is much lower than that obtained when the reaction
is carried out at pH 4 (57% instead of 90% yield).

1
618
M. Terreni et al. / Carbohydrate Research 337 (2002) 1615–1621
Furthermore, CRL showed the same regioselectivity
towards both substrates, allowing hydrolysis in the
In the hydrolysis of 1,2,3,4,6-penta-O-acetyl-a- and
-galactopyranoses (compounds 7 and 8), CRL
b-D
6
-position to give methyl 2,3,4-tri-O-acetyl-a-
D
-
-
confirmed the results obtained in the hydrolysis of
substrates 1 and 2 concerning both activity and regiose-
lectivity. Hydrolysis in the 6-position was in fact ob-
served to obtain 1,2,3,4-penta-O-acetyl-a- and
glucopyranoside (3a) and methyl 2,3,4-tri-O-acetyl-b-
glucopyranoside (4a) in ꢀ90% yield. PFL was found to
be completely inactive towards both substrates 3 and 4.
D
In the hydrolysis of butyl 2,3,4,6-tetra-O-acetyl-b-
glucopyranoside (5), butyl 2,3,4-tri-O-acetyl-b-
D
-
-
b- -galactopyranoses (products 7a and 8a) in 94 and
D
D
43% yield, respectively (Table 2). The reaction rate in
both examples was much higher for the a than for the
b anomer.
glucopyranoside (5a) was obtained surprisingly with
both lipases. With this substrate, PFL showed much
higher activity in the hydrolysis than that shown to-
wards substrate 4. The activity of PFL towards 5 also
resulted in much higher activity than that shown by
CRL. The presence of a long aliphatic chain at the
anomeric position probably enables PFL to hydrolyse
this substrate. With this enzyme 5 was completely hy-
drolysed in only 7 h, and product 5a was obtained in
One-pot chemoenzymatic synthesis of 1,2,3,6-tetra-O-
acetyl-h-
Scheme 1, CRL is able to hydrolyse in a regiospecific
manner the penta-O-acetyl-a- -glucopyranose (1) at the
D-glucopyranose.—As previously shown in
D
6-position. However depending on the pH of the reac-
tion, the 4-hydroxy derivative 1b can also be isolated. In
fact, both regioselectivity and yields decreased with
increasing the pH of the reaction probably as a conse-
quence of acetyl group migration from the 4-position to
the 6-position, which occurs in neutral or basic medium.
This migration has been better studied considering the
effect of pH and temperature, in order to explore the
possibility to control it to obtain the 4-hydroxy deriva-
tive 1b in good yield (Scheme 3). The best results have
been obtained at low temperature (4 °C) and moderately
basic pH. At pH 8 a controlled acyl group migration
ꢀ
80% yield.
The regioselectivity of CRL has been further investi-
gated considering other fully acetylated pyranoses
Scheme 2). In the hydrolysis of 2-acetamido-1,3,4,6-
-glucopyranose (6) CRL
(
tetra-O-acetyl-2-deoxy-a-
D
showed an activity and a regioselectivity close to that
observed in the hydrolysis of compound 1, allowing
2
-acetamido-1,3,4-tri-O-acetyl-2-deoxy-a-glucopyran-
ose (6a) to be obtained in good yield (78%).
Table 2
a
Hydrolysis of acetylated glucopyranoses and glucopyranosides using CRL and PFL
Substrate
Enzyme
Conversion (%)
t (h)
Yield (%)
Product
2
2
3
3
4
4
5
5
6
7
8
CRL
PFL
CRL
PFL
CRL
PFL
CRL
PFL
CRL
CRL
CRL
44
98
48
20
5
18
21
22
72
7
13
90
86
n.d.
92
n.d.
90
81
78
94
1a
1a
3a
n.d.
4a
n.d.
5a
5a
6a
100
B2
100
B2
100
100
100
100
51
9
19
48
7a
8a
43
a
Reaction conditions: phosphate buffer 25 mM, 25% acetonitrile pH 5; substrate concentration=20 mM; 40 U of immobilised
lipase in 20 mL of reaction mixture; n.d.=not determined.
Scheme 3.

M. Terreni et al. / Carbohydrate Research 337 (2002) 1615–1621
1619
Table 3
pH 4 and 25 °C allowed 1a to be obtained in very good
yield (90%). After enzyme derivative filtration, the reac-
tion conditions were modified by lowering the tempera-
ture to 4 °C and increasing the pH to pH 8. Under
these conditions a controlled acetyl group migration
from the 4-position to the 6-position gave 1b (70%
overall yield after isolation). The same chemoenzymatic
procedure has been applied to compound 4. After a
purification step, the pure regioisomer, methyl 2,3,6-tri-
Effect of pH on acetyl group migration from the 4-position to
the 6-position at 4 °C
a
Compound
pH
Time (h)
Yield (%)
1a
1a
1a
1a
1a
4a
4a
7
7.5
8
8.5
9
7
12
12
7
4
4
52
75
79
48
44
44
70
O-acetyl-b-
D
-glucopyranoside (4b), has been isolated in
4
4
a yield of 68% (Scheme 4).
8
a
Yield of reaction evaluated by HPLC analysis.
3
. Conclusions
migration occurs allowing compound 1a to be obtained
in ꢀ80% yield in 7 h (Table 3).
In this work we have described the regioselective
In order to evaluate the possibility to obtain a- and
b-protected glucopyranoses bearing a free secondary
C-4 hydroxyl group, we have extended the acyl migra-
tion studies to methyl 2,3,4-tri-O-acetyl-b-D-glucopy-
ranoside (4a). Also in this case, low temperature and
hydrolysis of different acetylated pyranoses and pyran-
osides. The hydrolysis of a and b acetyl pyranoses
catalysed by lipases isolated from Candida rugosa
(CRL) and Pseudomonas fluorescens (PFL), afforded
APs with only a free hydroxyl group in the 6-position
or 1-position, respectively. By using CRL, the regiose-
lective enzymatic hydrolysis of many acetylated pyran-
oses including a and b alkyl glucopyranosides was
achieved Generally, this enzyme showed much more
activity towards the a rather than the b acetylated
pyranosides.
basic pH allowed us to control the migration from the
4
-position to the 6-position resulting in the isolation of
methyl 2,3,6-tri-O-acetyl-b- -glucopyranoside (4b). As
reported in Table 3, by performing the reaction at pH
, after 4 h we obtained the pure regioisomer 4b in
D
8
ꢀ
70% yield.
Further studies on 2-acetamido-1,3,4-tri-O-acetyl-2-
By a new one-pot chemoenzymatic approach, a free
hydroxyl group in the 4-position was also obtained.
This approach involved a temperature- and pH-con-
trolled acyl migration performed on the product ob-
tained by regioselective enzymatic hydrolysis in the
deoxy-a-
tyl-a- -galactopyranose (7a) are in progress.
Considering the good results obtained in the hydroly-
sis of a- -glucose pentaacetate (1), as well as those
D
-glucopyranose (6a) and 1,2,3,4-tetra-O-ace-
D
D
obtained in the studies of acetyl group migration, we
have investigated the possibility of the synthesis of 1b
by a new one-pot chemoenzymatic approach (Scheme
6-position catalysed by CRL of acetylated a- and b-
glucose derivatives. According to this, 1,2,3,6-tetra-O-
acetyl-a- -glucopyranose (1b) can be easily synthesised
D-
D
4
). This synthesis has been developed by a correct
in good overall yield, starting from a commercially
available substrate and avoiding any intermediate
purification. In the case of b anomers, glucopyranosides
alkylated at the anomeric position must be used be-
cause of the low activity showed by CRL towards
combination of regioselective enzymatic hydrolysis of 1
and pH/temperature-controlled acyl group migration.
In fact, starting from compound 1, using CRL immo-
bilised on octyl agarose, the hydrolysis performed at
Scheme 4.

1
620
M. Terreni et al. / Carbohydrate Research 337 (2002) 1615–1621
penta-O-acetyl-b-
,3,6-tri-O-acetyl-b-
prepared in good yield starting from methyl 2,3,4,6-
tetra-O-acetyl-b- -glucopyranoside (4). The results re-
ported in this work are of general interest for developing
industrial processes for the preparation of sugar build-
ing blocks useful for the synthesis of oligosaccharides
and other sugar derivatives. In fact, very easy-to-execute
chemical procedures are used for the acetylation of
sugars, while immobilised lipases are used to perform
reactions that are difficult to develop using classical
chemical reactions, i.e., the preparation of protected
sugars having only one free hydroxyl group.
D
-glucose (2). As an example methyl
strate (20 mM) in phosphate buffer 25 mM (80%) and
2
D
-glucopyranoside (4b) has been
acetonitrile (20–30%) at the desired pH value. The
hydrolytic reaction was carried out under mechanical
stirring, and pH was controlled by automatic titration.
Hydrolysis reactions were followed by HPLC. Finally,
after filtration of the immobilised enzyme, the products
were isolated by flash chromatography.
D
1,2,3,4-Tetra-O-acetyl-h-
D
-glucopyranose
(1a).
3
1
HPLC analysis: t =8.7 min. H NMR (CDCl ): l
R
1.93–2.1 (4 s, CH , 12H), 3.59–3.78 (2 m, ABX system,
3
2H-6), 3.61–3.75 (AB part of ABX system, J1,3 4.16 Hz,
J1,3 2.36 Hz, J1,2 12.86 Hz, 2H-6), 3.95 (dq, J 2.36 Hz,
J 4.16 Hz, J 10.31 Hz, 1H-5), 5.1 (dd, J 3.7 Hz, J 9.9 Hz,
1H-2), 5.14 (d, J 3.69 Hz, 1H-4), 5.56 (t, J 9.9 Hz,
1H-3), 6.38 (d, J 3.69 Hz, 1H-1). Anal. Calcd for
C H O : C, 48.28; H, 5.79; O, 45.94. Found: C, 48.60;
The results achieved in this work are strictly depend-
ing on a careful design and selection of the enzyme
derivative used. The influence of the enzyme derivative
preparation will be matter of forthcoming publications.
14
20 10
H, 5.40; O, 46.0.
,3,4,6-Tetra-O-acetyl-h-
HPLC analysis: t =7.1/8.2 min. H NMR (CDCl ): l
2
D
-glucopyranose
(1c).
3
1
R
4. Experimental
0.79 (t, J 7.3 Hz, CH , 3H), 1.29–1.43 (m, CH , 1H),
3
2
1
.48–1.6 (m, CH , 1H), 2.01 (4 s, CH , 12H), 3.42–3.81
2 3
General procedure.—CRL was obtained from Sigma
Chemical Co., while PFL (Lipase AK Amano 20) was
generously donated by Amano Enzyme Europe Ltd
(2 m, ABX system, 2H), 3.44 (m, J 9.6 Hz, 1H-5), 3.48
and 3.64 (AB part of ABX system, J1,3 3.54 Hz, J1,3 2.4
Hz, J1,2 12.4 Hz, 2H-6), 4.42 (d, J 8 Hz, 1H-1), 4.76 (dd,
J 7.9 Hz, J 9.7 Hz, 1H-2), 4.92 (t, J 9.8 Hz, 1H-4), 5.13
(t, J 9.6 Hz, 1H-3). Anal. Calcd for C H O : C, 48.28;
(
Milton Keynes MK9; UK). Octyl-agarose (Sepharose
CL-4B) was obtained from Amersham Pharmacia Bio-
tech (Uppsala, Sweden). All reagents were analytical
grade.
The pH of the solutions during the enzymatic hydrol-
ysis was kept constant using an automatic titrator 718
Stat Tritino from Metrohm (Herisau, Switzerland).
HPLC analyses were performed using an HPLC
Merck–Hitachi L-7100 (E. Merck, Darmstadt, Ger-
many). The column was a Kromasil-C18 (250×4.6 and
14
20 10
H, 5.79; O, 45.94. Found: C, 48.50; H, 5.56; O, 45.94.
Methyl 2,3,4-tri-O-acetyl-h- -glucopyranose (3a).
HPLC analysis: t =6.8 min. H NMR (CDCl ): l 1.56
D
1
R
3
(s, CH , 3H), 1.99–2.08 (4 s, CH , 12H), 3.97 (m, 1H-5),
3
3
4.08 and 4.27 (2 m, ABX system, 2H-6), 4.1–4.25 (AB
part of ABX system, J1,3 7.7 Hz, J1,3 4.59 Hz, J1,2 12.3
Hz, 2H-6), 4.89 (dd, J 10 Hz, J 6.6 Hz, 1H-2), 4.94 (d,
J 3.51 Hz, 1H-1), 5.05 (t, J 9.8 Hz, 1H-4), 5.47 (t, J 9.9
Hz, 1H-3). Anal. Calcd for C H O : C, 49.72; H,
5
mm) from Analisis Vinicos (Tomelloso, Spain). Analy-
15
22 10
ses were run at 25 °C using an L-7300 column oven and
UV detector L-7400 at 220 nm. The eluent was a
mixture of 30% acetonitrile in phosphate buffer (10
mM) at pH 4; flow rate 1.0 mL/min.
6.12; O, 44.16. Found: C, 49.32; H, 6.76; O, 43.92.
Methyl 2,3,4-tri-O-acetyl-i- -glucopyranoside (4a).
D
1
HPLC analysis: t =6.7 min. H NMR (CDCl ): l
R
3
1.9–2.01 (3 s, CH , 9H), 3.52 (s, CH , 3H), 3.53 (m,
3
3
Substrates that were not commercially available were
synthesised using well-known chemical procedures, and
their structures were confirmed by H NMR spec-
troscopy. Columns for flash chromatography were made
up with Silica Gel 60 (E. Merck) 60–200 mm or 40–63
1H-5), 3.6 and 3.8 (AB part of ABX system, J1,3 7.9 Hz,
J1,3 2 Hz, J1,2 12.5 Hz, 2H-6), 4.46 (d, J 7.9 Hz, 1H-1),
4.94 (dd, J 9.7 Hz, J 8 Hz, 1H-2), 5.04 (t, J 9.5 Hz,
1H-3). Anal. Calcd for C H O : C, 49.72; H, 6.12; O,
1
15
22 10
44.16. Found: C, 48.60; H, 6.10; O, 45.30.
Butyl 2,3,4-tri-O-acetyl-i- -glucopyranoside (5a).
mm. For 2-acetamido-1,3,4-tri-O-acetyl-2-deoxy-a-
D
-
D
glucose (6a) elution of the flash chromatography column
was performed with 30:70 acetone–dichloromethane;
HPLC analysis: 40% acetonitrile in phosphate buffer (10
1
mM) at pH 4, flow rate 1.5 mL/min; t =8.3 min. H
R
otherwise 40:60 hexane–ethyl acetate was used.
NMR (CDCl ): l 0.83 (t, J 7.4 Hz, CH , 3H), 1.22–1.32
3
3
1
H NMR were recorded in CDCl (l=ppm) on a
(m, CH , 1H), 1.41–1.55 (m, CH , 2H), 1.93–2.01 (4 s,
3
2
2
Bruker AMX 400 instrument. The different products
obtained by enzymatic hydrolysis were characterised by
COSY 2D NMR studies in order to assign the exact
position of the hydrolysis.
Enzymatic hydrolyses.—All compounds were pre-
pared following a general procedure: a suitable amount
of immobilised lipase was added to a solution of sub-
CH , 12H), 3.42–3.81 (2 m, ABX system, 2H), 3.64 (m,
3
J 9.5 Hz, 1H-5), 4.05 and 4.20 (AB part of ABX system,
J1,3 4.77 Hz, J1,3 2.44 Hz, J1,2 12.2 Hz, 2H-6), 4.43 (d,
J 7.8 Hz, 1H-1), 4.90 (dd, J 7.9 Hz, J 9.6 Hz, 1H-2), 5
(t, J 9.8 Hz, 1H-4), 5.13 (t, J 9.5 Hz, 1H-3). Anal. Calcd
for C H O : C, 53.46; H, 6.98; O, 39.56. Found: C,
18
28 10
53.60; H, 6.60; O, 39.80.

M. Terreni et al. / Carbohydrate Research 337 (2002) 1615–1621
1621
2
-Acetamido-1,3,4-tri-O-acetyl-2-deoxy-h-
D
-gluco-
3.96 Hz, J1,2 12.6 Hz, 2H-6), 4.96 (dd, J 10.25 Hz, J
3.73 Hz, 1H-2), 5.28 (t, J 9.8 Hz, 1H-3), 6.23 (d, J 3.4
Hz, 1H-1). Anal. Calcd for C H O : C, 48.28; H,
pyranose (6a). HPLC analysis: 20% acetonitrile in phos-
phate buffer (10 mM) at pH 4, flow rate 1.0 mL/min;
t =6.8 min. H NMR (CDCl ): l 1.6 (s, CH , 3H),
.93–2.07 (4 s, CH , 12H), 3.97 (m, 1H-5), 4.03 and
.26 (2 m, ABX system, 2H-6), 4.05–4.24 (AB part of
14
20 10
1
5.79; O, 45.94. Found: C, 48.34; H, 5.54; O, 46.12.
Methyl 2,3,6-tri-O-acetyl-i- -glucopyranoside (4b).
R
3
3
1
4
D
3
1
R 3
HPLC analysis: t =7.12 min. H NMR (CDCl ): l
ABX system, J1,3 8.5 Hz, J1,3 3.92 Hz, J1,2 12.4 Hz,
H-6), 4.47 (dd, J 9.9 Hz, J 6.75 Hz, 1H-2), 5.1 (t, J 9.7
Hz, 1H-4), 5.23 (t, J 9.9 Hz, 1H-3), 5.53 (d, J 9 Hz,
H-NH), 6.1 (d, J 3.51 Hz, 1H-1). Anal. Calcd for
C H NO : C, 48.41; H, 6.09; N. 4.03; O, 41.46.
2.0–2.1 (3 s, CH , 9H), 3.495 (s, CH , 3H), 3.56 (m,
3 3
2
1H-4), 3.57 (m, 1H-5), 4.3 and 4.5 (2 dd, ABX system,
2H-6 J1,3 3.42 J1,3 3.9 J1,2 1.956), 4.45 (d, J 7.8 Hz,
1H-1), 4.9 (dd, J 9.7 Hz, J 8 Hz, 1H-2), 5.0 (t, J 9.5 Hz,
1
1H-3). Anal. Calcd for C H O : C, 49.72; H, 6.12; O,
1
4
21
9
15 22 10
Found: C, 48.40; H, 6.10; N. 4.03; O, 41.47.
,2,3,4-Penta-O-acetyl-h- -galactopyranose
HPLC analysis: t =8.6 min. H NMR (CDCl ): l
44.16. Found: C, 48.50; H, 6.20; O, 45.30.
1
D
(7a).
3
1
R
Acknowledgements
1
.98–2.15 (4 s, CH , 12H), 4.04–4.16 (2 m, ABX
3
system, 2H-6), 4.33 (t, J 6.45, 1H-5), 5.32 (m, 2H-2+
), 5.49 (t, J 8.7 Hz, 1H-4), 6.37 (d, J 2.8 Hz, 1H-1).
Anal. Calcd for C H O : C, 48.28; H, 5.79; O, 45.94.
We would like to thank Mr. Marco Filice and Dr.
Silvia Rocchietti for experimental assistance. We also
thank Amano Enzyme Europe Ltd for the generous gift
of PFL.
3
1
4
20 10
Found: C, 48.50; H, 5.90; O, 45.60.
,2,3,4-Penta-O-acetyl-i- -galactopyranose
HPLC analysis: t =8.4 min. H NMR (CDCl ): l
1
D
(8a).
3
1
R
1
.97–2.14 (4 s, CH , 12H), 3.51 and 3.8 (2 m, ABX
References
3
system, 2H-6), 3.54–3.76 (AB part of ABX system, J1,3
7
6
5
.64 Hz, J1,3 6.32 Hz, J1,2 11.7 Hz, 2H-6), 3.91 (dt, J
.45 Hz, 1H-5), 5.13 (dd, J 3.42 Hz, J 10.4 Hz, 1H-2),
.32 (t, J 8.37 Hz, 1H-3), 5.44 (d, J 3.39 Hz, 1H-4), 5.73
1. (a) Koto, S.; Morishima, N.; Kihara, Y.; Suziki, H.;
Kosugi, S.; Zen, S. Bull. Chem. Soc. Jpn. 1983, 56,
1
88–191;
(
b) Koto, S.; Morishima, N.; Zen, S. Bull. Chem. Soc.
(
d, J 8.26 Hz, 1H-1). Anal. Calcd for C H O : C,
14 20 10
Jpn. 1979, 52, 784–787;
4
4
8.28; H, 5.79; O, 45.94. Found: C, 48.40; H, 5.50; O,
6.10.
(
c) Leroux, F.; Perlin, A. S. Carbohydr. Res. 1978, 67,
163–167.
2
3
4
. Kirk, O.; Christensen, M. W.; Beck, F.; Damhus, T.
Biocatal. Biotransform. 1995, 12, 91–97.
. Hall, D. M.; Lawler, T. B.; Childress, B. C. Carbohydr.
Res. 1974, 38, 359–363.
Chemoenzymatic synthesis of AP bearing a free sec-
ondary C-4 hydroxyl group (compounds 1b and 4b).—
After the hydrolytic reaction catalysed by CRL at pH 4
were carried out following the procedure previously
described under ‘Enzymatic hydrolyses’, the enzyme
was filtered off. The solution was then cooled and step
by step adjusted to pH 8, all the while controlling the
temperature. The reaction was monitored by HPLC,
and when the maximum concentration of 1b (or 4b) was
. Koeppen, B. H. Carboydr. Res. 1972, 24, 154–158.
5. (a) Bnaszek, A.; Cornet, X. B.; Zamojski, A. Carbohydr.
Res. 1985, 144, 342–345;
(
b) Fiandor, J.; Garcia-Lopez, M. T.; De Las Heras, F.
G.; Mendez Castrillon, P. P. Synthesis 1985, 1121–1123.
. Hughes, A. B.; Ley, S. V.; Priepke, H. W. M.; Woods, M.
Tetrahedron Lett. 1994, 35, 773–776.
6
achieved, the solution was extracted with Et O. After
7. Shaw, J. F.; Klibanov, A. M. Biotechnol. Bioeng. 1987,
9, 648–652.
2
2
evaporation of the solvent under reduced pressure, the
residue was purified by flash chromatography (hexane–
ethyl acetate) to obtain the pure regioisomers 1b (70%
yield) or 4b (68% yield).
8
. Hennen, W. J.; Sweers, H. M.; Wang, Y. F.; Wong, C. H.
J. Org. Chem. 1988, 53, 4939–4945.
. Sweers, H.; Wong, C. H. J. Am. Chem. Soc. 1986, 108,
6421.
9
1
,2,3,6-Tetra-O-acetyl-h-
D
-glucopyranose
(1b).
3
10. Bastida, A.; Fernandez-Lafuente, R.; Fernandez-Lorente,
G.; Guisan, J. M.; Pagani, G.; Terreni, M. Biorg. Med.
Lett. 1999, 9, 633–636.
1. Bastida, A.; Sabuquillo, P.; Armisen, P.; Huget, J.; Fer-
nandez Lafuente, R.; Guisan, J. M. Biotechnol. Bioeng.
1998, 58, 486–490.
1
HPLC analysis: t =8.5 min. H NMR (CDCl ): l
.96–2.11 (4 s, CH , 12H), 3.59 (t, J 9.3 Hz, 1H-4), 3.92
m, 1H-5), 4.18 and 4.42 (2 m, ABX system, 2H-6),
.21–4.41 (AB part of ABX system, J1,3 8.6 Hz, J1,3
R
1
(
4
3
1