Candida antarctica lipase B catalyzed enantioselective acylation of pyrimidine acyclonucleoside

Article abstract of DOI:10.3109/10242422.2012.715637

The influence of solvent and acyl group donor on selectivity of the transesterification reaction of 1-[1′,3′-dihydroxy-2′- propoxymethyl]-5-methyluracil, a structural analogue of ganciclovir was examined. Lipase (EC 3.1.1.3) B from Candida antarctica (CAL

Full text of DOI:10.3109/10242422.2012.715637

Biocatalysis and Biotransformation, 2012; 30(4): 426–430
ORIGINAL ARTICLE
Candida antarctica lipase B catalyzed enantioselective acylation
of pyrimidine acyclonucleoside
RENATA KOŁODZIEJSKA1, ALEKSANDRA KARCZMARSKA–WÓDZKA1,
2
1
´
ANDRZEJ WOLAN & MARCIN DRAMINSKI
1Department of General Chemistry, Collegium Medicum Nicolaus Copernicus University, Bydgoszcz, Poland and
2Department of Organic Chemistry, Nicolaus Copernicus University,Torun´, Poland
Abstract
The influence of solvent and acyl group donor on selectivity of the transesterification reaction of 1-[1′,3′-dihydroxy-2′-
propoxymethyl]-5-methyluracil, a structural analogue of ganciclovir was examined. Lipase (EC 3.1.1.3) B from Candida
antarctica (CALB) enabled desymmetrization of prochiral hydroxyl groups when 1-butyl-3-methylimidazolium hexafluor-
ophosphate ([Bmim][PF6]) was used as a reaction medium. It was observed that CALB was up to 2.7–4 times more
enantioselective in the ionic liquid [Bmim][PF6] than in conventional organic solvents.
Keywords: lipase B from Candida antarctica, transesterification, desymmetrization, ionic liquids
Introduction
selectivity of the enzyme. Along with an increase in
medium polarity in an enzymatic reaction, the
amount of water needed to activate the enzyme
decreases because its molecules enter into interac-
tion with polar solvent. As a consequence, unfavour-
able conformational changes in the enzyme bring
about a decrease in activity and selectivity (Rantwijk
et al. 2006; Kaar et al. 2003). The removal of an
essential monolayer of water from the protein in
polar solvents is usually the reason for deactivation
of enzymes. Lipases are most selective in hydropho-
bic organic solvents, but their activity is typically
lower than in a suitable polar solvent (Lozano et al.
2001; Hernández-Fernández et al. 2007).
Increasingly, enzymatic reactions are being per-
formed in ionic liquids (Ha et al. 2008; Noël et al.
2004; Eckstein et al. 2002).The polarity of the major-
ity of imidazole based ionic liquids is comparable to
low molecular weight alcohols (methanol, ethanol,
1-butanol). The length of the alkyl chain attached to
the imidazolium ring as well as the accompanying
anion influences the hydrophilicity and hydrophobicity
of the liquid (Vidya & Chadha 2009). A properly
The catalytic potential of lipases has been the subject
of intensive investigation for many years. The chiral
structure of the enzyme facilitates enantioselective
reactions giving kinetic resolution of racemic mixtures
(Ha et al. 2008; Brem et al. 2009) or production of
enantiopure products from achiral precursors (Maha-
patra et al. 2008; Köhler & Wünsch 2006; Arai et al.
2004). This is particularly significant when the
enantiomer is a biologically active compound, and its
therapeutic properties depend on its optical purity.The
catalytic properties of lipases have been used to obtain
optically pure compounds by enantioselective hydroly-
sis, esterification and transesterification (Schöfer et al.
2001; Ducret et al. 2000; Utzig et al. 2001).
Lipases generally display a high tolerance towards
structural changes in substrates as well as a wide
spectrum of activity (they work not only in aqueous
systems but also in organic solvents).When planning
enzymatic synthesis, the choice of solvent used as a
reaction medium is very significant. The basic crite-
rion of this choice is its hydrophilic and hydrophobic
properties, which influence the activity, stability and
Correspondence: R. Kołodziejska, Department of General Chemistry, Collegium Medicum Nicolaus Copernicus University, 3 De˛bowa Street, 85-626
Bydgoszcz, Poland. Fax: (ϩ48)-52-585-25-86. Tel: (ϩ48)-52-585-25-84. E-mail: Renatakol@poczta.fm
(Received 11 January 2012; revised 23 March 2012; accepted 23 July 2012)
ISSN 1024-2422 print/ISSN 1029-2446 online © 2012 Informa UK, Ltd.
DOI: 10.3109/10242422.2012.715637

Enzymatic enantioselective acylation of pyrimidine acyclonucleoside 427
selected ionic liquid not only increases the activity of
enzymes but also improve stereoselectivity.
(1 mg), acyl donor (0.0001 mol) in a solvent (1 mL)
and were kept at constant temperature (37–50°C).
After the reaction methanol was added, the enzyme
removed by filtration and the resulting solution con-
centrated. In the case of ionic liquids, the mixture
was first extracted with hexane: 2-propanol (80:20),
and the organic phase concentrated.
The product in the residue was characterized by
1H NMR.The enantiomeric ratios were determined
on an HPLC system using a chiral column, using a
mobile phase of hexane: 2-propanol.
In this work, the enzymatic transesterification of
the acyclonucleoside 1-[1′,3′-dihydroxy-2′-propoxy-
methyl]-5-methyluracil (1) has been investigated.
Interest in acyclonucleosides arises from their
biologically active properties; 1-[1′,3′-dihydroxy-2′-
propoxymethyl]-5-methyluracil (1) is known as an E.
coli uridine phosphorylase inhibitor in the micromolar
range and compounds similar in structure inhibit the
growth of cancer cells in vitro (Drabikowska et al.
1987).
The influence of the solvent and the acylating
agent were examined for their effects on the enanti-
oselectivity of lipase B from Candida antarctica
(CALB), one of the few lipases that does not require
interfacial activation. Since the lipase is stable and
active in organic solvents, acylation reactions were
carried out under anhydrous conditions.
Analysis
HPLC analyses were performed on a Shimadzu
SCL–10A VP, analytical column Chiralcel OJ
250ϫ4, 6 mm, 10 μm, Daicel (France).
1H NMR spectra were recorded on Bruker
300 Hz apparatus using tetramethylsilane (TMS) as
an internal standard.
Materials and methods
Results and discussion
Lyophilized CALB was from Fluka (Switzerland),
10.9 U/mg.
Enzymatic transesterification of 1-[1′,3′-dihydroxy-2′-
propoxymethyl]-5-methyluracil (1)
Other chemicals of analytical grade were com-
mercially available: 1-phenylethanol from Fluka
(Switzerland),1-[1′,3′-dihydroxy-2-propoxymethyl]-
5-methyluracil was obtained from the Medical Acad-
emy (Poland) (Drabikowska et al.1987),vinyl acetate
from Fluka (Germany), vinyl butyrate and vinyl ben-
zoate from Fluka (Japan), vinyl laurate from Fluka
(Switzerland),1-butyl-3-methylimidazoliumhexaflu-
orophosphate and 1-butyl-3-methylimidazolium tet-
rafluoroborate from Fluka (Switzerland), pyridine,
cyclohexane,dioxane,n-hexaneforHigh-performance
liquid chromatography (HPLC), 2-propanol for
HPLC from POCH (Poland).
On the basis of a model reaction, solvents were
selected for transesterification of the prochiral diol
(Scheme 1).
In the transesterification reaction of compound
1 three different organic solvents (cyclohexane, pyri-
dine and dioxane) were tested and reactions were
carried out at 37 and 50°C. The reaction in cyclo-
hexane was carried out only at 50°C due to poor
solubility of the substrate. Vinyl acetate was used as
an acylating agent.
CALB allows regioselective acylation of primary
hydroxyl groups in nucleosides (Kołodziejska &
Dramin´ski 2004). Part of 1 imitates a sugar fragment
of nucleosides, however, in this case, the hydroxyl
groups are both primary. From the prochiral acyclo-
nucleoside, an optically pure compound could be
General procedure
Typical enzymatic reactions were performed in a
solution containing substrate (0.0001 mol), CALB
Scheme 1. Desymmetrization of the prochiral diol 1-[1′,3′-dihydroxy-2′-propoxymethyl]-5-methyluracil by enzymatic transesterification.

428
R. Kołodziejska et al.
Table II. Transesterification of diol with vinyl acetate in
pyridine.
obtained if CALB allowed desymmetrization of these
OH groups.
To investigate the course of the transesterification
of 1 and assess the optical purity of the resulting
homoesters, analyses were conducted on post-
reaction mixtures taken at specified intervals. The
analyses were carried out by HPLC on a Chiralcel
OJ column in hexane: 2-propanol (60:40 or 80:20)
system, with a flow rate of 1 mL/min.The results are
shown in Tables I—III. Transesterification of vinyl
acetate in the presence of lipase CALB proceeded at
a high rate. Apart from the monoacetylated deriva-
tive, completely acetylated acyclonucleoside was also
obtained. The maximum enantiomeric excess (ee)
(26%) was obtained in dioxane after 10 h at 37°C,
and at 50°C in dioxane after 23 h (26%) (Table I),
with a slightly lower excess (22%) in pyridine after
24 h of reaction (Table II).
The enzyme failed to give desymmetrization of
the prochiral hydroxyl groups in pyridine, with both
pro-(S) and pro-(R) groups being acetylated (race-
mic mixture after 1 h of reaction). Presumably, the
22% ee after 24 h was the result of different reaction
rates of the enantiomers (Table II).
Partial selectivity for acylation of only one pri-
mary –OH group in cyclohexane and dioxane made
it possible to obtain an excess of the enantiomer
of the opposite configuration. In this case, the ee
might have resulted from partial desymmetrization
of prochiral –OH groups.
T [°C]
Time [h]
Mono [%]
Di [%]
ee [%]
1
4
6
8
10
12
20.2
28.3
36.9
42.7
49.2
44.9
10.5
20.1
26.6
27.3
28.2
32.4
5
8
4
16
5
10
37
1
2
3
5
9
10
12
14
18
20
24
30
36
26.8
39.2
41.3
44.2
44.9
48.2
46.7
45.9
42.8
38.2
32.1
33.1
34.8
0
8.2
1
6
8
13
4
7
10
18
19
8
22
22
19
14.1
16.6
21.9
31.9
40.8
44.6
49.4
55.6
61.8
63.9
64.0
50
Additional attempts at kinetic resolution of the
racemic monoester failed to bring expected results.
In pyridine, CALB introduced the acetyl group into
the hydroxyl group of both enantiomers, 17% ee was
obtained.The reaction in pyridine proceeded rapidly
and after 24 h the diacetyl derivative was obtained
with a high yield (77%).
In order to improve the stereoselectivity of acyla-
tion of 1, the hydrophobic ionic liquid [Bmim][PF6]
was used and the results compared with those
obtained in pyridine. The reaction was carried out
for 24 h at a temperature of 50°C, with various acy-
lating agents. Apart from vinyl acetate, vinyl esters
of butyric, benzoic and lauric acids were used. The
application of [Bmim][PF6] increased the enantiose-
lectivity of the reaction, giving a higher ee than with
the less polar pyridine.
Lowering the temperature did not have a signifi-
cant influence on enantioselectivity (comparable ee
at 37 and 50°C). In the transesterification reaction
of 1 conducted in pyridine at room temperature,
after 2 months 17% ee was obtained (30% yield).
Table I. Transesterification of diol with vinyl acetate in dioxane.
T [°C]
Time [h]
Mono [%]
Di [%]
ee [%]
In pyridine, the best results were obtained in
transesterification with vinyl acetate and contrary to
expectations, elongation of the acyl donor chain did
not influence the enantioselectivity. Ionic liquid
[Bmim][PF6] appeared to be a much better medium
for the reaction. Acylation with vinyl acetate and
benzoate proceeded with about 60% ee.
1
4
6
8
10
12
26.1
43.6
46.2
43.1
39.2
34.8
4.1
23.5
27.6
32.2
44.6
56.1
18
18
14
15
26
22
37
1
2
4
7
9
11
13
16
21
23
26
33
31.8
38.2
43.6
44.5
30.1
36.4
34.9
28.2
20.8
18.4
18.2
8.6
13.8
22.9
23.5
27.1
54.3
54.4
55.8
69.7
76.6
79.4
79.6
89.3
10
5
18
18
16
17
13
24
23
26
18
24
Table III. Transesterification of diol with vinyl acetate in
cyclohexane.
T [°C]
Time [h]
Mono [%]
Di [%]
ee [%]
50
1
4
7
3.6
7.1
28.8
38.0
0
2.4
18.1
31.3
10
16
9
50
11
10
13
32.5
43.9
15

Enzymatic enantioselective acylation of pyrimidine acyclonucleoside 429
Table IV. Results of enzyme – catalyzed transesterification of diol.
Acyl agent
Solvent
Time [h]
Mono [%]
Di [%]
ee [%]
Vinyl acetate
Pyridine
[Bmim][PF6]
30
24
33.1
40.7
60.9
48.7
22
61
Vinyl butyrate
Vinyl benzoate
Vinyl laurate
Pyridine
[Bmim][PF6]
24
24
57.3
18.1
26.4
28.2
13
6
Pyridine
[Bmim][PF6]
24
24
13.9
45.1
63.2
0.6
14
57
Pyridine
24
24
38.9
9.8
5
5
[Bmim][PF6]
In [Bmim][PF6] the reaction with vinyl benzoate
yielded only monoesters after 24 h, whereas the same
reaction carried out in pyridine proceeded much
faster and mainly the diacetyl derivative was obtained
(monoester 14% ee). The results of transesterifica-
tion of diol in pyridine and [Bmim][PF6] are
demonstrated in Table IV and Figures 1 and 2.
Unexpectedly, transesterification reactions with
vinyl butyrate and laurate, proceeded almost com-
pletely non-selectively. In both pyridine and ionic
liquid, practically racemic mixture was obtained.
Ionic liquid [Bmim][PF6] is an ideal environment
for enzymatic transesterification reactions. [Bmim]
[PF6] undoubtedly has useful properties, it is as polar
as ethanol (identical value of ETN) and hydrophobic
at the same time (slightly soluble in water – 0.13%),
with the accompanying anion showing only weak
nucleophilic character. With imidazole based ionic
liquids of comparable polarity, the nucleophilic char-
acter of the accompanying anion mainly dictates the
properties of the liquid (hydrophobicity, miscibility
with organic solvents, solubility of organic and non-
organic substances) and consequently its usefulness
for a catalyzed reaction.Together with the increase in
nucleophilicity, the accompanying ion coordinates
more strongly in the active site of the enzyme con-
tributing to damaging conformation changes of a
catalyst, which influence enzyme activity and stability
(Kaar et al. 2003;Vidya & Chadha 2009). Consistent
with the argument, acetylation of 1 in another imida-
zolic ionic liquid derivative [Bmim][BF4], whose
accompanying anion is a stronger nucleophile than
Ϫ
PF6 ion, showed that the reaction gave a lower ee
(29%) and a low yield (28%).
[Bmim][PF6] provides an excellent microenvi-
ronment for the enzyme, leading to a more compact
conformation capable of giving both high activity
and stability;CALB was 2.7–4 times more in [Bmim]
[PF6] than in a conventional organic solvent.
Encouraged by these initial results, we are con-
tinuing our studies to look for a more efficient bio-
catalyst.Therefore, the work is being extended using
a variety of commercially available lipases.
Conclusions
We have reported desymmetrization of identical
hydroxyl groups of a prochiral diol in an enzymatic
Figure 1. The transesterification of diol catalyzed by CALB in
pyridine.
Figure 2. The transesterification of diol catalyzed by CALB in
[Bmim][PF6].

430
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Supporting information
Detailed information on product characterization
specifications (1H NMR spectra and retention time in
HPLC analysis) is available in the Supplementary
Supporting Information, at http://informahealthcare.
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Declaration of interest: The authors report no
conflicts of interest. The authors alone are respon-
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Supplementary material available online
Supplementary Supporting Information