Regioselective monohydrolysis of per-O-acetylated-1-substituted-β-glucopyranosides catalyzed by immobilized lipases

Article abstract of DOI:10.1016/j.tet.2008.08.099

The regioselective monohydrolysis of different peracetylated-β-glucopyranosides in aqueous media using immobilized preparations of three different lipases-those from Aspergillus niger (ANL), Candida rugose (CRL) and Candida antarctica B (CAL-B)-has been studied. Three very different immobilization strategies-covalent attachment, anionic exchange and interfacial activation on a hydrophobic support-were employed for each lipase. The role of the immobilization strategy and the effect of the presence of different moieties in the anomeric position of the substrate on the hydrolytic activities, specificities and regioselectivities of the lipases were investigated. For example, the PEI-ANL preparation exhibited 800 times higher activity than the octyl-ANL in the hydrolysis of 2-acetamido-2-deoxy-1-(4-nitrophenyl)-3,4,6-tri-O-acetyl-β-d-glucopyranoside-producing 4-OH derivative in 18% yield-whereas the octyl-ANL was five times more active than the PEI-ANL in the hydrolysis of 1-(4-nitrophenyl)-2,3,4-tri-O-acetyl-β-d-xylopyranoside, producing 4-OH monohydroxy product in >99% yield. The octyl-CRL preparation hydrolyzed regioselectively 3,4,6-tri-O-acetyl-glucal in position 6 in 68% yield while the PEI-CRL produced the 3-OH product in 11% yield, although with moderate specificity. The CNBr-CAL-B hydrolyzed specifically and regioselectively the glucal producing the 3-OH product in >99% yield.

Full text of DOI:10.1016/j.tet.2008.08.099

Tetrahedron 64 (2008) 10721–10727
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Regioselective monohydrolysis of per-O-acetylated-1-substituted-
b-glucopyranosides catalyzed by immobilized lipases
,
,
Adriano A. Mendes y z, Dasciana S. Rodrigues y z, Marco Filice, Roberto Fernandez-Lafuente,
*
*
Jose M. Guisan , Jose M. Palomo
´
´
Departamento de Biocatalisis, Instituto de Catalisis (CSIC), Campus UAM Cantoblanco, 28049 Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 July 2008
Received in revised form 21 August 2008
Accepted 29 August 2008
Available online 10 September 2008
The regioselective monohydrolysis of different peracetylated-b-glucopyranosides in aqueous media us-
ing immobilized preparations of three different lipasesdthose from Aspergillus niger (ANL), Candida
rugose (CRL) and Candida antarctica B (CAL-B)dhas been studied. Three very different immobilization
strategiesdcovalent attachment, anionic exchange and interfacial activation on a hydrophobic sup-
portdwere employed for each lipase. The role of the immobilization strategy and the effect of the
presence of different moieties in the anomeric position of the substrate on the hydrolytic activities,
specificities and regioselectivities of the lipases were investigated. For example, the PEI-ANL preparation
exhibited 800 times higher activity than the octyl-ANL in the hydrolysis of 2-acetamido-2-deoxy-1-(4-
Keywords:
Lipase
Regioselectivity
Immobilization
Glucopyranosides
Monohydrolysis
nitrophenyl)-3,4,6-tri-O-acetyl-
the octyl-ANL was five times more active than the PEI-ANL in the hydrolysis of 1-(4-nitrophenyl)-2,3,4-
tri-O-acetyl- -xylopyranoside, producing 4-OH monohydroxy product in >99% yield.
b-D-glucopyranosidedproducing 4-OH derivative in 18% yielddwhereas
b-D
The octyl-CRL preparation hydrolyzed regioselectively 3,4,6-tri-O-acetyl-glucal in position 6 in 68% yield
while the PEI-CRL produced the 3-OH product in 11% yield, although with moderate specificity. The
CNBr-CAL-B hydrolyzed specifically and regioselectively the glucal producing the 3-OH product in >99%
yield.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
different neo-glycoderivatives (oligosaccharides, glycolipids, gly-
copeptides, glycolipopeptides, etc.).10–12
Carbohydrates exist in very different forms in nature playing
a very important role in many biological processes.1 Furthermore,
many natural compounds with medical relevance are glycosylated.2
Specially, alkyl and aryl glycosides have attracted tremendous
interest in recent years in view of their diverse applications as food
emulsifiers, antimicrobial agents, drug carriers or inhibitors of
carbohydrate–lectin interactions.3–8
Fully acetylated alkyl/aryl glycosides are commonly used for the
chemical synthesis of O-glycosides in view of their ready avail-
ability, low cost and ease preparation.9 These per-O-acetyl-glyco-
sides could be used as raw materials to obtain regioisomers of
O-acetyl-anomeric substituted glycopyranosides presenting only
one free hydroxyl group, key intermediates in the preparation of
However, the synthesis of monohydroxy derivatives is very
difficult by classical chemical approaches, being necessary to use
many chemically selective protection/deprotection stepsdwith
a poor final overall yielddbecause of the low regioselectivity to
remove only one acetyl group.13 Consequently, the use of enzymes
in aqueous media could be an attractive alternative. However, in
most cases the enzymatic deacylation of fully acylated pyranoses is
very slow or proceeds with poor selectivity and yield.14
To perform the enzymatic synthesis of monodeacetylated car-
bohydrates following this strategy, it is necessary to find bio-
catalysts, specially in an immobilized form,15 exhibiting good
catalytic activity and a high regioselectivity. Moreover, the yield of
monodeacetylated product will be defined by the specificity of the
enzyme: the biocatalyst should prefer to hydrolyze the peracety-
lated substrate instead of hydrolyzing the monodeacetylated
product to accumulate the last.
* Corresponding authors. Tel.: þ34 91 585 54 78; fax: þ34 91 585 47 60.
E-mail addresses: jmguisan@icp.csic.es (J.M. Guisan), josempalomo@icp.csic.es
Lipases may be a good option to catalyze these processes, be-
cause they recognize a broad range of substrates with high regio
and enantioselectivity in many instances.13b,16,17
(J.M. Palomo).
y
´
Present address: Departamento de Engenharia Quımica, Universidade Federal
The mechanism of catalysis of lipases implies dramatic confor-
mational changes of the enzyme molecule between a ‘closed’ and
de Sa˜o Carlos Rodovia Washington Luis, Km 235-676, 13565-905 Sa˜o Carlos, Brazil.
z
Both authors contributed equally to this work.
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.08.099

10722
A.A. Mendes et al. / Tetrahedron 64 (2008) 10721–10727
an ‘open’ form.18,19 This mechanism of action produces that the use
of different immobilization protocols (involving different areas of
the lipase, rigidity, micro-environments, etc.)20 causes a strong
modulation of the lipase properties. In fact, this strategy has been
used to modulate the lipase properties.21–23 In a similar way, the
use of different immobilization protocols may be used to modulate
the lipase regioselectivity. For example, the hydrolysis of several
per-O-acetylated monosaccharides by different immobilized prep-
arations of lipasesdto deacetylate them at the primary and the
anomeric positionsdhas been recently studied.24 Now, differently
immobilized lipases have been used in the hydrolysis of alkyl or aryl
Using CAL-B, the highest activity for the octyl-CAL-B and CNBr-
CAL-B immobilized preparations was achieved in the hydrolysis of
19, more than 100 times higher, in the case of using the CNBr-CAL-B,
than the activity of this biocatalyst in the hydrolysis of peracetylated
glucose 22. In contrast, CAL-B immobilized on PEI-agarose displayed
its maximal activity in the hydrolysis of 22.
The CNBr-CAL-B was the most active biocatalyst in the hydrolysis
of 1, 9, 13, 17 and 19, 13 times more activedusing peracetylated
butyl-glucopyranoside 1dand 16 timesdusing 4-nitrophenyl glu-
cosamine derivative 13dwhen it was compared to the octyl-CAL-B
preparation. However, the octyl-CAL-B preparation exhibited the
highest activity when a phenyl moiety was linked in the anomeric
position (5), up to 300 times more active compared to the PEI-CAL-B
preparation. The PEI-CAL-B was always the less active preparation,
and product was not detected in the hydrolysis of 1, 9 and 17.
Using ANL as biocatalyst, the CNBr-ANL and PEI-ANL prepara-
tions exhibited the highest activity in the hydrolysis of the per-
acetylated product 22, while the octyl-ANL exhibited the highest
activity towards xylopyranoside 17, 80 times higher compared to
the activity displayed for it in the hydrolysis of 22.
The CNBr-ANL immobilized preparation was the most active
preparation in the hydrolysis of 1, 19 and 22, 40 timesdusing
peracetylated butyl-glucopyranoside 1dand 45 timesdusing glu-
cal 19dwhen it was compared to the octyl-ANL. However, the
octyl-ANL preparation showed the highest activity in the hydrolysis
of 9 and 17, e.g., more than 15 times highest compared to the ac-
tivity displayed by other preparations hydrolyzing 9.
The PEI-ANL preparation displayed the highest activity in the
hydrolysis of 5 and 13, around 180 times more active towards 5
when it was compared to the enzyme immobilized on octyl-aga-
rose. Interestingly, the activity of the octyl-ANL decreased more
than 800 times in the hydrolysis of glucosamine 13 whereas the
PEI-ANL activity slightly decreased.
anomeric substituted peracetylated-b-glucopyranosides in aque-
ous media to study if the immobilization protocol may alter the
lipase properties and if the substituent in the anomeric position
may also influence the lipase performance, producing new mono-
decatylated regioisomers.
Three different immobilization protocols have been applied: (i)
immobilization on hydrophobic supports at low ionic strength by
interfacial activation of the lipases, involving the hydrophobic area
surrounding the active site of the lipases;25 (ii) immobilization on
agarose activated with CNBr via covalent attachment at neutral pH
value throughout the most reactive amino group (usually the ter-
minal NH2) on the enzyme surface;26 (iii) immobilization via an-
ionic exchange through the areas with the highest negative charge
of the lipase on agarose beads coated with PEI.27
These different immobilization strategies were applied to three
very used lipases: those from Candida antarctica (isoform B) (CAL-
B), from Candida rugosa (CRL) and from Aspergillus niger (ANL). The
immobilized enzymes were utilized as biocatalysts in the hydro-
lysis of different 1-substituted per-O-acetylated glucopyranosides
in aqueous media.
2. Results and discussion
When using CRL, the highest activity for the octyl-CRL was
achieved in the hydrolysis of 22. The CNBr-CRL preparation
exhibited the highest activity using xylopyranoside 17 while the
PEI-CRL was the most active catalyst towards glucal 19.
2.1. Effect of the immobilization protocol on the lipase
activity in the hydrolysis of different peracetylated-
b-1-substituted-glucopyranosides
The octyl-CRL preparation was the most active catalyst in the
hydrolysis of 1, 5, 9 and 19, 18 timesdusing peracetylated butyl-
glucopyranoside 1dand 10 timesdusing glucal 19dwhen it was
compared to the CNBr-CRL.
However, the CNBr-CRL was the most active catalyst in the hy-
drolysis of 13 and 19, with three fold highest activity than the octyl-
CRL preparation.
The activity of the CRL immobilized on octyl-agarose decreased
more than 100 times when the anomeric position was blocked in
substrates 1, 5, 9 and 13. However, the CNBr-CRL showed the same
activity using 22 and 5 and more than 100 times higher activity
when the xylopyranoside was used.
The PEI-CRL was always the less active preparation, and any
products were not detected in the hydrolysis of 1, 5 and 17. How-
ever, this lipase immobilized preparation showed better activity
(four fold) towards glucal 19 than using 22.
The specific activity displayed by different immobilized
preparations from CAL-B, ANL and CRL in the hydrolysis of 1-butyl-
2,3,4,6-tetra-O-acetyl- -glucopyranoside (1),1-phenyl-2,3,4,6-
tetra-O-acetyl- -glucopyranoside (5), 1-(4-nitrophenyl)-2,3,4,
6-tetra-O-acetyl- -glucopyranoside (9), 2-acetamido-2-deoxy-
1-(4-nitrophenyl)-3,4,6-tri-O-acetyl- -glucopyranoside (13), 1-
(4-nitrophenyl)-2,3,4-tri-O-acetyl- -xylopyranoside (17) and
b-D
b-D
b-D
b-D
b-D
3,4,6-tri-O-acetyl-glucal (19) are shown in Table 1. The activities
were compared with that obtained in the hydrolysis of the
peracetylated glucopyranose (22).
The presence of different groups in the anomeric position of
peracetylated glucose affected in a different way to the enzyme
activity.
Table 1
Specific activity of different immobilized preparations of lipases in the hydrolysis of
per-O-acetylated 1-substituted-b
-glucopyranosidesa
In this way, the modification in the anomeric position of
different peracetylated-
b-1-substituted-glucopyranosides pre-
Enzyme
CAL-B
Support
1
5
9
13
0.003
0.075
0.004
17
19
22
sented a very different effect on the activity exhibited by li-
pases immobilized following different protocols. That way,
lipases specificity was strongly affected by the immobilization
protocol.
Octyl
CNBr
PEI
0.9
12
Nd
8
6
Nd
1.20
Nd
25
70
Nd
486
1940
23
124
132
29
0.024
ANL
CRL
Octyl
CNBr
PEI
15
630
540
48
5370
8640
390
25
21
0.45
7
15
53,340
27,150
13,710
60
2790
1410
660
374,400
99,930
2.2. Specificity and regioselectivity of the different
immobilized lipases preparations
Octyl
CNBr
PEI
0.75
0.04
Nd
1.80
0.21
Nd
0.28
0.06
0.06
0.002
0.006
0.004
8
23
Nd
52
5
4
187
0.2
1
Recently we found that different immobilized biocatalysts of
these three lipases exhibited a regioselectivity towards anomeric or
6-OH position in the hydrolysis of the peracetylated 22.24 In this
Nd: activity not detected.
a
ꢁ1
The initial rate in nmolꢀmg ꢀminꢁ1. It was calculated at 10–15% conversion.
prot

A.A. Mendes et al. / Tetrahedron 64 (2008) 10721–10727
10723
OAc
O
OH
OAc
OAc
AcO
AcO
AcO
AcO
Immobilized lipase
OR1 pH 5, 25 °C
HO
AcO
AcO
HO
O
O
O
+
OR1
+
OR1
R2
OR1
R2
2
R2
R2
4
R2 = OAc
R2 = OAc
1
5
R1
R1
=
=
3
6
7
8
R1
R1
=
=
9
10
R2 = OAc
NO2
NO2
11
15
12
16
R2 = NHAc
13
14
Scheme 1. Specific and regioselective hydrolysis of different 1-substituted 2,3,4,6-tetra-O-acetyl-b-D-glucopyranosides by different immobilized lipases.
study, the anomeric position was blocked expecting that to alter
these previous results.
6 (6). The PEI-CAL-B only gave 5% conversion after 168 h producing
6 (Scheme 1, Table 3).
Using ANL, the immobilization on CNBr-agarose or PEI-agarose
was not very specific, producing 61% and 49% of 6-OH product 6,
respectively. The octyl-ANL was specific, although hydrolyzing at
position 6 (6, 38%) and also, interestingly, at position 4, but with
a low yield (7, 6%) (Table 3).
In the case of CRL, the immobilization on octyl-agarose per-
mitted to obtain a not very specific catalyst to produce 6 in 35%
yield. The other CRL preparations hydrolyzed at positions 6 and 4
gave a mixture of 6 and 7 (Table 3, entries 8 and 9). The production
of 7deven although at low yieldsdopens the possibility of
obtaining this interesting product.
2.2.1. Hydrolysis of 1
When CAL-B was studied, the octyl-CAL-B preparation was not
specific towards the peracetylated substrate, giving the non-de-
sired multi-hydrolyzed products (data not shown). The CNBr-CAL-B
preparation was also not specific, giving 11% of monodeacetylated
product, although accumulating the 6-OH product 2 (Scheme 1,
Table 2).
Table 2
Specificity and regioselectivity of different immobilized preparations of lipases in
the hydrolysis of 1
2.2.3. Hydrolysis of 9
Entry
Enzyme
CAL-B
Support
Reaction
time [h]
c
a [%]
2b [%]
3b [%]
Using CAL-B, the CNBr-CAL-B preparation was the unique
preparation active of this lipase towards 9. It was non-specific,
producing 14% of 6-OH monodeacetylated product 10 (Scheme 1,
Table 4).
1
2
3
Octyl
CNBr
PEI
168
90
168
51
0
11
d
0
100
d
d
4
5
6
ANL
CRL
Octyl
CNBr
PEI
168
6
24
43
96
96
10
77
73
5
Table 3
5
Specificity and regioselectivity of different immobilized preparations of lipases in
the hydrolysis of 5
7
8
9
Octyl
CNBr
PEI
168
168
168
90
7
0
47
4
8
3
d
Entry
Enzyme
CAL-B
Support
Reaction time [h]
ca [%]
6b [%]
7b [%]
d
1
2
3
Octyl
CNBr
PEI
168
168
168
100
100
5
17
3
5
a
Conversion.
Yield of the monodeacetylated product.
b
4
5
6
ANL
CRL
Octyl
CNBr
PEI
168
5
5
44
100
100
38
61
49
6
Using ANL, the CNBr-ANL immobilized preparation was quite
7
8
9
Octyl
CNBr
PEI
160
168
168
100
36
3.3
35
19
1.8
specific (accumulating the monodeacetylated products) and
regioselective (hydrolyzing at position 6), producing the tri-acety-
lated product 2 in 77% yield (Scheme 1, Table 2). ANL immobilized
on PEI-agarose was also quite specific but not totally regioselective,
producing 73% of 2 but also 5% of 4-OH product 3 was achieved
(entry 6, Table 2). The hydrolysis at position 4, although still mi-
nority, may be interesting. However, the octyl-ANL preparation
showed low specificity towards the peracetylated 1, giving only 15%
yield of monodeacetylated products 2 and 3 (2:1) (entry 4, Table 2).
When CRL immobilized preparations were used, the octyl-CRL
was not very specific with 55% yield in monohydroxy product at
100% conversion, neither it was regioselective, because both
monohydroxy 6-OH (2) and 4-OH (3) products were obtained (47%
2, 8% 3). The hydrolysis at position 4dalthough minoritydmay be
interesting. The CNBr-CRL preparation was not regioselective, with
the production of both 2 and 3 in similar amounts (Table 2).
6
1.5
a
Conversion.
Yield of the monodeacetylated product.
b
Table 4
Specificity and regioselectivity of different immobilized preparations of lipases in
the hydrolysis of 9
Entry
Enzyme
CAL-B
Support
Reaction time [h]
ca [%]
10b [%]
11b [%]
1
2
3
Octyl
CNBr
PEI
168
168
168
0
96
0
14
d
d
4
5
6
ANL
CRL
Octyl
CNBr
PEI
54
168
168
100
100
73
42
50
13
5
8
6
7
8
9
Octyl
CNBr
PEI
168
168
168
90
33
89
6
10
7
5
12
5
2.2.2. Hydrolysis of 5
When CAL-B was used, the octyl-CAL-B or CNBr-CAL-B prepa-
rations were not specific, giving 17% or 3% of monodeacetylated
product, although accumulating the product hydrolyzed at position
a
Conversion.
Yield of the monodeacetylated product.
b

10724
A.A. Mendes et al. / Tetrahedron 64 (2008) 10721–10727
Table 5
production of the interesting product 4-OH 18. Using the octyl-ANL
preparationdsuch as optimal catalystdwas possible to obtain 18 in
>99% yield in 25 h (Table 6).
Any of the CRL immobilized preparations did not hydrolyze this
substrate.
Specificity and regioselectivity of different immobilized preparations of lipases in
the hydrolysis of 13
Entry
Enzyme
CAL-B
Support
ca [%]
14b [%]
15b [%]
16b [%]
1
2
3
Octyl
CNBr
PEI
5
24
3
1
12
3
2.2.6. Hydrolysis of 19
4
5
6
ANL
CRL
Octyl
CNBr
PEI
6
84
92
5
51
37
1
7
3
Using CAL-B, all the three immobilized preparations hydrolyzed
specifically and regioselectively the glucal 19d1,2-anhydrosugar
with great synthetic potential as building blocks in oligosaccharide
synthesisdat position 3 (21) in >99% yield (Table 7, Scheme 3).
The ANL preparations were also regioselective producing the
monohydrolyzed 3-OH derivative 21 although with different yields
for the three ANL immobilized preparations. Using the PEI-ANL
preparation, 75% of 21 was obtained whereas only 34% of 21 was
achieved employing the CNBr-ANL preparation (Table 7, entries 5
and 6).
11
18
7
8
9
Octyl
CNBr
PEI
2
9
5
2
3
5
a
Conversion at 168 h.
Yield of the monodeacetylated product.
b
Table 6
Specificity and regioselectivity of different immobilized preparations of lipases in
the hydrolysis of 17
Table 7
Entry
Enzyme
CAL-B
Support
Reaction time [h]
c [%]
18a [%]
Specificity and regioselectivity of different immobilized preparations of lipases in
the hydrolysis of 19
1
2
3
Octyl
CNBr
PEI
168
168
168
14
41
0
14
41
Entry
Enzyme
CAL-B
Support
Reaction time [h]
ca [%]
20b [%]
21b [%]
d
1
2
3
Octyl
CNBr
PEI
17
5
120
100
100
100
>99
>99
>99
4
5
6
ANL
Octyl
CNBr
PEI
25
35
74
100
100
100
>99
>99
>99
a
4
5
6
ANL
CRL
Octyl
CNBr
PEI
168
24
168
27
100
100
10
34
75
Yield of the monodeacetylated product.
When ANL was employed, all the preparations showed different
specificity (Table 4) producing an interesting mixture of mono-
deacetylated 6-OH (10) and 4-OH (11) products. Using the CNBr-
ANL immobilized preparation was possible to achieve a yield of 50%
of 10 and 8% of 11 (Table 4, entry 5).
In the case of CRL, the octyl-CRL or PEI-CRL preparation showed
very low specificity. The CNBr-CRL immobilized preparation pre-
sented better specificity producing in the same amounts 10 and the
interesting 11 (Table 4).
7
8
9
Octyl
CNBr
PEI
24
168
168
100
100
90
68
9
11
a
Conversion.
Yield of the monodeacetylated product.
b
When CRL was used, the octyl-CRL preparation showed good
specificity and regioselectivity, hydrolyzing exclusively at position 6
of 19, producing the monodeacetylated product 20 in 68% yield. The
other CRL biocatalysts gave low yield in the monodeacetylation of
19, accumulating the 3-OH product 21 in 10% yield (Table 7, entries
7–9).
2.2.4. Hydrolysis of 13
In the hydrolysis of 4-nitrophenyl glucosamine derivative (13),
the CAL-B immobilized on octyl-agarose or CNBr-agarose yielded
less than 25% of monodeacetylated sugar but only producing the
very interesting product 16. However, the PEI-CAL-B preparation
hydrolyzed the acetyl group producing only the 6-OH 14 although
with low yield (Table 5).
Employing ANL, the CNBr-ANL preparation was highly specific in
the hydrolysis of peracetylated 13, producing 6-OH product 14 in
51% yield, and also the interesting 4-OH 15 in 11% and 3-OH 16 in 7%
yield. Using the PEI-ANL, the results were quite similar (entry 6,
Table 5). The octyl-ANL hydrolyzed specifically at positions 6 and 3
in a relation 5:1.
3. Conclusion
In this manuscript, we have presented the enzymatic deacety-
lationdusing three different lipasesdof different 1-substituted-
peracetylated glucopyranosides by hydrolysis in aqueous media.
The immobilization strategy was a key point to determine the op-
timal catalysts in each case. The immobilization strategy defines
the activity, specificity and regioselectivity of the final biocatalyst.
The presence of different moieties in the anomeric position of the
substrate altered the catalytic properties of these immobilized li-
pases compared to the use of sugars with an acetyl group in the
anomeric position.
Using CRL, all immobilized preparations exhibited very low ac-
tivity and the regioselectivity was not analyzed.
2.2.5. Hydrolysis of 17
The variation of the specific activity caused by the immobiliza-
tion strategy may be exemplified by the ANL. The PEI-ANL immo-
bilized preparation exhibited 800 times higher activity in the
hydrolysis of 13 compared with the activity displayed by the octyl-
In the regioselective hydrolysis of the xylopyranoside 17 (Table
6, Scheme 2), all ANL and the octyl- and CNBr-CAL-B preparations
displayed a very high specificity and regioselectivity towards the
AcO
O
HO
O
AcO
O
NO2
AcO
O
NO2
Immobilized lipase,
buffer: CH3CN (90:10)
pH 5, 25 °C
AcO
AcO
17
18
Scheme 2. Regioselective hydrolysis of 17 by different immobilized preparations of lipases.

A.A. Mendes et al. / Tetrahedron 64 (2008) 10721–10727
10725
OAc
O
OH
OAc
O
AcO
AcO
AcO
AcO
Immobilized lipase
AcO
HO
O
+
pH 5, 25 °C
19
20
21
Scheme 3. Regioselective hydrolysis of 19 by different immobilized preparations of lipases.
ANL whereas the octyl-ANL preparation was five times more active
than the PEI-ANL in the hydrolysis of 17. About the modulation of
the regioselectivity by the immobilization strategy, for example, the
octyl-CRL hydrolyzed regioselectively the position 6 of 19, pro-
ducing the monodeacetylated product 20 in 68% yield while the
PEI-CRL produced the 3-OH product 21 in 11% yield, although with
moderate specificity.
Using this biocatalytic approach, the 4-OH monohydroxy de-
rivative 18 was produced in >99% yield using the octyl-ANL.
Moreover, the monodeacetylated 3-OH 16 and 21 was also obtained
in 12% and >99% yields using the CNBr-CAL-B preparation. These
monodeacetylated glucosides are very interesting and useful
building blocks for carbohydrate chemistry and are not produced
using the unmodified peracetylated sugars. Thus, the combination
of combinatorial biocatalysis (use of different enzymes immobi-
lized following different strategies) and a small collection of
modified substrates may permit to produce monodeacetylated
sugars in no standard positions, with a high interest in the pro-
duction of neo-glycoderivatives or glycoconjugates for biological
studies.
hydrolyzed p-NPB per minute per mg of enzyme (IU) under the
conditions described above.
4.3. Purification of lipases
The purification of CAL-B and CRL was performed using a pre-
viously described protocol, based on the selective adsorption of
lipases on hydrophobic supports at low ionic strength.25 CRL
commercial solid powder (0.16 g, 30 mg protein) or 2.5 mL CAL-B
commercial solution (12 mg protein/mL)28 was offered to 95 mL of
10 mM sodium phosphate at pH 7.0. In each case, 5 g of octyl-
agarose support was added. After 4 h, the enzyme immobilized
preparation was filtered under vacuum using a sintered glass fun-
nel and abundantly washed with distilled water. In all cases, more
than 95% of lipase was adsorbed on the support. ANL was purified
from the commercial extract crude as previously described.29 Six
milligrams lipase per gram of support was prepared. The SDS–PAGE
analysis of the proteins after the purification treatment showed
only a single band with a molecular weight corresponding to that of
the different native lipases. These adsorbed lipases were used in
some instances directly as biocatalysts in some of the studies.
In some other cases, the purified lipases were finally immobilized
in other supports (see below). To have a pure lipase from the octyl
immobilized preparations, the adsorbed enzyme was added to
a solution of 10 mM sodium phosphate containing 1% triton (v/v) at
pH 7.0 and 4 ꢂC for 1 h, obtaining a purified lipase solution with
a final concentration of 0.6 mg lipase/mL. Then, the enzymatic
solution was used for immobilization in the other supports.
4. Experimental
4.1. General
Lipase from A. niger (ANL) was purchased from Fluka (Neu Ulm,
Germany). Lipase from C. antarctica B (CAL-B) was kindly supplied
by Novo Nordisk (Denmark). Octyl-agarose (4BCL) and cyanogen
bromide (CNBr-activated Sepharose 4BCL) beads were purchased
from GE-Healthcare (Uppsala, Sweden). Polyethyleneimine (PEI)
(Mr 25,000), Triton X-100, p-nitrophenyl butyrate (p-NPB), lipase
4.4. Immobilization of lipases on other supports
from C. rugosa (CRL), 1-butyl-2,3,4,6-tetra-O-acetyl-
anoside (1), 1-phenyl-2,3,4,6-tetra-O-acetyl- -glucopyranoside
(5), 1-(4-nitrophenyl)-2,3,4,6-tetra-O-acetyl- -glucopyranoside (9),
2-acetamido-2-deoxy-1-(4-nitrophenyl)-3,4,6-tri-O-acetyl- -glu
copyranose (13), 1-(4-nitrophenyl)-2,3,4-tri-O-acetyl- -xylopyr-
anoside (17), 3,4,6-tri-O-acetyl-glucal (19) and 1,2,3,4,6-penta-
O-acetyl- -glucopyranoside (22) were from Sigma Chem. Co.
b-D-glucopyr-
Lipases were immobilized in the presence of 1% triton, as
obtained after the enzyme purification, to prevent any lipase–lipase
interaction that could produce the immobilization of lipase dimers
with altered properties.30 The further exhaustive washing with
distilled water of the immobilized lipases permitted to fully elim-
inate the detergent.
b-D
b-D
b-D
b-D
b-D
Agarose beads coated with PEI were prepared as previously de-
4.4.1. Immobilization of lipases on CNBr-activated support
scribed.27 Columns for flash chromatography were made up with
Commercial agarose support activated with CNBr was sus-
pended in an acidic aqueous solution (pH 2–3) for 1 h. After that,
the solid support was dried by filtration under vacuum using
a sintered glass funnel. Lipase solution (10 mL, 0.6 mg lipase/mL)
was added to 30 mL of 10 mM sodium phosphate solution at pH 7.
After that, 1 g of the CNBr-agarose support was added. The mixture
was then stirred at 25 ꢂC and 250 rpm for 2 h. After, the superna-
tant was removed by filtration using a sintered glass funnel and the
supported lipase added to 40 mL of 3 M glycine at pH 8 for 1 h.
Finally, the immobilized lipases were filtered using a sintered glass
funnel and washed several times with distilled water. The immo-
bilization yield wasdin all casesdmore than 95%.
Silica Gel 60 (Merck) 60–200 or 40–63
formed with 40:60 hexane–ethyl acetate. 1H NMR were recorded in
CDCl3
products obtained by enzymatic hydrolysis were characterized by
COSY 2D NMR homocorrelation studies in order to assign the exact
position of the hydrolysis.
mm. The elution was per-
(d
¼ppm) on a Bruker AMX 400 instrument. The different
4.2. Standard enzymatic activity assay determination
In order to follow the immobilization process, the activities of
the soluble lipases and their immobilized preparations were ana-
lyzed spectrophotometrically measuring the increment in absor-
bance at 348 nm produced by the release of p-nitrophenol (p-NP)
4.4.2. Immobilization of lipases on PEI-agarose support
(
3
¼5.150 Mꢁ1 cmꢁ1) in the hydrolysis of 0.4 mM p-NPB in 25 mM
Lipase solution (10 mL, 0.6 mg lipase/mL) was added to 30 mL of
10 mM sodium phosphate solution at pH 7. After that, 1 g of the
glyoxyl-agarose beads coated with polyethyleneimine (PEI) was
added. The mixture was then stirred at 25 ꢂC and 250 rpm for 4 h.
sodium phosphate at pH 7 and 25 ꢂC. To initialize the reaction,
0.05–0.2 mL of lipase solution or suspension was added to 2.5 mL of
substrate solution. Enzymatic activity is given as
mmol of

10726
A.A. Mendes et al. / Tetrahedron 64 (2008) 10721–10727
After, the supernatant was removed by filtration and the supported
lipase was washed off several times with distilled water. The im-
mobilization wasdin all casesdmore than 95%.
4.11. 1-(4-Nitrophenyl)-2,3,6-tri-O-acetyl-b-D-
glucopyranoside (11)
1H NMR (CDCl3):
d
(ppm): 8.14 (d, 2H, J¼8.6 Hz, H-30, H-50), 6.98
(d, 2H, J¼8.5 Hz, H-20, H-60), 5.18 (t, 1H, J¼9.6 Hz, H-3), 5.06 (m, 1H,
H-2), 4.77 (d, 1H, J¼7.8 Hz, H-1), 4.34–4.12 (m, 2H, 2ꢀH-6), 3.91 (t,
1H, J¼9.5 Hz, H-4), 3.69 (m, 1H, J¼9.5 Hz, H-5), 2.09–1.96 (s, 9H,
3ꢀCH3). The NMR data are in agreement with the reported
values.32
4.5. Enzymatic hydrolysis of peracetylated-
glucopyranosides
b-
Compounds 1, 5, 19 and 22 (2 mM) or 0.2 mM of 9, 13 and 17 in
50 mM sodium acetate with 20% acetonitrile at pH 5 and 25 ꢂC were
prepared. Biocatalyst (0.5 g) was added to 3 mL (1, 5, 9, 13, 17) or
10 mL (19, 22) of the previous solution to initialize the reaction. The
pH value was selected to avoid the chemical acyl-migration in the
per-O-acetylated carbohydrates hydrolysis.31 The hydrolytic re-
action was carried out under mechanical stirring, and the pH value
was kept constant using an automatic titrator 718 Stat Tritino from
Metrohm (Herisau, Switzerland). Reactions were followed by HPLC
using a HPLC spectrum P100 (Thermo Separation products). At least
triplicates of each assay were made. The column was a Kromasil-C18
4.12. 2-Acetamido-2-deoxy-1-(4-nitrophenyl)-3,4-di-O-
acetyl-
b-D-glucopyranoside (14)
1H NMR (CDCl3):
d
(ppm): 8.14 (d, 2H, J¼8.6 Hz, H-30, H-50), 7.1
(d, 2H, J¼8.5 Hz, H-20, H-60), 5.28 (t, 1H, J¼9.5 Hz, H-3), 4.85 (d, 1H,
J¼7.7 Hz, H-1), 4.79 (m, 1H, H-2), 4.66 (t, 1H, J¼9.5 Hz, H-4), 3.85–
3.70 (m, 2H, 2ꢀH-6), 3.65 (m, 1H, H-5), 2.20 (s, 6H, 2ꢀCH3), 1.84 (s,
3H, CH3).
(250ꢀ4.6 and 5
mm) from Analisis Vinicos (Spain). Analyses were
4.13. 2-Acetamido-2-deoxy-1-(4-nitrophenyl)-3,6-di-O-
run at 25 ꢂC using an L-7300 column oven and UV detector L-7400 at
215 nm. The mobile phase was an isocratic mixture of 40% acetoni-
trile–60% 10 mM sodium phosphate at pH 4; flow rate 1.0 mL/min.
Finally, the products were isolated and identified by 1H NMR.
acetyl-
b-D-glucopyranoside (15)
1H NMR (CDCl3):
d
(ppm): 8.15 (d, 2H, J¼8.4 Hz, H-30, H-50), 6.9
(d, 2H, J¼8.6 Hz, H-20, H-60), 4.85 (d, 1H, J¼8.0 Hz, H-1), 4.80 (t, 1H,
J¼9.3 Hz, H-3), 4.65 (m, 1H, H-2), 4.20–4.08 (m, 2H, 2ꢀH-6), 3.90
(m, 1H, H-4), 3.75 (m, 1H, H-5), 2.17 (s, 6H, 2ꢀCH3), 1.85 (s, 3H, CH3).
4.6. 1-Butyl 2,3,4-tri-O-acetyl-
b-D-glucopyranoside (2)
1H NMR (CDCl3):
d
(ppm): 5.25 (t, 1H, J¼9.5 Hz, H-3), 5.00 (t, 1H,
4.14. 2-Acetamido-2-deoxy-1-(4-nitrophenyl)-4,6-di-O-
J¼9.8 Hz, H-4), 4.98 (dd, 1H, J¼7.9, 9.6 Hz, H-2), 4.43 (d, 1H,
J¼7.8 Hz,1H-1), 3.90–3.79 (m, 2H, 2ꢀH-6), 3.64 (m, 1H, J¼9.5 Hz, H-
5), 3.46–3.41 (m, 2H, CH2a), 2.01–1.93 (s,12H, 4ꢀCH3),1.55–1.46 (m,
2H, CH2), 1.32–1.22 (m, 2H, CH2), 0.90 (t, 3H, J¼7.4 Hz CH3). The
NMR data are in agreement with the reported values.24c
acetyl-
b-D-glucopyranoside (16)
1H NMR (CDCl3):
d
(ppm): 8.12 (d, 2H, J¼8.4 Hz, H-30, H-50), 7.0
(d, 2H, J¼8.5 Hz, H-20, H-60), 4.85 (d, 1H, J¼7.8 Hz, H-1), 4.66 (m, 1H,
H-2), 4.4 (t, 1H, J¼9.5 Hz, H-4), 4.30–4.09 (m, 2H, 2ꢀH-6), 3.99 (t,
1H, J¼9.0 Hz, H-3), 3.80 (m, 1H, H-5), 2.16–2.00 (s, 6H, 2ꢀCH3), 1.89
(s, 3H, CH3).
4.7. 1-Butyl-2,3,6-tri-O-acetyl-
1H NMR (CDCl3):
1H, J¼7.9, 9.6 Hz, H-2), 4.42 (d, 1H, J¼7.8 Hz, 1H-1), 4.34–4.09 (m,
2H, 2ꢀH-6), 3.98 (t, 1H, J¼9.8 Hz, H-4), 3.64 (m, 1H, J¼9.5 Hz, H-5),
3.45–3.40 (m, 2H, CH2a), 2.10–2.03 (s, 12H, 4ꢀCH3), 1.46–1.45 (m,
2H, CH2), 1.35–1.27 (m, 2H, CH2), 0.94 (t, 3H, J¼7.4 Hz CH3).
b-D-glucopyranoside (3)
d
(ppm): 5.23 (t, 1H, J¼9.5 Hz, H-3), 4.97 (dd,
4.15. 1-(4-Nitrophenyl)-2,3-di-O-acetyl-b-D-
xylopyranoside (18)
1H NMR (500 MHz, CDCl3):
d
(ppm): 8.10 (d, 2H, J¼8.4 Hz, H-30,
H-50), 6.88 (d, 2H, J¼8.31 Hz, H-20, H-60), 5.21 (d, 1H, J¼5.3 Hz, H-1),
5.16 (m, 1H, H-2), 4.78 (m, 1H, H-3), 4.12 (m, 1H, H-5), 3.92 (m, 1H,
H-4), 3.55 (m, 1H, H-5), 2.10 (s, 3H, CH3), 2.05 (s, 3H, CH3). The NMR
data are in agreement with the reported values.33
4.8. 1-Phenyl-2,3,4-tri-O-acetyl-
1H NMR (CDCl3):
J¼9.5 Hz, H-3), 5.16 (dd, 1H, J¼7.9 Hz, J¼9.6 Hz, H-2), 4.80 (d, 1H,
J¼7.8 Hz, H-1), 4.65 (t, 1H, J¼9.8 Hz, H-4), 3.90–3.82 (m, 2H, 2ꢀH-
6), 3.64 (m, 1H, J¼9.5 Hz, H-5), 2.11–1.98 (s, 9H, 3ꢀCH3).
b-D-glucopyranoside (6)
d
(ppm): 7.35–7.11 (m, 5H, Ar–H), 5.26 (t, 1H,
4.16. 3,4-Di-O-acetyl-glucal (20)
1H NMR (500 MHz, CDCl3),
d
(ppm): 6.49 (dd, 1H, J¼6.1 Hz, H-1),
5.41–5.50 (m, 1H, H-3), 5.22 (dd, 1H, J¼9.0, 6.5 Hz, H-4), 4.81 (dd,
1H, J¼5.9, 2.8 Hz, H-2), 3.98–4.09 (m, 1H, H-5), 3.66–3.86 (m, 2H, H-
6A,B), 2.07–2.13 (2s, 6H, 2ꢀCH3). The NMR data are in agreement
with the reported values.34
4.9. 1-Phenyl 2,3,6-tri-O-acetyl-
b-D-glucopyranoside (7)
1H NMR (CDCl3):
d
(ppm): 7.34–7.12 (m, 5H, Ar–H), 4.79 (t, 1H,
J¼9.4 Hz, H-3), 5.14 (dd, 1H, J¼7.8, 9.5 Hz, H-2), 4.75 (d, 1H,
J¼7.8 Hz, H-1), 4.34–4.09 (m, 2H, 2ꢀH-6), 3.91 (t, 1H, J¼9.5 Hz, H-
4), 3.65 (m, 1H, J¼9.5 Hz, H-5), 2.09–1.96 (s, 9H, 3ꢀCH3).
4.17. 4,6-Di-O-acetyl-glucal (21)
1H NMR (500 MHz, CDCl3),
d
(ppm): 6.35 (dd, 1H, J¼6.2 Hz, H-1),
4.95 (dd, 1H, J¼8.4, 6.2 Hz, H-4), 5.41–5.50 (m, 1H, H-3), 4.84 (dd,
1H, J¼6.2, 5.2, 3, H-2), 4.27 (dd, J¼12.9, 6.2 Hz, 1H-3), 4.11 (ddd, 1H,
J¼3.1 Hz, H-5), 4.20–4.37 (m, 2H, H-6A,B), 2.55 (br s, 1H, OH), 2.16
(s, 3H, CH3), 2.11 (s, 3H, CH3). The NMR data are in agreement with
the reported values.34
4.10. 1-(4-Nitrophenyl)-2,3,4-tri-O-acetyl-b-D-
glucopyranoside (10)
1H NMR (CDCl3):
d
(ppm): 8.10 (d, 2H, J¼8.5 Hz, H-30, H-50), 6.88
(d, 2H, J¼8.3 Hz, H-20, H-60), 5.25 (t, 1H, J¼9.4 Hz, H-3), 5.14 (dd, 1H,
J¼7.8, 9.5 Hz, H-2), 4.81 (d, 1H, J¼7.7 Hz, H-1), 4.66 (t, 1H, J¼9.7 Hz,
H-4), 3.92–3.84 (m, 2H, 2ꢀH-6), 3.65 (m, 1H, J¼9.4 Hz, H-5), 2.19 (s,
3H, CH3), 2.11–2.09 (s, 6H, 2ꢀCH3). The NMR data are in agreement
with the reported values.32
Acknowledgements
This research was supported by the Spanish CICYT (projects BIO-
2005-8576). We gratefully recognize CSIC by an I3P contract for JMP

A.A. Mendes et al. / Tetrahedron 64 (2008) 10721–10727
10727
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