Synthesis of enantiomerically pure glycidol via a fully enantioselective lipase-catalyzed resolution

Article abstract of DOI:10.1016/j.tetasy.2004.12.027

The efficient enzymatic synthesis of enantiopure 2,3-epoxypropanol (glycidol) has been achieved. The racemic glycidyl butyrate was successfully resolved by enzymatic hydrolysis using a strategy that combines different immobilization protocols and different experimental reaction conditions. A new enzyme (25 kDa lipase)-which is a lipase-like enzyme purified from the pancreatic porcine lipase (PPL) extract-immobilized on DEAE-Sepharose was selected as the optimal biocatalyst. The optimal results were obtained at pH 7, 25°C and 10% dioxane using this biocatalyst and a very high enantioselectivity for the enzyme was displayed, obtaining both (R)-(-)-glycidyl butyrate and (R)-(+)-glycidol with enantiomeric excesses >99% (E >100). The hydrolysis of (R)-(-)-glycidyl butyrate produced pure (S)-(-)-glycidol.

Full text of DOI:10.1016/j.tetasy.2004.12.027

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 16 (2005) 869–874
Synthesis of enantiomerically pure glycidol via a fully
enantioselective lipase-catalyzed resolution
a
Jose M. Palomo, Rosa L. Segura, Cesar Mateo, Marco Terreni,
a
a
b
a,
Jos e´ M. Guisan and Roberto Fern a´ ndez-Lafuente
a,
*
*
a
Departamento de Biocat a´ lisis, Instituto de Cat a´ lisis (CSIC), Campus UAM Cantoblanco, 28049 Madrid, Spain
b
Dipartimento di Chimica Farmaceutica, Universit a` di Pavia, via Taramelli 12, 27100 Pavia, Italy
Received 8 November 2004; accepted 16 December 2004
Available online 1 February 2005
Abstract—The efficient enzymatic synthesis of enantiopure 2,3-epoxypropanol (glycidol) has been achieved. The racemic glycidyl
butyrate was successfully resolved by enzymatic hydrolysis using a strategy that combines different immobilization protocols and
different experimental reaction conditions. A new enzyme (25 kDa lipase)—which is a lipase-like enzyme purified from the pancreatic
porcine lipase (PPL) extract—immobilized on DEAE–Sepharose was selected as the optimal biocatalyst. The optimal results were
obtained at pH 7, 25 ꢀC and 10% dioxane using this biocatalyst and a very high enantioselectivity for the enzyme was displayed,
obtaining both (R)-(ꢀ)-glycidyl butyrate and (R)-(+)-glycidol with enantiomeric excesses >99% (E >100). The hydrolysis of (R)-
(
ꢁ
ꢀ)-glycidyl butyrate produced pure (S)-(ꢀ)-glycidol.
2005 Elsevier Ltd. All rights reserved.
1
. Introduction
enantiomeric excess is slightly low (ee = 91% at 65%
8
yield). Recently, Jacobsen et al. have described the
use of (salen) Co III complex as catalyst in the prepara-
tion of highly enantioenriched terminal epoxides.
Optically pure epoxides are versatile intermediates in
organic synthesis because the epoxide ring is very reactive
towards nucleophiles and easily yields asymmetric alco-
hols. Particularly, optically active 2,3-epoxypropanol
(glycidol) and its derivatives have a compact skeleton
of glycerol and wide potential for synthesis. They are
Using biological methods, the resolution of racemic
epoxides as glycidyl derivatives has been performed by
a biocatalytic process using enzymes, especially lip-
1
–3
9–11
considered to be versatile chiral synthesis units.
this sense, both enantiomers of glycidol 1 have become
In
ases.
was developed using a commercial lipase preparation
Indeed, the resolution of (±)-glycidyl esters
9
widely used as starting materials for the synthesis of
4
from Porcine pancreas (PPL). However, high conver-
many interesting compounds, such as anticancer drugs,
5
sions were necessary to achieve an appropriate enantio-
meric excess of (R)-glycidyl butyrate 2 for industrial
application, which decreased the yield of the enantio-
merically pure enantiomer that remained.
protein synthesis inhibitors, as well as a 2-oxazolidi-
6
none derivative used against depression.
7
,8
9–11
Therefore, different chemical and biological
meth-
ods have been developed in order to get these alcohol
epoxides in their enantiomerically enriched form. Thus,
from the chemical point of view the asymmetric epoxi-
dation of allyl alcohol with peroxide developed by
Sharpless allowed both enantiomers to be obtained
and this was a breakthrough in the industrial production
of optically active C3 building blocks. However the
Herein, we report an improvement in this area using a
new strategy based on the preparation of several bio-
catalysts from the same lipase with very different immo-
bilization protocols, involving different areas of the
enzyme surface, conferring different rigidity levels to
the enzyme structure or even generating a special micro-
environment surrounding the enzyme, which could
somehow modify the shape and features of the lipase
active centre (Scheme 1). Moreover, the combination
of these methods with the appropriate reaction
conditions could improve the selectivity. This strategy
7
*
Corresponding authors. Tel.: +34 91 585 48 09; fax: +34 91 585 47
0; e-mail addresses: jmguisan@icp.csic.es; rfl@icp.csic.es
6
0
957-4166/$ - see front matter ꢁ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.12.027

870
J. M. Palomo et al. / Tetrahedron: Asymmetry 16 (2005) 869–874
Scheme 1. General representation of how to modulate the catalytic properties of lipases using different immobilization strategies.
has been already successfully used in the design of
biotransformations catalyzed by enzymes, which suffer
arctica (CAL-B) displays low E value (Table 1, entry 1)
with ee of 31%. With the lipase isolated from Rhizomu-
s
1
2,13
significant changes during catalysis.
In fact, this
cor miehei (RML) better results was obtained E = 5
(Table 1, entry 2) although the highest selectivity
towards this substrate was found using a lipase purified
technique has demonstrated that the properties of the
same lipase immobilized on different supports could be
completely different.
1
6
from PPL crude preparation (25L) reaching 43% yield
in 15 min with ee of 54% (Table 1, entry 3). Therefore,
the latter was selected to perform the studies in order
to get a high enantioselectivity.
This strategy is based on the drastic conformational
changes that lipases undergo during catalysis. In aque-
ous media, they exist mainly in a closed and inactive
1
4
conformation, where the active site is separated from
the reaction medium by an oligopeptide chain acting
as a ÔlidÕ, in equilibrium with an open and active conform-
ation, where the lid is displaced permitting access of
substrates to the active site. However, upon exposure
to a hydrophobic interface, the lipase is adsorbed onto
it provoking a conformational change shifting the equi-
librium to the open form, a phenomenon known as
2.2. Enantioselective resolution of (± ±-2 under different
conditions using different 25L-immobilized preparations
The initial rate and enantiomeric ratio (E) values were
found to depend on the biocatalyst and the experimen-
tal conditions used (Table 2). In this sense, lipase
immobilized on octyl agarose (25L-1 biocatalyst) pre-
sented the highest initial rate when the reaction was
performed at pH 7 and 25 ꢀC (Table 2, entry 1). The
activity of the other biocatalysts—prepared using other
immobilization strategies—was at least 10-fold lower in
relation to the one of the 25L-1 biocatalyst (Table 2,
entries 2–4). However, with the adsorption of the lipase
on commercial DEAE–Sepharose (25L-3 biocatalyst) it
was possible to obtain the most enantioselective cata-
1
5
interfacial activation.
Herein, we describe the kinetic resolution of racemic
glycidyl butyrate (±)-2 in aqueous media catalyzed by
different lipases. Several immobilized biocatalysts from
1
6
a 25 kDa lipase (25L) —which is a lipase-like enzyme
purified from the pancreatic porcine lipase (PPL) ex-
tract—were prepared.
lyst with ee of 81% at 52% yield under these experi-
s
mental conditions (Table 2, entry 3). It is worth
noting that this lipase covalently attached on amino-
1
8
2
. Results and discussion
agarose activated with glutaraldehyde (25L-2 biocata-
lyst) did not exhibit any discrimination between iso-
mers, and 55% of the substrate was converted into a
virtually racemic product after 6 h at 25 ꢀC (Table 2,
entry 2).
2
.1. Enzymatic resolution of (± ±-2 in aqueous media
We screened different lipases for their ability to catalyze
the hydrolysis of racemic 2 (Table 1). The lipases were
immobilized on agarose activated with octyl groups on
the surface. It was found that the reactivity and the
enantioselectivity depended largely on the enzyme
used. For instance, the lipase isolated from Candida ant-
When the pH of the reaction medium was changed
from 7 to 5, the activity of the biocatalysts did not
improve (Table 2, entries 5–8), even the initial rate for
the lipase immobilized on dextran-sulfate agarose
1
7

J. M. Palomo et al. / Tetrahedron: Asymmetry 16 (2005) 869–874
Table 1. Enzymatic resolution screening of (±)-2 in aqueous media
871
a
b
m
c
d
Entry
Enzyme
T (ꢀC)
pH
(g)
V (mL)
t (min)
c (%)
Ee
s
(%)
E
1
2
3
CAL-B
RML
25L
25
25
25
7
7
7
0.01
0.05
0.03
8
20
5
15
5
58
41
43
31*
39
2
5
10
15
54
a
b
c
Lipases were immobilized on octyl-agarose support.
m: amount of catalyst added.
Determined by chiral HPLC: ee
s
= ee [(R)-2 or *(S)-2].
d
Determined from c and ee
s
as in Ref. 17.
Table 2. Kinetic resolution of (±)-2 catalyzed by different 25L-biocatalysts
O
O
O
H O
2
O
OH
H
+
biocatalyst
O
H
H
O
O
(± ±)-
(R)-(+)-1
(R)-(-)-2
c (%)
a
b
c
d
E
Entry
Biocatalyst
T (ꢀC)
pH
Initial rate
t (h)
Ee
s
(%)
1
2
3
4
5
6
7
8
25L-1
25L-2
25L-3
25L-4
25L-1
25L-2
25L-3
25L-4
25
25
25
25
25
25
25
25
7
7
7
7
5
5
5
5
1200
81
0.30
61
55
52
56
53
51
50
47
89
06
81
50
94
0
10
1.1
6
6
63
17
4
114
850
51
3
0.33
8
38
1
54
8
60
47
38
4
3.6
5.4
a
b
c
Different biocatalysts were prepared as described in experimental.
ꢀ1
ꢀ1
The initial rate (lmol · mgprot · min ) was calculated at 10–15% of conversions.
Determined by chiral HPLC: ee = ee [(R)-2].
as in Ref. 17.
s
d
Determined from c and ee
s
(25L-4 biocatalyst) decreased up to 20-fold (Table 2,
entry 8). Nevertheless, the 25L-1 biocatalyst displayed
Therefore, these immobilization strategies combined
with the appropriate reaction conditions have permitted
to obtain E values very different from 1 (25L-2 biocata-
lyst) to >100 (25L3 biocatalyst) for the same lipase.
the highest E value when the reaction was performed
at pH 5 with 94% of ee at 53% conversion (Table 2,
s
entry 5). The selectivity in the case of the other biocata-
lysts was similar or lower.
On the other hand, the use of different percentages of
solvent (1,4-dioxane) in the aqueous solution displayed
interesting results on the enantioselectivity of the bio-
catalysts (Fig. 1). The addition of 10% solvent to the
aqueous phase promoted an improvement in the E value
for most of the biocatalysts (Fig. 1), emphasizing the
excellent enantioselectivity reached by the 25L-3 biocata-
lyst in these conditions (E P100, in fact we cannot
detect hydrolysis of the (R)-enantiomers of remaining
glycidyl butyrate after 24 h even using pure com-
pounds). The increase in dioxane concentration to 20%
promoted a very dissimilar effect on the enantioselecti-
vity of the different biocatalysts. The 25L-4 biocatalyst
improved the E value slightly from 4 (0% dioxane)
to 11 (Fig. 1). However, the 25L-3 biocatalyst suffered
a considerable decrease in the enantiomeric ratio
from P100 (10% dioxane) to 4.
100
8
0
0
6
-5L)1
-
-
5L)-
5L)3
40
-5L)4
-0
0
0
5
10
15
-0
Dioxane (%±
Figure 1. Diagram of the co-solvent effect on the enantiomeric ratio of
5L-biocatalysts in the resolution of (±)-2. Experiments were per-
formed at pH 7 using 1,4-dioxane as co-solvent. E values were
determined from c and ee as in Ref. 17.
A similar study was performed at pH 5, although the re-
sults did not improve on the addition of dioxane (results
not shown).
2
s

872
J. M. Palomo et al. / Tetrahedron: Asymmetry 16 (2005) 869–874
2
.3. Preparative resolution of (R±-glycidol
3. Conclusions
Taking into account the fact that when the 25L-3 biocata-
lyst was used none of the (S)-product was detected
at 50% conversion as previously described (even when
the reaction time was double), we carried out the hydro-
lytic resolution of (±)-2 (3 mmol) using 10% dioxane
These results show the full resolution of (±)-glycidyl
butyrate (±)-2 via a hydrolysis reaction. This was possi-
ble by selecting the best lipase (25L), the best biocatalyst
from different ones, 25L immobilized on DEAE–Sepha-
rose, and the best reaction medium conditions—pH 7,
25 ꢀC and 10% 1,4-dioxane. Under these conditions,
both products, (R)-(+)-glycidol as well as (R)-(ꢀ)-glyc-
idyl butyrate were obtained with enantiomeric excesses
>99% (E >100). Moreover, the hydrolytic reaction of
(R)-(ꢀ)-glycidyl butyrate permitted to obtain the (S)-
(ꢀ)-glycidol in enantiopure form (Scheme 2). This easy
and rapid biocatalytic method competes with the results
(
Scheme 2). Hydrolyses were carried out up to 50%
conversion (over 10 h when using 1 g of 25L-biocatalyst
and 100 mL of substrate) and the ee and ee were ana-
p
s
lyzed at different conversions (Table 3). Thus, the ee-1
was always >99% at 10–50% conversions (Table 3,
entries 1–3) whereas the ee-2 was >99% at 50% conver-
sion (Table 3). They were isolated in enantiopure form
from the same reaction by semi-preparative HPLC and
8
in enantiomeric purity obtained by Jacobsen or by
9,10
other enzymatic processes.
20
characterized, obtaining (R)-(+)-1 ð½aꢁ ¼ 36:9Þ in 45%
D
overall yield.
Moreover, a simple hydrolytic reaction, using, for exam-
ple, 25L-2 biocatalyst, converted all of the (R)-2 enan-
tiomer into (S)-1 providing the other enantiomer of
glycidol in enantiopure form (Scheme 2). Nine cycles
could be performed with identical results with respect
to the catalytic properties of the biocatalyst (activity,
enantioselectivity).
4. Experimental
4.1. General
p-Nitrophenyl propionate (pNPP), 1,4-dioxane, dextran
sulfate (Mr 10,000), glutaraldehyde, Porcine pancreatic
lipase (type II) (PPL) and Triton X-100 were obtained
from Sigma–Aldrich St. Louis, EEUU. Fully purified
2
5 kDa lipase from PPL crude was prepared as previ-
1
6
ously described. The lipase from Candida antarctica
fraction B) (Novozym 525 L) and from Rhizomucor
(
Table 3. Hydrolytic resolution of (±)-2 catalyzed by 25L-3 immobi-
lized preparation under the optimized reaction conditions
Miehei (Novozym 388) were from Novo Nordisk (Den-
mark). Glyoxyl-agarose 6BCL and 10 BCL was kindly
donated by the company Hispanagar SA (Burgos,
Spain). Octyl-agarose 4BCL and DEAE–Sepharose
were purchased from Pharmacia Biotech (Uppsala, Swe-
a
b
p
c
d
20
½aꢁD
Entry Solvent pH
%)
T
Ee
Ee
s
c
(%) (%)
E
(
(ꢀC) (%)
1
2
3
10
10
10
7.00 25
7.00 25
7.00 25
>99 22
>99 66
18
40
>100
>100
—
—
1
8,19
den). Glutaraldehyde agarose
was prepared as previ-
>99 >99 50
>100 +36.9
ously described. (±)-Glycidyl butyrate (±)-2 was kindly
donated by Dr. Pregnolato (University of Pavia, Italy).
Protein concentration was determined by the BradfordÕs
a
b
c
The solvent was 1,4-dioxane.
Determined by chiral GC: ee
Determined by chiral HPLC: ee
p
= ee[(R)-1].
20
= ee [(R)-2].
method. pH-stat Mettler Toledo DL50 graphic was
s
d
Determined from c and ee
s
as in Ref. 17.
used to maintain a constant pH value during the
O
O
O
H O
2
O
OH
H
+
2
5L-3 lipase
O
H
H
O
O
(±)-2
(R)-(+)-1
(R)-(-)-2
lipase
O
H O
2
H
OH
(S)-(-)-1
Scheme 2. Synthesis of (R)- and (S)-glycidol by using a biocatalytic process.

J. M. Palomo et al. / Tetrahedron: Asymmetry 16 (2005) 869–874
873
reactions. Conversion was analyzed by RP-HPLC
Spectra Physic SP 100 coupled with an UV detector
Spectra Physic SP 8450) using kromasil C18
pH 7. The mixture was then shaken at 4 ꢀC and 250 rpm
for 24 h. After that, the solution was removed by filtra-
tion. NaBH4 (2.65 mmol) dissolved in 100 mL of
100 mM sodium carbonate buffer at pH 8.5 was added
to the solid supported lipase. The mixture was then sha-
ken at 4 ꢀC for 30 min. After that, the solution was re-
moved by filtration and the supported lipase washed
properly with distilled water to remove the reduction
agent excess and keep it at 4 ꢀC.
(
a
(
25 · 0.4 cm) column. Products were eluted at flow rate
ꢀ
1
of 1.5 mL min using acetonitrile–10 mM ammonium
phosphate buffer at pH 2.95 (35:65, v/v) and UV detec-
tion performed at 225 nm. The enantiomeric excess
(
ee) of the remaining ester was determined by Chiral
Phase HPLC analysis. The column was a Chiracel
OD, the mobile phase was an isocratic mixture of iso-
propanol and hexane (2:98 v/v) at a flow rate of
4.6. 25L3 Biocatalyst: immobilization on DEAE–
Sepharose
0
2
.4 mL/min and UV detection was performed at
25 nm. The retention times of the enantiomers were:
(
(
S)-2 (17.32 min), (a = 0.92); (R)-2 (18.60 min),
a = 1.08). Otherwise, the enantiomeric excess of the
Ten grams of DEAE–Sepharose gel was added to 50 mL
of 25 mM sodium phosphate buffer lipase solution
(0.1 mg protein/mL) at pH 7. The mixture was then sha-
ken at 4 ꢀC and 250 rpm for 4 h. After that, the solution
was removed by filtration and the supported lipase
washed several times with distilled water.
released glycidol was evaluated by gas chromatographic.
GC Varian 3800 analyses were performed using a Cyclo-
silb 112-6632 chiral column (30 m · 0.25 mm, I&J Scien-
tific). Chromatographic conditions were: injector
temperature 230 ꢀC, initial oven temperature 60 ꢀC for
3
min to 76 ꢀC (1 ꢀC/min) and then to 240 ꢀC (25
C/min). Flame Ionization detector was maintained at
00 ꢀC. Gas carrier:Helio (1.5 mL/min). The retention
times of the enantiomers were: (R)-1 (9.8 min),
4.7. 25L4 Biocatalyst: immobilization on dextran sulfate
agarose
ꢀ
2
Nine grams of activated agarose gel with amino groups
22
coated with dextran sulfate was added to 45 mL of
1
(
a = 0.965); (S)-1 (10.1 min) (a = 1.036), (R = 1.02). H
s
13
and C NMR data were recorded on a Varian Mercury
00 spectrometer. Optical rotations were measured with
a Perkin–Elmer Polarimeter 341.
25 mM sodium acetate buffer lipase solution (0.1 mg
protein/mL) at pH 5. The mixture was then shaken at
4 ꢀC and 250 rpm for 3 h. After that, the solution was
removed by filtration and the supported lipase washed
several times with distilled water.
4
4
.2. Enzymatic activity assay
This assay was performed by measuring the increase in
the absorbance at 348 nm produced by the released p-
nitrophenol in the hydrolysis of 0.4 mM pNPP in
4.8. Enzymatic hydrolysis of (± ±-glycidyl butyrate (± ±-2
(±)-2 (0.6 mmol) was dissolved in 25 mM sodium phos-
phate buffer (6 mL) at pH 7 or 25 mM sodium acetate
buffer (6 mL) at pH 5 with different percentage of 1,4-
dioxane from 0% to 20% (v/v) and biocatalyst (60 mg)
was added. The mixture was then shaken at 25 ꢀC and
250 rpm for the time shown in Table 2. The biocatalyst
was filtered and the conversion was analyzed by RP-
HPLC. Enantiomeric excesses were determined using a
chiral phase-column previous extraction 0.2 mL of
aqueous phase with hexane in HPLC or ethyl acetate
in GC (4 · 0.2 mL). After that, the enantiomeric ratio
(E) was calculated in all cases using the equations re-
2
5 mM sodium phosphate buffer at pH 7 and 25 ꢀC.
To initialize the reaction, 0.05 mL of lipase solution or
suspension was added to 2.5 mL of substrate solution.
One international unit of pNPP activity was defined as
the amount of enzyme necessary to hydrolyze 1 lmol
of pNPP per minute (IU) under the conditions described
above.
4
.3. Biocatalysts preparation
Four different immobilization protocols were used. The
yields from the immobilization process were >95% in all
cases.
1
7
ported by Chen et al.
4.9. Separation protocol of compounds (R±-1 and (R±-2
4
.4. 25L1 Biocatalyst: immobilization on octyl agarose
One hundred millilitres of reaction mixture at 50% of
conversion (432 mg of initial substrate) was separated
of immobilized preparation by vacuum filtration. The
reaction mixture was extracted with dichloromethane
(3 · 100 mL) and the organic solvent was dried over so-
dium sulfate and evaporated under vacuum. The residue
was dissolved in acetonitrile and chiral products, alcohol
as well as ester were isolated by reverse-phase HPLC on
a 10–250 mm C18 column (Vydac, 5 lm particles and
Ten grams of octyl-agarose support was added to
00 mL of 25 mM sodium phosphate buffer lipase solu-
tion (0.1 mg protein/mL) at pH 7. The mixture was
then shaken at 4 ꢀC and 250 rpm for 3 h. After that,
the solution was removed by filtration and the sup-
ported lipase washed several times with distilled water.
1
2
1
4.5. 25L2 Biocatalyst: immobilization on glutaraldehyde
agarose
˚
300 A pore size) using a Bio-Rad model 2800 HPLC sys-
tem equipped with a Linear model 205 dual wavelength
UV detector with an acetonitrile–water (65:35) mobile
phase. Reversed-phase HPLC was used to confirm the
purity of the products (>99%).
Eight grams of activated agarose gel modified with glu-
1
8,19
taraldehyde was added to 40 mL of 25 mM sodium
phosphate buffer lipase solution (0.1 mg protein/mL) at

874
J. M. Palomo et al. / Tetrahedron: Asymmetry 16 (2005) 869–874
The remaining (R)-2 was hydrolyzed using the 25L-2
biocatalyst to get enantiomerically pure (S)-1.
Biochem. Biotech. 2003, 104, 69–79; (c) Palomo, J. M.;
Segura, R. L.; Fern a´ ndez-Lorente, G.; Guis a´ n, J. M.;
Fern a´ ndez-Lafuente, R. Tetrahedron: Asymmetry 2004,
15, 1157–1161.
2
D
0
1
(
R)-glycidol 1: (45%). ½aꢁ ¼ þ36:9 (c 1, CHCl ) H
3
1
1
1. Kloosterman, M.; Elferink, V. H. M.; Iersel, J van.;
Roskam, J.-H.; Meijer, E. M.; Hulshof, A.; Sheldon, R. A.
TIBTECH. 1988, 6, 251.
2. (a) Palomo, Jose M.; Fernandez-Lorente, G.; Rua, M. L.;
Guisan, J. M.; Fernandez-Lafuente, R. Tetrahedron:
Asymmetry 2003, 14, 3679–3687; (b) Palomo, J. M.;
Fernandez-Lorente, G.; Mateo, C.; Fuentes, M.; Fernan-
dez-Lafuente, R.; Guisan, J. M. Tetrahedron: Asymmetry
NMR (400 MHz, CDCl ): d = 3.78 (dd, J = 4.89 Hz,
3
1
H, CH ), 3.75 (dd. J = 5.47 Hz, 1H, CH ), 2.85 (s,
2
2
1
J = 4.89 Hz, 1H, CH oxi).
H; OH), 2.67 (m, 2H; CHoxi, CH oxi), 2.57 (dd,
2
1
3
C NMR (100 MHz,
2
CDCl ): d = 63.6, 52.6, 44.5.
3
2
002, 13, 1337–1345.
Acknowledgements
1
3. (a) Palomo, J. M.; Fern a´ ndez-Lorente, G.; Mu n˜ oz, G.;
Mateo, C.; Fuentes, M.; Guis a´ n, J. M.; Fern a´ ndez-
Lafuente, R. J. Mol. Catal. B: Enzym. 2003, 21, 201–
This work is supported by the Spanish CICYT (project
BIO2001-2259). Authors would like to thank Hispana-
gar SA for the gift of glyoxyl agarose.
2
10; (b) Palomo, Jose M.; Fern a´ ndez-Lorente, G.; Mateo,
C.; Ortiz, C.; Fern a´ ndez-Lafuente, R.; Guis a´ n, J. M.
Enzyme. Microb. Technol. 2002, 31, 775–783.
1
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