Regioselective hydrolysis of different peracetylated β-monosaccharides by immobilized lipases from different sources. Key role of the immobilization

Article abstract of DOI:10.1002/adsc.200700055

The effect of the immobilization strategy on the activity, specificity and regioselectivity of three different lipases [those from Thermomyces lanuginose (TLL), Aspergillus niger (ANL) and Candida antarctica B (CAL-B)] in the hydrolysis of peracetylated β-monosaccharides has been evaluated. Three very different immobilization strategies were utilized, covalent attachment, anionic exchange and interfacial activation on a hydrophobic support. The octyl-TLL immobilized preparation was the most efficient biocatalyst in the hydrolysis of 1,2,3,4,6-pentaO-acetyl-β-D-galactopyranose, producing specifically 6-hydroxy-l,2,3,4-tetra-O-acetyl-β-D-galactopyranose in 95 % overall yield, whereas the CNBr-TLL preparation was 48 times slower and regioselective towards the anomeric position, producing the 1-hydroxy derivative in 70% yield. The PEI-TLL immobilized preparation was the most efficient catalyzing the hydrolysis of 1,2,3,4,6-penta-O-acetyl-β-D-glucopyranose, permitting us to obtain up to 70% of the 6-hydroxy product. In the hydrolysis of 2-acetamido2-deoxy-l,3,4,6-tetra-0-acetyl-β-D-glucopyranose, the octyl-CALB preparation was not selective at all for the production of monohydroxy products whereas when CAL-B was immobilized on PEI-agarose, the enzyme was highly specific and regioselective producing the 6-hydroxy-2- acetamido-2-deoxy-l,3,4-tri-O-acetyl-β-D-glucopyranose in 70 % yield.

Full text of DOI:10.1002/adsc.200700055

FULL PAPERS
DOI: 10.1002/adsc.200700055
Regioselective Hydrolysis of Different Peracetylated
b-Monosaccharides by Immobilized Lipases from Different
Sources. Key Role of The Immobilization
Jose M. Palomo,a,* Marco Filice,b Roberto Fernandez-Lafuente,a Marco Terreni,b
and Jose M. Guisana,*
a
Departamento de Biocatµlisis, Instituto de Catµlisis (CSIC), Campus UAM Cantoblanco, 28049 Madrid, Spain
Phone: (+34)-91-585-5478; fax: (+34)-91-585-4760; e-mail: josempalomo@icp.csic.es or jmguisan@icp.csic.es
Dipartimento di Chimica Farmaceutica, Università di Pavia, via Taramelli 12, 27100 Pavia, Italy
b
Received: January 24, 2007; Revised: May 17, 2007
Abstract: The effect of the immobilization strategy droxy derivative in 70% yield. The PEI-TLL immo-
on the activity, specificity and regioselectivity of bilized preparation was the most efficient catalyzing
three different lipases [those from Thermomyces la- the hydrolysis of 1,2,3,4,6-penta-O-acetyl-b-d-gluco-
nuginose (TLL), Aspergillus niger (ANL) and Candi- pyranose, permitting us to obtain up to 70% of the
da antarctica B (CAL-B)] in the hydrolysis of perace- 6-hydroxy product. In the hydrolysis of 2-acetamido-
tylated b-monosaccharides has been evaluated. 2-deoxy-1,3,4,6-tetra-O-acetyl-b-d-glucopyranose, the
Three very different immobilization strategies were octyl-CALB preparation was not selective at all for
utilized, covalent attachment, anionic exchange and the production of monohydroxy products whereas
interfacial activation on a hydrophobic support. The when CAL-B was immobilized on PEI-agarose, the
octyl-TLL immobilized preparation was the most ef- enzyme was highly specific and regioselective pro-
ficient biocatalyst in the hydrolysis of 1,2,3,4,6-penta- ducing the 6-hydroxy-2-acetamido-2-deoxy-1,3,4-tri-
O-acetyl-b-d-galactopyranose, producing specifically O-acetyl-b-d-glucopyranose in 70% yield.
6-hydroxy-1,2,3,4-tetra-O-acetyl-b-d-galactopyranose
in 95% overall yield, whereas the CNBr-TLL prepa-
ration was 48 times slower and regioselective to- Keywords: immobilization; lipase; lipase modula-
wards the anomeric position, producing the 1-hy- tion; peracetylated monosaccharides; regioselectivity
Introduction
overall yield – because of the low regioselectivity to
remove only one acetyl group among different esters
Carbohydrates exist in very different forms in nature with similar reactivity.
and are playing a very important role in many biologi-
Consequently, the use of enzymatic catalysts could
cal processes.[1]. Furthermore, many compounds with be an attractive and important alternative. However,
medical relevance are glycosylated.[2]
in most cases the enzymatic deacylation of fully acy-
Pure regioisomers of O-acetyl-glycopyranoses pre- lated pyranoses is very slow or proceeds with poor se-
senting only one free hydroxy group may be em- lectivity and yield. In addition, these reactions often
ployed as key intermediates in the preparation of dif- afford complex mixtures of tetra-, tri-, di-, and mono-
ferent glyco derivatives, such as oligosaccharides, gly- acetylated derivatives, along with free monosacchar-
colipids or glycopeptides.[3–7]
ides.[10]
To perform the enzymatic synthesis of mono-deace-
Per-O-acetyl-glycopyranoses could be used as raw
material to obtain monohydroxy peracetylated carbo- tylated carbohydrates following this strategy, it is nec-
hydrates, although the reported procedures of chemi- essary to find biocatalysts exhibiting good catalytic ac-
cal hydrolysis usually afford the deacetylation at the tivity and a high regioselectivity. Moreover, in case
anomeric position.[8] The synthesis of the monohy- that the enzyme is able to hydrolyze in several posi-
droxy derivatives in primary or secondary positions is tions – producing a mixture of monodeacetylated
very difficult by classical chemical approaches, products – it would be convenient that the enzyme
making it necessary to use many chemically selective preparation prefers the per-acetylated monosacchar-
protection/deprotection steps,[9] – with a poor final ide instead of the mono-deacetylated one, in order to
Adv. Synth. Catal. 2007, 349, 1969 – 1976
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1969

Jose M. Palomo et al.
FULL PAPERS
accumulate the latter (that is, to exhibit a high specif- of the lipase,[21] generating a highly hydrophobic envi-
icity for the per-acetylated sugar). ronment around the enzyme active site. (ii) Immobili-
Lipases recognize a broad range of substrates with zation on agarose activated with CNBr via covalent
high regio- and enantioselectivity in different process- attachment throughout the most active amino group
es such as racemic mixture resolutions and asymmet- (usually the terminal NH2) on the enzyme surface.[22]
ric reactions.[9b,11–13]. Indeed, lipase-catalyzed hydroly- (iii) Immobilization on agarose beads coated with PEI
ses of 1,2,3,4,6-penta-O-acetyl-a-d-glucopyranose via anionic exchange through the areas with highest
have been reported to yield deacetylation at the pri- negative net charge of the lipase.[23]
mary and the anomeric positions.[14]
These different immobilization strategies were used
The mechanism of catalysis of lipases implies dra- with lipases from Candida antarctica (isoform B)
matic conformational changes of the enzyme mole- (CAL-B),[24] Thermomyces lanuginose (TLL)[25] and
cule between a ꢁclosedꢂ and an ꢁopenꢂ form.[15,16] This Aspergillus niger (ANL),[26] applying them as biocata-
mechanism of action has permitted the use of differ- lysts in the hydrolysis of several per-O-acetylated
ent immobilization protocols (involving different monosaccharides in aqueous media.
areas of the lipase, rigidity, micro-environments,
etc.)[17] causing a strong alteration of the lipase prop-
erties in the kinetic resolution of racemic mix- Results and Discussion
tures[17–19] and asymmetric hydrolysis.[20] Here, we hy-
pothesize that, in a similar way, the use of different Effect and Key Role of the Immobilization Protocol
immobilization protocols may be used to modulate on the Lipase Activity in the Hydrolysis of
the regioselectivity of lipases in the hydrolysis of per- Acetylated b-Glycopyranoses
acetylated carbohydrates in aqueous media.
Herein, in order to demonstrate this hypothesis, The specific activity displayed by different immobi-
three very different immobilization protocols have lized preparations from TLL, CAL-B and ANL in the
been applied to the immobilization of different lipas- hydrolysis of 1,2,3,4,6-penta-O-acetyl-b-d-galactopyra-
es: (i) Immobilization on supports coated with a nose (1), 1,2,3,4,6-penta-O-acetyl-b-d-glucopyranose
dense layer of hydrophobic groups at low ionic (4) and 2-acetamido-2-deoxy-1,3,4,6-tetra-O-acetyl-b-
strength by interfacial activation of the lipases, involv- d-glucopyranose (7) are shown in Table 1, Table 2,
ing the hydrophobic area surrounding the active site and Table 3.
Table 1. Specificity and regioselectivity of different immobilized preparations of lipases in the hydrolysis of 1.
Entry
Enzyme
TLL
Support
Initial rate[a]
Reaction time [h]
Yield[b] [%]
2[c] [%]
3 [%]
Others
1
2
3
4
5
6
7
8
9
Octyl
BrCN
PEI
Octyl
BrCN
PEI
Octyl
BrCN
PEI
0.48
0.01
0.02
0.77
45
23
168
84
29
1.5
1
96
15
49
99
96
25
75
100
100
70
100
100
99
77
14.5
75
100
97
58
19
3
7.5
3
ANL
31
CAL-B
0.01
0.06
0.08
12
15
85
89
12
[a]
ꢀ1
The initial rate in mmol ” mgprot ” hꢀ1. It was calculated at 10–15% conversion.
Yield of the monohydroxy peracetylated product at 100% conversion.
The proportion of the anomers was a/b (1.3:1).
[b]
[c]
Table 2. Specificity and regioselectivity of different immobilized preparation of lipases in the hydrolysis of 4.
Entry
Enzyme
TLL
Support
Initial Rate[a]
Reaction time [h]
Yield[b] [%]
5[c] [%]
6 [%]
1
2
3
Octil
BrCN
PEI
0.0022
0.0022
0.0165
193
193
25
33
100
43
9
28
15
24
72
28
[a]
ꢀ1
The initial rate in mmol ” mgprot ” hꢀ1. It was calculated at 10–15% conversion.
Yield of the monohydroxy peracetylated product at 100% conversion
The proportion of the anomers was a/b (1.1:1)
[b]
[c]
1970
asc.wiley-vch.de
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2007, 349, 1969 – 1976

Regioselective Hydrolysis of Different Peracetylated b-Monosaccharides
FULL PAPERS
Table 3. Specificity and regioselectivity of different immobilized preparation of lipases in the hydrolysis of 7.
Entry
Enzyme
TLL
Support
Initial Rate[a]
Reaction time [h]
Yield[b] [%]
8
[c] [%]
9 [%]
Other[d]
1
2
3
4
5
6
7
8
9
Octyl
BrCN
PEI
Octyl
BrCN
PEI
Octyl
BrCN
PEI
0.004
0.002
0.006
0.10
29
96
194
68
95
90
26
20
33
33
30
33
69
70
67
63
70
67
100
96
100
100
0
ANL
84
0.5
0.42
312
102
122
32
CAL-B
0.003
0.095
0.074
80
65
35
100
15
30
20
70
[a]
ꢀ1
The initial rate in mmol ” mgprot ” hꢀ1. It was calculated at 10-15% conversion.
Yield of the monohydroxy peracetylated product at 100% conversion.
The proportion of the anomers was a/b (1.1:1).
[b]
[c]
[d]
Di-deacetylated product.
First, we will comment the results obtained with on agarose activated with CNBr presented the highest
TLL: In the hydrolysis of 1, TLL immobilized on activity value, around 30-fold higher than using the
octyl-agarose presented the highest activity value octyl-CAL-B preparation (Table 3).
compared with the other immobilized preparations
Taken together, the above results clearly show that
from this lipase, even up to 48 times higher compared the use of different immobilization protocols greatly
with that one of the CNBr-TLL preparation or 24 alters the catalytic activity of the enzyme for different
times higher in relation of the activity of PEI-TLL per-O-acetylated carbohydrates. This may be clearly
preparation (Table 1, entries 1–3). However, the PEI- appreciated because, for each particular lipase, the
TLL immobilized preparation was the most active most active immobilized preparation was different
catalyst in the hydrolysis of 4 (Table 2), with a specific when the substrate was different, although all prepa-
activity seven times higher with respect to the other rations presented the same lipase.
TLL preparations. In the hydrolysis of 7 (Table 3), the
PEI-TLL preparation was again the most active bio-
catalyst (more than three times compared with the Specificity and Regioselectivity of the Different
other preparations). Thus, the presence of an acetami- Immobilized Lipase Preparations Catalyzing the
do group in position 2 (7) (compared with 4) produ- Hydrolysis of 1
ces a very different effect on the activity of the differ-
ent TLL preparations. The activity of the PEI-TLL The different immobilized preparations of the lipases
preparation decreased by a factor of almost three presented a different specificity and regioselectivity in
times whereas the activity of the octyl-TLL immobi- the hydrolysis of 1 (Table 1, Scheme 1).
lized preparation increased by a factor of almost two
times while the activity of the CNBr-TLL preparation ose preparation was very specific towards the perace-
remained very similar. tylated monosaccharide and very regioselective to-
When TLL preparations were used, the octyl-agar-
Using ANL as biocatalyst, the CNBr-immobilized wards the 6 position (Scheme 1), permitting us to
preparation presented the highest activity in the hy- obtain 99% of the 6-OH free monohydroxyperacety-
drolysis of 1, being more than 50 times more active lated sugar 3 (entry 1, Table 1). However, this enzyme
than the octyl-ANL immobilized preparation. The immobilized on CNBr-agarose produced 96% of
PEI-ANL preparation also showed a very high activi- monohydroxy products of which 77% was in the
ty, although lower than that of the CNBr-ANL prepa- anomeric position (a/b, 56%/44%) (2) (entry 2,Ta-
ration (Table 1). In the hydrolysis of 7, the immobili- ble 1). On the other hand, the PEI-TLL immobilized
zation of ANL on PEI-agarose permitted us to obtain preparation was neither specific towards peracetylat-
the most active preparation (around 300 times more ed 1 – with only a 25% yield in monohydroxy product
active compared to the enzyme immobilized on octyl- – nor totally regioselective, giving a mixture of mono-
agarose) (Table 3).
hydroxy products deprotected in the anomeric and 6-
In the case of CAL-B, the lipase showed the highest OH positions (entry 3, Table 1). Thus, Figure 1 shows
activity after immobilization on PEI-agarose in the how, depending of the immobilization strategy, the
hydrolysis of 1, being eight times more active than profiles of the course in the hydrolysis of 1 catalyzed
the octyl-CAL-B immobilized preparation (Table 1). by the TLL were quite different.
However, in the hydrolysis of 7, CAL-B immobilized
Adv. Synth. Catal. 2007, 349, 1969 – 1976
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
1971

Jose M. Palomo et al.
FULL PAPERS
Scheme 1. Specific and regioselective hydrolysis of different 1,2,3,4,6-penta-O-acetyl-b-d-glycopyranoses.
a mixture of monohydroxy products, 97% of 2 and
3% of 3 (entry 6 , Table 1).
When CAL-B was studied, the octyl-CAL-B and the
CNBr-CAL-B immobilized preparations were regiose-
lective, preferentially hydrolyzing the anomeric position.
The octyl-CAL-B preparation hydrolyzed 1 producing
70% of monohydroxy compound of which 58% was
monohydroxy 2 whereas this enzyme immobilized on
CNBr-agarose was specific for the mono-deacetylation
of 1 with a 100% yield of which 85% was monohy-
droxy 2 (entries 7 and 8, Table 1). The immobilization
of CAL-B on PEI-agarose permitted us to get a high
specific biocatalyst towards the peracetylated monosac-
charide, but the enzyme was not totally regioselective,
giving 89% of 2 and 12% of 3 (entry 9, Table 1).
For a more high scale production, the hydrolysis
was performed using 8 g/L substrate concentration
using octyl-TLL immobilized preparation as catalyst.
3 was isolated in 95% overall yield. A similar result
was obtained in the production of 2 using the CNBr-
ANL immobilized preparation as catalyst.
Specificity and Regioselectivity of the Different
Immobilized TLL Preparations Catalyzing the
Hydrolysis of 4
Figure 1. Reaction course of different immobilized prepara-
tions of TLL in the hydrolysis of 1: A) octyl-agarose prepa-
ration; B) PEI-agarose preparation (squares=conversion,
triangles=yield of monoprotected product).
The different preparations of TLL presented a differ-
ent specificity and regioselectivity in the hydrolysis of
4 (Table 2). TLL immobilized on CNBr-agarose was
quite specific, producing only monohydroxy product,
and yielding 24% of 5 and 72% of 6 (Scheme 1).
However, when the enzyme was immobilized on
In the case of using ANL, the different immobilized octyl-agarose or on PEI-agarose, the production of
preparations preferentially hydrolyzed in the anome- mono-deacetylated product decreased with yields
ric position producing the tetra-acetylated product 2 lower than 50% (entry 1, 3, Table 2), suggesting a
with a different specificity (Scheme 1); 100% yield lower preference by the peracetylated substrate. The
using the CNBr-ANL preparation and 75% yield regioselectivity was similar to that observed with the
using the octyl-ANL preparation. (Table 1). However, CNBr-TLL preparation.
when ANL was immobilized on PEI-agarose, the
Using 8 g/L substrate, the hydrolysis of 4 was per-
enzyme was completely specific towards the peracety- formed using the CNBr-TLL immobilized prepara-
lated sugar although not totally regioselective, giving tion, and 6 was isolated in 63% overall yield.
1972
asc.wiley-vch.de
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2007, 349, 1969 – 1976

Regioselective Hydrolysis of Different Peracetylated b-Monosaccharides
FULL PAPERS
Specificity and Regioselectivity of the Different
Immobilized Lipase Preparations Catalyzing the
Hydrolysis of 7
the PEI-TLL immobilized preparation was the most
active one catalyzing the hydrolysis of 4. Furthermore,
the octyl-TLL was the most specific and regioselective
producing exclusively the 6-OH monoxydroxy deriva-
Table 3 summarizes the results observed using this tive 3 in 95% overall yield, whereas the CNBr-TLL
substrate. When TLL was used, the immobilization on preparation was regioselective towards the anomeric
octyl-agarose or PEI-agarose permitted us to obtain a position, producing 2 from the hydrolysis of 1. The
highly specific catalyst with a yield of monohydroxy octyl-CAL-B immobilized preparation was not selec-
products 8 and 9 (Scheme 1) near to 100% whereas tive at all for the production of monohydroxy deriva-
the enzyme immobilized on CNBr-agarose gave 70% tives in the hydrolysis of 7, whereas when CAL-B was
yield (Table 3). It is interesting to note that the most immobilized on PEI-agarose, the enzyme was highly
specific preparations in the hydrolysis of 2-acetamido- specific and regioselective, producing 70% of 9.
2-deoxy-1,3,4,6-tetra-O-acetyl-b-d-glucopyranose (7) Therefore, the use of this lipase engineering strategy
(Table 3) presented
a
poor specificity towards via immobilization have permitted to obtain an opti-
1,2,3,4,6-penta-O-acetyl-b-d-glucopyranose
(4) mal biocatalyst for the regioselective hydrolysis of dif-
(Table 2).
ferent peracetylated b-monosaccharides producing
When using ANL, the three immobilized prepara- only one free hydroxy group in different positions in
tions were quite specific in the hydrolysis of the per- high overall yields and suitable reaction times.
acetylated compound, with a regioselectivity towards
anomeric (30%, 8) and 6-OH (70%, 9) positions.
The most interesting results were found when we Experimental Section
used the different preparations of CAL-B as biocata-
lysts (entries 7–9, Table 3). Here, the immobilization General Remarks
of CAL-B on octyl-agarose produced a biocatalyst
with no specificity towards 7, and in fact the main
products were di-deacetylated compounds (80%)
Lipase from Aspergillus niger (ANL) was purchased from
Fluka (Neu Ulm, Germany). The lipases from T. lanuginose
(TLL) and C. antarctica B (Novozym 525 L) (CAL-B) were
kindly supplied by Novo Nordisk company. PEI-agarose[23]
were prepared as previously described. Octyl-agarose
(4BCL) and cyanogen bromide (CNBr-activated Sepharose
4BCL) were purchased from Pharmacia Biotech (Uppsala,
Sweden). Polyethyleneimine (PEI) (Mr 25000), Triton X-
100, p-nitrophenyl propionate (pNPP), 1,2,3,4,6-penta-O-
acetyl-b-d-galactopyranose (1), 1,2,3,4,6-penta-O-acetyl-b-d-
glucopyranose (4) and 2-acetamido-2-deoxy-1,3,4,6-tetra-O-
acetyl-b-d-glucopyranose (7) were from Sigma Chem. Co.
The pH of the solutions during the enzymatic hydrolysis was
kept constant using an automatic titrator 718 Stat Tritino
from Metrohm (Herisau, Switzerland). HPLC analyses were
performed using a HPLC spectra P100 (Thermo Separation
products). The column was a Kromasil-C18 (250”4.6 and
5 mm) from Analisis Vinicos (Tomelloso, Spain). Analyses
were run at 258C using an L-7300 column oven and UV de-
tector L-7400 at 215 nm. The eluent was an isocratic mixture
of 30% acetonitrile in phosphate buffer (10 mM) at pH 4
for 1 and 4, and 20% acetonitrile in phosphate buffer for 7;
flow rate 1.0 mLminꢀ1. The retention times of the substrates
in these conditions were; 1: 30 min, 4: 25 min, 7: 15.5 min.
Columns for flash chromatography were made up with
Silica Gel 60 (Merck) 60–200 or 40–63 mm. The elution was
(entry 7, Table 3). When the enzyme was immobilized
on CNBr-agarose, it presented a certain specificity to-
wards the production of mono-deacetylated product
(35%) with a regioselectivity towards 8 and 9,
(entry 8, Table 3) although still 65% were di-deacety-
lated products. However, the immobilization of CAL-
B on PEI-agarose permitted us to obtain a highly spe-
cific biocatalyst, with 100% yield of a mixture of two
monohydroxy derivatives: 8 (30%) and 9 (70%)
A
(entry 9, Table 3). When the hydrolysis was performed
using 8 g/L substrate and using the CNBr-ANL im-
mobilized preparation, 9 was isolated in 65% overall
yield.
Conclusions
In this paper, we have described a simple approach
for preparing monohydroxy peracetylated monosac-
charides, very interesting and useful building blocks
for carbohydrate chemistry, by specific and regioselec-
tive hydrolysis catalyzed by immobilized lipases.
Here, we presented the key role of the immobilization
strategy in the hydrolytic activity, specificity and re-
gioselectivity of three different lipases in the hydroly-
sis of peracetylated sugars. Thus, different immobi-
lized preparations of different lipases presented quite
different behaviour in the hydrolysis of several fully
protected monosaccharides. For example, with respect
to the reaction rate, the octyl-TLL preparation was
1
performed with 40:60 hexane-ethyl acetate. H NMR were
recorded in CDCl3 (d=ppm) on a Bruker AMX 400 instru-
ment.
Standard Enzymatic Activity Assay
In order to follow the immobilization process, the activities
of the soluble lipases and their immobilized preparations
were analyzed spectrophotometrically by measuring the in-
crement in absorbance at 348 nm ( 2 =5.150Mꢀ1 cmꢀ1) pro-
the most active biocatalyst in the hydrolysis of 1 while duced by the release of p-nitrophenol (pNP) in the hydroly-
Adv. Synth. Catal. 2007, 349, 1969 – 1976
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
1973

Jose M. Palomo et al.
FULL PAPERS
sis of 0.4 mM pNPP in 25 mM sodium phosphate buffer at
pH 7 and 258C. To initialize the reaction, 0.05–0.2 mL of
lipase solution (blank or supernatant) or suspension was
added to 2.5 mL of substrate solution. Enzymatic activity
was determined as mmol of hydrolyzed pNPP per minute
per mg of enzyme (IU) under the conditions described
above.
Immobilization of Lipases on CNBr-Activated
Support
Commercial agarose support activated with CNBr was sus-
pended in an acidic aqueous solution (pH 2–3) during one
hour. After that the solid support was dried by filtration
under vacuum.
10 mL (for TLL or CAL-B) and 4 mL (for ANL) of the
purified lipase solution (1.2 mgmLꢀ1) were added to 8 mL
of 10 mM sodium phosphate buffer solution at pH 7. After
that, 1 g of the CNBr-agarose support was added. The mix-
ture was then shaken at 258C and 250 rpm for 18 h. After
that, the solution was removed by filtration and the support-
ed lipase was washed several times with distilled water. The
percentage of immobilization and the amount of immobi-
lized lipase in each case are shown in Table 4. The immobili-
zation was followed by the assay described above.
Purification of Lipases
The purification of the lipases was performed using a previ-
ously described protocol, based on the interfacial activation
of lipases on hydrophobic supports at low ionic strength.[21]
0.5 g of ANL commercial solid powder (22 mg protein)[27]
and 5 mL TLL or CAL-B commercial solution (12 mg pro-
tein/mL)[27] were dissolved in 95 mL of 10 mM sodium phos-
phate buffer at pH 7.0. In each case 5 g of octyl-agarose sup-
port were added. The supernatant and suspension activities
were periodically checked by the method described above
and the immobilization was terminated after 5 h by filtra-
tion. In all cases, more than 90% of lipase was immobilized.
Following this protocol, the SDS-PAGE analysis of the pro-
tein adsorbed to the octyl-Sepharose[21] only showed a single
band with a molecular weight corresponding to that of the
different native lipases. These adsorbed lipases were used as
the biocatalyst in some studies. The percentage of immobili-
zation and the enzyme loading in each case are shown in
Table 4.
Immobilization of Lipases on PEI-Agarose Support
20 mL (for TLL or CAL-B) and 8 mL (for ANL) of the pu-
rified lipase solution (1.2 mgmLꢀ1) were added to 16 mL of
10 mM sodium phosphate buffer solution at pH 7 for TLL,
ANL and pH 9 for CAL-B. After that, 2 g of the glyoxyl-
agarose beads coated with polyethyleneimine (PEI) were
added. The mixture was then shaken at 258C and 250 rpm
for 4 h. After that, the supernatant was removed by filtra-
tion and the supported lipase washed several times with dis-
tilled water. The percentage of immobilization and the
enzyme loading in each case are shown in Table 4.
If the lipases need to be released from the octyl-agarose,
the preparation was added to a solution of 1% Triton (v/v)
in 10 mM sodium phosphate buffer at pH 7.0 and 48C for
1 h obtaining a purified lipase solution with a final concen-
tration of 1.2 mg lipase/mL. Then the enzymatic solution
was used for immobilization on the different supports.
Enzymatic Hydrolysis of Peracetylated
Monosaccharides
Standard assay was performed as follows: 1,
4 or 7
(0.02 mmol, 8 mg) was added to 10 mL solution of phos-
phate buffer 25 mM with 10% acetonitrile at pH 5, 258C
and the reaction was initialized by adding 0.8 g (1) or 1 g (4,
7) of the biocatalyst. The reaction was performed at pH 5 in
order to avoid the chemical acyl-migration in the per-O-ace-
tylated carbohydrates hydrolysis.[28] The hydrolytic reaction
was carried out under mechanical stirring, and the pH value
was controlled by automatic titration. Hydrolysis reactions
were followed by HPLC. Finally, the optimization of the re-
action in each case was performed using a 8 g/L substrate
Table 4. Immobilization yield of lipases on different sup-
ports.
Lipase Support[a] Immobilization Protein
Recovered
loading[c] activity [%]
yield [%][b]
TLL
soluble
octyl
CNBr
PEI
-
-
100
1800
40
100
100
100
-
100
48
48
-
12
12
12
-
4.5
2.4
2.4
-
1
and the products were isolated and identified by H NMR.
60
ANL soluble
octyl
100
115
100
95
100
100
90
2,3,4,6-Tetra-O-acetyl-a/b-d-galactopyranose (2)
Substrate 1 (390 mg, 1 mmol) was hydrolyzed in 50 mL solu-
tion of phosphate buffer 50 mM (80%) and acetonitrile
(20%) using 5 g CNBr-ANL preparation at pH 5. When the
substrate had disappeared (checked by TLC and HPLC),
the aqueous solution was filtered and saturated with NaCl
and then extracted with ethyl acetate (5”50 mL). The col-
lected organic layers were washed with a 5% NaHCO3 solu-
tion (2”10 mL), separated and dried over anhydrous
Na2SO4, which was then removed by filtration and concen-
trated under vacuum to afford 2 as a white solid; yield:
370 mg (95%). HPLC analysis: tR =8.3 (b anomer), 9.8 min
CNBr
PEI
CAL- soluble
B
octyl
CNBr
PEI
100
16
42
12
2
5
95
[a]
[b]
Soluble is referred to the purified lipase by the method-
ology described in Experimental Section.
The percentage of immobilized enzyme was determined
comparing the enzymatic activity left in the supernatant
with the activity of free enzyme – which is 100% – using
the pNPP assay.
1
(a-anomer). H NMR (400 MHz, CDCl3): d=5.52 (bd, 1H,
J=3, 4 Hz; H-1), 5.48 (dd, 1H, J=1.25 Hz, H-4), 5.41 (dd,
1H, J=3.4 Hz, H-3), 5.19 (dd, 1H, J=3, 4 Hz, H-2), 4.72
[c]
As mg of purified lipase/g of support.
1974
asc.wiley-vch.de
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2007, 349, 1969 – 1976

Regioselective Hydrolysis of Different Peracetylated b-Monosaccharides
FULL PAPERS
(dt, 1H, J=6.5 Hz, H-5), 4.12–4.08 (dd, 2H, J=11.5 Hz, H-
6a,b), 2.15–1.99 (s, 12H ,4CH3). The NMR data are in
agreement with the reported values.[29]
3H, CH3), 1.99 (s, 3H, CH3). The NMR data are in agree-
ment with the reported values.[30]
2-Acetamido-2-deoxy-3,4,6-tri-O-acetyl-a/b-d-
glucopyranose (8)
1,2,3,4-Tetra-O-acetyl-b-d-galactopyranose (3)
Substrate 7 (390 mg, 1 mmol) was hydrolyzed in 50 mL solu-
tion of phosphate buffer 50 mM (80%) and acetonitrile
(20%) using 5 g CNBr-ANL preparation at pH 5. When the
substrate had disappeared (checked by TLC and HPLC),
the aqueous solution was filtered and saturated with NaCl
and then extracted with ethyl acetate (5”50 mL). The col-
lected organic layers were washed with a 5% NaHCO3 solu-
tion (2”10 mL), separated and dried over anhydrous
Na2SO4, which was then removed by filtration and concen-
trated under vacuum. After that, purification by the flash
chromatography column was performed with 95:5 dichloro-
methane:methanol as eluent; yield: 97.5 mg (25%). HPLC
Substrate 1 (390 mg, 1 mmol) was hydrolyzed in 50 mL solu-
tion of phosphate buffer 50 mM (80%) and acetonitrile
(20%) using 5 g octyl-TLL preparation at pH 5. When the
substrate had disappeared (checked by TLC and HPLC),
the aqueous solution was filtered and saturated with NaCl
and then extracted with ethyl acetate (5”50 mL). The col-
lected organic layers were dried over anhydrous Na2SO4,
which was then removed by filtration and concentrated
under vacuum to afford 3 as a white solid; yield: 370 mg
(95%). HPLC analysis: tR =10.6 min. 1H NMR (500 MHz,
CDCl3): d=5.73 (d, J=8.26 Hz, H-1), 5.44 (d, J=3.39 Hz,
H-4), 5.32 (t, J=8.37 Hz, H-3), 5.13 (dd, J=3.42 Hz, J=
10.4 Hz, H-2), 3.91 (dt, J=6.45 Hz, H-5), 3.8–3.51 (m, ABX
system, 2H, H-6), 2.14–1.97 (s, 12H, 4CH3).
1
analysis: tR =6.5 min. H NMR (500 MHz, CDCl3): d=6.04
(d, J=9.5 Hz, NH), 5.25 (dd, J3,2 =10.0, J3,4 =9.5 Hz, H-3),
5.15 (d, J=3.5 Hz, H-1), 5.07 (t, J=9.5 Hz, H-4), 4.73 (d, J=
8.5 Hz, H-1, b-anomer), 4.22 (m, H-5), 4.16 (m, 2H, H-2, H-
6), 4.02 (m, H-6), 2.03 (s, 3H, CH3), 1.97 (s, 3H, CH3), 1.96
(s, 3H, CH3), 1.90 (s, 3H, N-2 CH3). The NMR data are in
agreement with the reported values .[29]
2,3,4,6-Tetra-O-acetyl-a/b-d-glucopyranose (5)
Substrate 4 (390 mg, 1 mmol) was hydrolyzed in 50 mL solu-
tion of phosphate buffer 50 mM (80%) and acetonitrile
(20%) using 5 g CNBr-TLL preparation at pH 5. When the
substrate had disappeared (checked by TLC and HPLC),
the aqueous solution was filtered and saturated with NaCl
and then extracted with ethyl acetate (5”50 mL). The col-
lected organic layers were dried over anhydrous Na2SO4,
which was then removed by filtration and concentrated
under vacuum. After that, purification by the flash chroma-
tography column was performed with 40:60 hexane-ethyl
acetate as eluent;yield: 78 mg (20%). HPLC analysis: tR =
2-Acetamido-2-deoxy-1,3,4-tri-O-acetyl-b-d-
glucopyranose (9)
Substrate 7 (390 mg, 1 mmol) was hydrolyzed in 50 mL solu-
tion of phosphate buffer 50 mM (80%) and acetonitrile
(20%) using 5 g CNBr-ANL preparation at pH 5. When the
substrate had disappeared (checked by TLC and HPLC),
the aqueous solution was filtered and saturated with NaCl
and then extracted with ethyl acetate (5”50 mL). The col-
lected organic layers were washed with a 5% NaHCO3 solu-
tion (2”10 mL), separated and dried over anhydrous
Na2SO4, then filtered and reduced under vacuum. After
that, purification by the flash chromatography column was
performed with 95:5 dichloromethane:methanol; yield:
253 mg (65%). HPLC analysis: tR =8.4 min. 1H NMR
(400 MHz, CDCl3): d=5.80 (d, J=3.51 Hz, H-1), 5.50 (d,
J=9 Hz, 1H-NH), 5.30 (t, J=9.9 Hz, H-3), 5.10 (t, J=
9.6 Hz, H-4), 4.35 (dd, J=9.8 Hz, J=6.70 Hz, H-2), 4.28–
4.20 (m, 2H, H-6), 4.19–4.10 (m, H-5), 2.21 (s, 9H, 3CH3),
1.96 (s, 3H, CH3).
1
8.2 min. H NMR (500 MHz, CDCl3): d=5.47 (t, J=9.7 Hz,
H-3, a anomer), 5.38 (d, J=3.5 Hz, H-1, a-anomer), 5.18 (t,
J=9.6 Hz, H-3, b-anomer), 5.03 (t, J=9.7 Hz, H-4, a/b
anomers), 4.86 (dd, J=8.1, 9.7 Hz, H-2, b-anomer), 4.83 (dd,
J=3.6, 10.1 Hz, H-2, a-anomer), 4.70 (d, J=8.0 Hz, H-1, b-
anomer), 3.95–4.25 (m, 3H, H-5, a anomer, H-6, a/b anom-
ers), 3.71 (m, ABX system, H-5, b-anomer), 1.90–2.20 (s,
12H, 4CH3, a/b anomers). The NMR data are in agreement
with the reported values .[29]
1,2,3,4-Tetra-O-acetyl-b-d-glucopyranose (6)
Substrate 4 (390 mg, 1 mmol) was hydrolyzed in 50 mL solu-
tion of phosphate buffer 50 mM (80%) and acetonitrile
(20%) using 5 g CNBr-TLL preparation at pH 5. When the
substrate had disappeared (checked by TLC and HPLC),
the aqueous solution was filtered and saturated with NaCl
and then extracted with ethyl acetate (5”50 mL). The col-
lected organic layers were dried over anhydrous Na2SO4,
which was then removed by filtration and concentrated
under vacuum. After that, purification by the flash chroma-
tography column was performed with 40:60 hexane-ethyl
acetate as eluent; yield: 246 mg (63%). HPLC analysis: tR =
10 min. 1H NMR (500 MHz, CDCl3): d=5.70 (d, 1H, J=
Acknowledgements
This research was supported by the Spanish CICYT (projects
BIO-2005–8576). We gratefully recognize CSIC by an I3P
contract for JMP (FEDER founds). Dr. Angel Berenguer is
gratefully recognized for his help during the writing of this
manuscript.
8.4 Hz, H-1), 5.27 (t, J=9.7 Hz, H-3), 5.07 (m, J=8.4 and References
9.7 Hz, 2H, H-2, H-4), 3.73 (dd, J=2.3 and 12.5 Hz, H-6),
3.62 (ddd, J=2.3, 4.2 and 9.7 Hz, H-5), 3.55 (dd, J=4.2 and
12.5 Hz, H-6), 2.08 (s, 3H, CH3), 2.03 (s, 3H, CH3), 2.00 (s,
[1] a) A. Varki, Glycobiology 1993, 3, 97–130; b) C. A.
Ryan, Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 1–2;
Adv. Synth. Catal. 2007, 349, 1969 – 1976
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
1975

Jose M. Palomo et al.
FULL PAPERS
c) C. R. Bertozzi, L. L. Kiessling, Science. 2001, 291,
2357–2364; d) A. Giannis, Angew. Chem., Int. Ed.
Engl.1994, 33, 178–180.
[18] a) A. Chaubey, R. Parshad, S. Koul, S. C. Taneja, G. N.
Qazi, J. Mol. Catal. B: Enzym. 2006, 42,39–44; b) Y.
Nakamura, T. Matsumoto, F. Nomoto, M. Ueda, et al.
Biotechnol. Progr. 2006, 22, 998–1002; c) J. M. Palomo,
R. L Segura, C. Mateo, M. Terreni, J. M. Guisµn, R.
Fernµndez-Lafuente. Tetrahedron: Asymmetry 2005,
16, 869–874.
[2] a) S. Blanchard, J. S. Thorson, Curr. Opin Chem. Biol.
2006, 10, 263–271; b) E. Borges de Melo, A. da Silveira
Gomes, I. Carvalho, Tetrahedron 2006, 62, 10277–
10302.
[3] a) K. J. Doores, D. P. Gamblin, B. G. Davis, Chem. Eur.
J. 2006, 12, 656–665; b) T. K.-K. Mong, L. V. Lee, J. R.
Brown, J. D. Esko, C.-H. Wong, ChemBioChem. 2003,
4, 835–840; c) M. Filice, D. Ubiala, G. Pagani, M. Ter-
reni, M. Pregnolato, Arkivoc 2006, 2006, 66–73.
[4] D. Disney, P. H. Seeberger, Chem. Biol. 2004, 11, 1701–
1707.
[5] S. A. Svarovsky, M. B. Taraban, J. J. Barchi, Org.
Biomol. Chem. 2004, 2, 3155–3161.
[6] K. C. Nicolaou, S. Y. Cho, R. Hughes, N, Winssinger,
C. Smethurst, H. Labischinski, R. Endermann, Chem.
Eur. J. 2001, 7, 3798–3823.
[7] A. Nikolakakis, K. Haidara, F. Sauriol, O. Mamer,
L. O. Zamir, Bioorg Med Chem. 2003, 11, 1551–1556.
[8] R. Khan, P. A. Konowicz, L. Gardossi, M. Matulova, S.
de Genaro, Aust. J. Chem. 1996, 49, 293–298.
[9] a) A. Ghanem, H. Y. Aboul-Enein, Chirality 2005, 17,
44–50; b) C.-H. Wong, G. M. Whitesides Enzymes in
synthetic organic chemistry, Pergamon Press, Oxford,
1994; c) B. La Ferla, Monatsh. Chem. 2002, 133, 1–18.
[10] a) W. J. Hennen, H. M. Sweers, Y. F. Wang, C. H.
Wong, J. Org. Chem. 1988, 53, 4939; b) J. F. Shaw,
A. M. Klibanov, Biotechnol. Bioeng. 1987, 29, 648–651.
[11] a) U. T. Bornscheuer. Curr. Opin. Biotechnol. 2002, 13,
543–547; b) N. J. Turner. Curr. Opin. Biotechnol. 2003,
14, 401–406.
[12] S. Akai, K. Tanimoto, Y. Kanao, M. Egi, T. Yamamoto,
Y. Kiia, AngewChem. Int. Ed. 2006, 45, 2592–2595.
[13] B. Larissegger-Schnell, S. M. Glueck, W. Kroutil, K.
Faber, Tetrahedron 2006, 62, 2912–2916.
[14] G. Fernµndez-Lorente, J. M. Palomo, J. Cocca, C.
Mateo, R. Fernµndez-Lafuente, P. Moro, M, Terreni,
J. M. Guisµn, Tetrahedron 2003, 59, 5705–5711.
[19] a) H. Yu, J. Wu, B. C. Chi, Biotechnol. Lett. 2004, 26,
629–633; b) J. M. Palomo, G. Fernµndez-Lorente, C.
Mateo, C. Ortiz, R. Fernµndez-Lafuente, J. M. Guisµn,
Enzyme Microb. Technol. 2002, 31, 775–783; c) G. Fer-
nandez-Lorente, J. M. Palomo, C. Mateo, R. Munilla,
C. Ortiz, Z. Cabrera, J. M. Guisan, R. Fernandez-La-
fuente, Biomacromolecules. 2006, 7, 2610–2615.
[20] Z. Cabrera, J. M. Palomo, G. Fernµndez-Lorente, J. M.
Guisan, R. Fernµndez-Lafuente, Enzyme Microb. Tech-
nol. 2007, 40, 1280–1285.
[21] a) A. Bastida, P. Sabuquillo, P. Armisen, R. Fernµndez-
Lafuente, J. Huguet, J. M. Guisµn, Biotechnol. Bioeng.
1998, 58, 486–493; b) J. M. Palomo, G. MuÇoz, G.
Fernµndez-Lorente, C. Mateo, G. Fernµndez-Lafuente,
J. M. Guisµn, J. Mol. Cat. B: Enzym. 2002, 19–20, 279–
286.
[22] C. Mateo, O. Abian, M. Bernedo, E. Cuenca, M.
Fuentes, G. Fernµndez-Lorente, J. M. Palomo, V.
Grazu, B. C. C. Pessela, C. Giacomini, G. Irazoqui, A.
Villarino, K. Ovsejevi, F. Batista-Viera, R. Fernµndez-
Lafuente, J. M. Guisµn, Enzyme Microb. Technol. 2005,
37, 456–462.
[23] C. Mateo, O. Abian, R. Fernµndez-Lafuente, J. M.
Guisµn, Biotechnol Bioeng. 2000, 68, 98–105.
[24] J. Uppenberg, M. T. Hansen, S. Patkar, T. A. Jones,
Structure 1994, 2, 293–308.
[25] F. Ganske, U. T. Bornscheuer, J. Mol. Catal. B: Enzym.
2005, 36,40–42.
[26] M. Drescher, F. Hammerschmidt, H. Kahlig, Synthesis
1995, 1267–1272.
[27] M. M. Bradford, Anal Biochem. 1976, 72, 248–254.
[28] a) T. Horrobin, Ch. H. Tran, D. Crout, J. Chem. Soc.,
Perkin Trans. 1 1998, 1069–1080; b) M. Terreni, R. Sal-
vetti, L. Linati, R. Fernandez-Lafuente, G. Fernµndez-
Lorente, A. Bastida, J. M. Guisan, Carbohydr. Res.
2002, 337, 1615–1621.
[15] L. Brady, A. M. Brzozowski,
Derewenda, Z. S.
Dodson, E. Dodson, G. Tolley, S. Turkenburg, J. P.
Christiansen, L. Huge-Jensen, B. Norskov, L. Thim, L.
Menge, Nature 1990, 43, 767–770.
[16] A. Aloulou, J. A. Rodriguez, S. Fernandez, D. van Oos-
terhout, D. Puccinelli, F. Carri›re, Biochim. Biophys
Acta 2006, 1761, 995–1013.
[17] J. M. Palomo, G. Fernµndez-Lorente, C. Mateo, M.
Fuentes, R. Fernµndez-Lafuente, J. M. Guisµn, Tetrahe-
dron: Asymmetry 2002, 13, 1337–1345.
[29] M. M. Sing, H. Kondo, C.-H. Wong, J. Am. Chem. Soc.
1993, 115, 2260–2267.
[30] X. Ding, W. Wang, F. Wong, Carbohydr. Res. 1997, 303,
1354–1358.
1976
asc.wiley-vch.de
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2007, 349, 1969 – 1976