Enantioselective enzymatic desymmetrization of the prochiral pyrimidine acyclonucleoside

Article abstract of DOI:10.1016/j.tetasy.2012.05.003

The effect of the solvent and the acyl group donor on the selectivity of the transesterification reaction of 1-{[(1,3-dihydroxypropan-2-yl)oxy]methyl}-5- methylpyrimidine-2,4(1H,3H)-dione was examined. Lipase (EC 3.1.1.3) Amano PS from Burkholderia cepacia (BCL) enabled desymmetrization of prochiral hydroxyl groups and gave the (R)-monoester in high enantiomeric excess (ee 88-99%). The best selectivity was obtained for the transesterification reaction with vinyl benzoate as the acylating agent (only monoester, 99% ee). The absolute configuration of the newly formed stereogenic center was determined with a high degree of confidence on the basis of the combined experimental and theoretical electronic circular dichroic (ECD) studies. The hydrolysis of prochiral diesters formed during the transesterification reaction in the presence of BCL provided the opposite enantiomer, that is, the (S)-monoester.

Full text of DOI:10.1016/j.tetasy.2012.05.003

Tetrahedron: Asymmetry 23 (2012) 683–689
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Tetrahedron: Asymmetry
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Enantioselective enzymatic desymmetrization of the prochiral pyrimidine
acyclonucleoside
Renata Kołodziejska a, , Marcin Górecki , Jadwiga Frelek , Marcin Draminski
⇑
b
b
a
´
a Department of General Chemistry, Collegium Medicum Nicolaus Copernicus University, De˛bowa 3, 85-626 Bydgoszcz, Poland
b Institute of Organic Chemistry of the Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
The effect of the solvent and the acyl group donor on the selectivity of the transesterification reaction of 1-
{[(1,3-dihydroxypropan-2-yl)oxy]methyl}-5-methylpyrimidine-2,4(1H,3H)-dione was examined. Lipase
(EC 3.1.1.3) Amano PS from Burkholderia cepacia (BCL) enabled desymmetrization of prochiral hydroxyl
groups and gave the (R)-monoester in high enantiomeric excess (ee 88–99%). The best selectivity was
obtained for the transesterification reaction with vinyl benzoate as the acylating agent (only monoester,
99% ee). The absolute configuration of the newly formed stereogenic center was determined with a high
degree of confidence on the basis of the combined experimental and theoretical electronic circular
dichroic (ECD) studies. The hydrolysis of prochiral diesters formed during the transesterification reaction
in the presence of BCL provided the opposite enantiomer, that is, the (S)-monoester.
Received 5 April 2012
Accepted 3 May 2012
Available online 8 June 2012
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
cleosides, we examined both the acylation (transesterification) of
the prochiral 1-{[(1,3-dihydroxypropan-2-yl)oxy]methyl}-5-meth-
The use of enzymatic reactions in synthetic organic chemistry is
constantly growing and has become increasingly popular. This is
undoubtedly related to the ecological aspect of this type of reaction
and therefore syntheses carried out using the catalytic properties
of enzymes have earned the status of green chemistry. Another
equally important characteristic of enzymatic reactions is that
the enzymatic proteins often allow us to obtain chiral products
with high enantiomeric purity. This feature is particularly signifi-
cant for biologically active compounds because of the dependence
of their therapeutic properties on their enantiomeric purity. In this
context, it is not surprising that the synthetic potential of catalytic
lipases has been the subject of intensive explorations over recent
years. As a result, it turned out that the lipases are attractive bio-
catalysts both in the kinetic resolution of racemic mixtures[1,2]
and in the generation of asymmetry by the selection of an enantio-
topic group of prochiral compounds.[3–5]
The interest in acyclonucleosides arises from their biological
activity. The compound 1-{[(1,3-dihydroxypropan-2-yl)oxy]-
methyl}-5-methylpyrimidine-2,4(1H,3H)-dione {common name:
1-[(10,30-dihydroxy-20-propoxy)methyl]-5-methyluracil} is known
as a micromole uridine phosphorylase inhibitor from Escherichia
coli and compounds with a similar structure are reported to inhibit
in vitro growth of cancer cells.[6,7]
ylpyrimidine-2,4(1H,3H)-dione 1 and the hydrolysis of the corre-
sponding diesters in the presence of a lipase under various
conditions (Scheme 1).
2. Results and discussion
2.1. Optimization of the reaction conditions using BCL (Amano
PS from Burkholderia cepacia)
2.1.1. The influence of the solvent on the enantioselectivity of
the enzymatic reaction
While planning an enzymatic synthesis, the choice of the sol-
vent used as the reaction medium is often very significant. The sol-
vent properties influence the activity, stability, and selectivity of
the enzyme. In a polar organic solvent, a decrease in the enzymatic
activity and the selectivity of the enzymatic protein was observed.
This is probably caused by the interaction of a solvent molecule
with a monomolecular layer of water that is essential for the acti-
vation of enzyme. In a hydrophobic organic solvent, an improve-
ment of enzymatic enantioselectivity is usually observed.[8–10]
In the transesterification reaction of compound 1, eight different
solvents were tested (Table 1). The solvents were put in order
according to the increase of their hydrophobic properties (logP).
The reactions were carried out at 50 °C for 24 h in the presence
of lipase Amano PS from Burkholderia cepacia (BCL). Vinyl acetate
was used as the acylating agent.
As part of our interest in the development of enzymatic stereo-
selective methods for the preparation of optically active acyclonu-
The transesterification reaction of diol 1 generally proceeded as
expected. The BCL selectively acetylated enantiotopic hydroxyl
⇑
Corresponding author.
E-mail address: renatakol@poczta.fm (R. Kołodziejska).
0957-4166/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetasy.2012.05.003

684
R. Kołodziejska et al. / Tetrahedron: Asymmetry 23 (2012) 683–689
O
N
O
O
N
HN
HN
HN
lipase
O
O
O
N
O
O
RCO2CH=CH2, 50 °C
solvent
O
O
O
(R)
OH
OH
O
OH
O
O
O
R
R
R
1
2'-5'
2''-5''
O
2'
2''
3''
Mono ; Di : R= -CH3
HN
3'
Mono ; Di : R= -(CH2)3CH3
4'
4''
Mono ; Di : R= -C6H5
lipase
Mono 5'; Di 5'': R= -(CH2)10CH3
TMBE/H2O
50 °C
O
O
N
O
(S)
O
OH
R
Scheme 1. A strategy to obtain (R)- and (S)-mono-derivatives.
Table 1
in the active site of the enzyme contributing to the disadvanta-
geous conformation changes of a catalyst.9,15
Transesterification of diol 1 with vinyl acetate in different organic solvents
Solvent
logP
Mono
200 (%)
Ra
eeb (%)
In the case of hydrophobic organic solvents, such as cyclohex-
ane and hexane, an improvement of reaction selectivity was ex-
pected. The degree of conversion was high. Monoesters 20 were
obtained with 40% and 49% yield, respectively. However, along
with an increase of hydrophobicity of the reaction medium, a dras-
tic decrease of enantioselectivity was observed. In hexane and
cyclohexane, the monoester was obtained with a low enantiomeric
excess. Under those conditions, BCL did not allow sufficient diver-
sification of the prochiral –OH groups.
(S)-20 (%)
(R)-20 (%)
[BMIM][BF4]
[BMIM][PF6]
Dioxane
THF
Pyridine
TBME
ꢀ2.5111
ꢀ2.399
ꢀ1.1012
0.4913
0.7114
1.30
4.1
1.3
4.3
9.6
1.9
3.3
19.4
21.4
17.0
18.2
32.9
44.5
17.8
52.1
21.0
27.9
4.6
–
–
–
–
44.0
44.0
26.0
0.24
0.07
0.13
0.22
0.11
0.06
0.92
0.77
61
86
77
65
81
88
4
Cyclohexane
Hexane
3.4012
3.5013
13
The relationship between the solvent hydrophobicity and its
activity and BCL enantioselectivity was not straightforward. In
standard hydrophobic organic solvents, such as cyclohexane or
hexane (logP >3), an increase in the BCL catalytic activity was ob-
served, with a simultaneous decrease of enantiomeric selectivity.
Lowering the hydrophobic properties of the solvents led to an in-
crease in the enantiomeric properties of BCL as well as a decrease
in lipase reactivity in the majority of solvents with a low logP va-
lue. On the basis of these experiments, it can be concluded that
TBME was the optimal solvent for the enzymatic transesterification
reaction of compound 1 by vinyl acetate.
For the purpose of investigating the course of the transesterifica-
tion reaction of 1 and an assessment of the enantiomeric purity of
the resulting monoesters, an analysis of the reaction mixtures was
conducted at specified intervals. The reaction was carried out for
24 h at 50 °C (Table 2, Fig. 1) and samples were analyzed by HPLC.
The transesterification reaction of 1 in TBME proceeded at a
high rate and almost entirely stereoselectively. After 10 min,
monoester 20 was obtained in 22% yield and with 82% ee. After
one hour, the degree of conversion of 1 into the respective mono-
acetylated derivative reached 47.9% yield and enantiomeric purity
was 88% ee. The diacetyl derivative 200 was detected as 5% of the
reaction mixture. For the next 23 h of the reaction, no lowering
of the enantioselectivity was observed. Finally, 55.4% of monoester
20 and 44.0% diacetyl derivative 200 were obtained.
a
Where
R
= [ratio of the (S)-enantiomer]/[ratio of the (R)-enantiomer].
b
The ee was determined by HPLC.
groups to give the (R)-monoester. The determination of the abso-
lute configuration will be discussed in Section 2.4.
The best results of the transesterification were obtained using
tert-butyl methyl ether (TBME). After 24 h, monoester 20 was ob-
tained with high yield (55.4%) and enantioselectivity (88% ee), as
can be seen in Table 1. In addition to the monoester, the diacetyl
derivative 200 was obtained with a 44.0% yield. In the two remaining
ethers (dioxane or THF), the enantiomeric purity of monoester 20
was lower. In dioxane, it was 77% ee, but in the less polar THF, it
was 65% ee. The BCL in pyridine and ionic liquid [Bmim][PF6] dis-
played lower activity than in TBME, but the selectivity was similar.
Monoacetylated derivative 20 was obtained with an enantiomeric
excess similar to that observed in TBME. The reaction in pyridine,
similar to [Bmim][PF6], provided the monoester in 20% yield. How-
ever, pyridine was a better medium for the reaction, due to the bet-
ter solubility of the substrate.
In ionic liquid [Bmim][BF4] a decrease in enantioselectivity was
observed. The solvent with a nucleophilic character of the accom-
panying anion probably had an influence on the lipase activity and
enantioselectivity in ionic liquids. Ionic liquid [Bmim][PF6] practi-
cally does not mix with water (slightly soluble in water—0.13%)
while [Bmim][BF4] has unlimited solubility. Moreover, BFꢀ ion is
On the basis of the results presented, it can be concluded that
BCL in TBME allows for the enantiofacial differentiation of prochi-
4
a stronger nucleophile than PFꢀ6 ion and thus has more strength

R. Kołodziejska et al. / Tetrahedron: Asymmetry 23 (2012) 683–689
685
Table 2
The BCL enzymatic activity was higher in TBME than in pyridine.
The degree of conversion of monoester 40 in TBME was 48.9% and
in pyridine 13.2%.
The transesterification of 1 catalyzed by BCL in TBME
Time (h)
20 (%)
200 (%)
eea (%)
In the acylations using either vinyl acetate or vinyl butyrate the
monoester 20 or 30, respectively, was obtained with a similar enan-
tiomeric purity (in pyridine 81% ee, in TBME 88–90% ee). The
transesterification reaction with vinyl butyrate was also faster than
the other acylation reaction regardless of the solvent used.
In the reactions with vinyl laurate in TBME, a decrease in both
the activity and the selectivity of the lipase was observed. Monoes-
ter 50 was obtained in 31.0% yield and with 81% ee. Generally, the
catalytic activity of BCL in pyridine was lower than in TBME. Only
in the transesterification reaction of vinyl laurate was the yield of
monoacetyl derivative 50 higher than that in TBME. As in TBME,
when in pyridine with an elongation of the acyl donor chain, the
selectivity of the reaction became lower (Fig. 2).
0.17
0.33
0.50
0.67
0.83
1.00
1.50
2.50
3.50
6.00
18.00
24.00
22.0
25.0
36.2
37.3
41.1
47.9
53.7
56.4
55.4
51.4
51.4
55.4
2.2
2.1
3.4
3.6
4.6
82
87
87
86
87
88
90
91
88
85
87
88
5.0
25.7
33.0
34.0
41.6
44.0
44.0
a
The ee was determined by HPLC.
2.2. The comparison of the enantioselective properties of BCL
with lipase B from Candida antarctica (CALB)
The transesterification reactions of compound 1 were also car-
ried out in the presence of lipase B from Candida antarctica (CALB)
with different acyl donors in two solvents: pyridine and TBME.
CALB is on sn-3 stereospecific lipase that is able to hydrolyze the
ester bonds at the third carbon atom of acylglycerols.1–5,16 The
reactions were carried out for 24 h at 50 °C; the results are summa-
rized in Table 4.
Generally, the speed of acylation of compound 1 in the presence
of CALB was higher in pyridine than in TBME. The same reaction
carried out in pyridine with vinyl acetate and vinyl benzoate pro-
ceeded much faster and mainly diacyl derivatives were obtained
(yield was approximately 60%). The presence of lipase CALB failed
to provide the selective acylation of the enantiotopic groups of the
prochiral diol. Regardless of the solvent or acyl donor, the monoa-
cyl derivatives were obtained with unsatisfactory enantiomeric ex-
cesses. In Table 5 the comparison of both lipases in pyridine and
TBME depending on the acyl agent used is shown.
.
.
.
.
.
.
.
.
1
(S)-2'
(R)-2'
2''
Time [h]
Figure 1. Progress of diol 1 acylation with vinyl acetate in TBME at 50 °C.
ral diol sites by the almost entirely selective acetylation of one of
the primary hydroxyl groups. Regardless of the solvent used, in
the presence of BCL, the (R)-ester was obtained in excess.
On the basis of the results presented it can be concluded that
the catalytic activity of CALB in TBME was lower than that of
BCL. The yields of monoacyl derivatives in TBME, regardless of
the acyl donor used, were higher than the transesterification reac-
tion in the presence of BCL. Moreover, the acyl reaction catalyzed
by lipase CALB both in pyridine and TBME was almost entirely
non-selective. The expected improvement of the enantioselective
properties of lipase CALB corresponding to the donor0s acyl chain
elongation was not found.
The presence of CALB was unsuccessful in giving a selective
acylation of enantiotopic hydroxyl groups: pro-R and pro-S. In or-
der to conduct the desymmetrization of chemically identical hy-
droxyl groups of compound 1, BCL should be used.
2.1.2. The influence of the acylating agents on the
enantioselectivity of the enzymatic reaction
The acylation reaction of 1 was carried out in TBME for 24 h
with the set of different acylating agents. The results obtained
were compared with the course of the reaction in pyridine (Ta-
ble 3). Four different acylating agents were used: vinyl acetate,
butyrate, benzoate, and laurate.
It was found that the highest selectivity and reactivity for the
BCL-catalyzed acylation of 1 was observed with vinyl benzoate.
The reaction with vinyl benzoate in TBME and pyridine, after
24 h gave only the monoester 40 with high enantiomeric excess.
Table 3
2.3. The enzymatic hydrolysis of diesters 200, 300,and 500
The influence of the type of acyl donor on the selectivity for the enzymatic acylation
of 1 by BCL
The enzymatic hydrolysis of prochiral diesters obtained during
the transesterification reaction 200, 300, and 500 was performed in
TBME–water (80:20 v/v) in the presence of BCL. The reaction was
carried out at 50 °C for 24 h. The results are shown in Table 6.
In the enzymatic hydrolysis, the expected monoesters were ob-
tained in high yield and with high enantioselectivity. The enantio-
meric purity of the monoesters depended on the length of the acyl
donor chain. The best results were observed during the hydrolysis
of diacyl derivatives 200 and 300. All of the monoesters obtained were
found to be the respective (S)-enantiomer.
Acyl agent
Solvent
Mono (20–50)
Di (200–500)
(%)
Ra
eeb
(%)
(S)
(%)
(R)
(%)
Vinyl acetate
Pyridine
TBME
1.9
17.8
52.1
28.6
63.5
13.1
48.7
32.7
28.1
0.11 81
0.06 88
0.10 81
0.05 90
0.01 99
>0.01 99
0.32 52
0.10 81
3.3
3.0
3.3
0.1
0.2
34.0
16.3
Vinyl butyrate Pyridine
TBME
Vinyl
benzoate
Vinyl laurate
Pyridine
TBME
Pyridine 10.4
TBME 2.9
2.8
In the acylation and hydrolysis the BCL favors the same prochi-
ral groups (pro-R). It selectively introduces an acyl group onto the
pro-R hydroxyl group in the transesterification reaction. As a result
a
Where
R
= [ratio of the (S)-enantiomer]/[ratio of the (R)-enantiomer].
b
The ee was determined by HPLC.

686
R. Kołodziejska et al. / Tetrahedron: Asymmetry 23 (2012) 683–689
Figure 2. The transesterification of 1 catalyzed by BCL in TBME and pyridine.
Table 4
Table 6
Enantioselective desymmetrization of prochiral 200, 300 and 500 by enzymatic hydrolysis
The influence of the type of acyl donor on the selectivity for the enzymatic acylation
of 1 by CALB
Compound
1
Mono (20, 30, 50)
(S) (%) (R) (%)
eea (%)
Acyl agent
Solvent
Mono (20–50)
Di (200-500) (%) Ra
eeb (%)
(S) (%) (R) (%)
200
300
500
21.5
—
26.1
71.4
99
54.7
7.1
1
19.2
82
98
48
Vinyl acetate
Pyridine 20.2
TBME 8.5
Pyridine 24.9
12.7
12.3
32.4
20.9
7.9
60.9
1.3
0.64 22
0.69 18
Vinyl butyrate
26.4
11.4
63.2
—
0.77 13
0.74 14
0.75 14
0.69 12
a
The ee was determined by HPLC.
TBME
15.6
6
Vinyl benzoate Pyridine
TBME
Vinyl laurate
12.8
16.4
18.5
13.5
Pyridine 20.4
TBME 10.5
0.6
0.91
5
4.5
0.78 15
a
Where
R
= [ratio of the (S)-enantiomer]/[ratio of the (R)-enantiomer].
b
The ee was determined by HPLC.
of this reaction (R)-monoester was obtained in high enantiomeric
purity while in the deacylation reaction, the monoester was ob-
tained with the opposite configuration.
2.4. Determination of the absolute configuration
Our goal was to provide a reliable assignment of the absolute
configuration of the newly formed stereogenic center, thus provid-
ing a solid basis for the stereocontrol observed in the enzymatic
transesterification reaction. In order to achieve this, we selected
electronic circular dichroism spectroscopy (ECD) from within the
set of available modern chiroptical methods. ECD spectroscopy is
very fast, convenient, sensitive to even the smallest changes in
geometry and electronic structure of molecules, as well as a pow-
erful technique that can be used to explore the asymmetric envi-
ronment of chiral non-racemic molecular species.[17]
Figure 3. ECD spectra of compounds
measured in CH3CN.
(
)-20,
(
)-30,
(
)-40, and
(
)-50
All spectra of compounds 20–50 with an undefined absolute con-
figuration were measured in CH3CN. As evident from Figure 3, the
ECD spectra of all four compounds were very similar in shape. Each
spectrum exhibits three bands; two negative at 265 and 205 nm,
and a positive one at 230 nm. These bands are associated with
the transitions of the thymine ring affected by the chiral ester
moiety.
The same signs of all of the bands observed in the experimental
ECD spectra clearly indicate the same absolute configuration for
the newly formed stereogenic center, as expected in reactions with
enzymes. The difference in intensity with regard to the ECD bands
of the individual compounds is related to their different enantio-
meric purities (see Table 3), as well as their different conforma-
Table 5
Comparison of the processes catalyzed by CALB and BCL
Solvent
Lipase
Mono products
20
30
40
50
Yield (%)
eea (%)
Yield (%)
eea (%)
Yield (%)
eea (%)
Yield (%)
eea (%)
Pyridine
TBME
BCL
CALB
BCL
19.7
32.9
35.4
20.8
81
22
88
18
31.6
57.3
66.8
36.5
81
13
90
14
13.2
13.9
48.9
29.2
99
14
99
12
43.1
38.9
31
52
5
81
15
CALB
24
a
The ee was determined by HPLC.

R. Kołodziejska et al. / Tetrahedron: Asymmetry 23 (2012) 683–689
687
Table 7
reactions influenced the enantioselectivity of the obtained mono-
derivatives. TBME provided an optimal environment for the en-
zyme. In TBME, BCL selectively acylated the enantiotopic hydroxyl
groups and gave (R)-monoesters with high enantiomeric purity
(81–99% ee). The (S)-monoesters were obtained from the prochiral
diesters as a result of the enzyme-catalyzed enantiomeric selective
hydrolysis. A combined analysis of the experimental and simulated
ECD spectra in conjunction with a thorough conformational analy-
sis allowed us to unambiguously assign the absolute configuration
of the newly formed stereogenic center in the transesterification
reaction to be (R).
Overview of the conformer analysis for the (R)-enantiomer of compound 20
Conformer
D
G (kcal/mol)
Population (%)
1
2
3
4
5
6
7
8
9
10
0.0
0.4
0.7
0.9
1.0
1.6
1.7
1.9
2.1
2.3
41.5
20.9
12.6
9.1
7.3
2.7
2.2
1.7
1.2
0.9
Conformer populations were calculated using the Gibbs free energy.
4. Experimental
tional flexibilities. Their relatively unrestricted mobility is limited
to the side chain substituent and is evident by considering the
dependence of the intensity of the ECD bands on the length of
the side chain, that is the longer the side chain, and thus the higher
number of possible rotamers, the lower the intensity of the bands.
The absolute configuration of the newly formed stereogenic
center was determined on the basis of the comparison of the
experimental and computed spectra for an arbitrarily chosen enan-
tiomer. Compound 20 was chosen as the model compound for com-
putations with application of Time Dependent Density Functional
Theory (TD DFT) because of its relatively short side chain and
therefore relatively high-intensity of ECD bands. Compound 40,
characterized by the highest intensity of cotton effects (CE0s) in
its ECD spectrum, was excluded due to the presence of an addi-
tional chromophoric system in the skeleton. The deconvolution
of the overlapping contributions from both aromatic systems
would not only hamper the interpretation of the results, but would
also be time-consuming.
A fundamental prerequisite for the computational calculation of
ECD spectra is the knowledge of all ECD-relevant conformational
species of the respective molecule. Thus, in order to make a reliable
assignment, very careful conformational analysis by molecular
mechanics must first be carried out in order to determine the low-
est energy conformers. Then, all of the conformers found can be
reoptimized on the B3LYP/6-31G(d) level. As a result, ten conform-
ers of compound 20 were found within the range of 5 kcal/mol (Ta-
ble 7). It is important to note that for both the conformational
analysis and calculations of the ECD spectra, the (R)-enantiomer
of compound 20 was arbitrarily selected (all details are contained
in the Section 4).
4.1. Analytical methods
The HPLC experiments were performed on a Shimadzu SCL-10A
VP, analytical column Chiralcel OJ 250 ꢁ 4.6 mm, 10
lm, Daicel
(France). 1H NMR spectra were recorded on a Bruker 300 Hz appa-
ratus (TMS as an internal standard). Enantiomeric separation of 20–
50 was performed using the Chiralcel OJ and a mixture of hexane
and propan-2-ol 60:40 (v/v) or 80:20 (v/v) as eluant, at 1 mL/min
or 0.5 mL/min flow rate (Table 8).
4.2. Reagents and solvents
Lyophilized lipase B from Candida antarctica (Fluka, Switzer-
land), 10.9 U/mg. Lyophilized lipase Amano PS from Burkholderia
cepacia (Aldrich, Japan), 30 U/g. Other chemicals of analytical grade
were commercially available: 1 was obtained according to litera-
ture procedure,[6] vinyl acetate from Fluka (Germany), vinyl buty-
rate and vinyl benzoate from Fluka (Japan), vinyl laurate from
Fluka (Switzerland), 1-butyl-3-methylimidazolium hexafluoro-
phosphate ([BMIM][PF6]) and 1-butyl-3-methylimidazolium tetra-
fluoroborate ([BMIM][BF4]) from Fluka (Switzerland), pyridine,
cyclohexane, dioxane, n-hexane for HPLC, propan-2-ol for HPLC
from POCH (Poland).
4.3. General procedures
4.3.1. Analytical scale enzymatic acylation of diol 1
In a typical experiment, an enzymatic reaction was performed
in a solution containing 1 (1 ꢁ 10ꢀ4 mol), BCL (350 mg) or CALB
(1 mg), acyl donor (1 ꢁ 10ꢀ4 mol) and solvent (1 mL). The reaction
mixture was kept at a constant temperature (50 °C). After the reac-
tion, the enzyme was removed by filtration and the filtrate was
concentrated. In the case of ionic liquid, the solution mixture
was first extracted with hexane–propan-2-ol (80:20), and the or-
ganic phase was concentrated. The enantiomeric ratios were deter-
mined on an HPLC system using a chiral column. The mobile phase
consisted of hexane–propan-2-ol.
The geometry of the three lowest energy conformers of the (R)-
enantiomer of compound 20, namely conformers 1, 2, and 3, is
shown in Figure 4.
The next step in the stereochemical analysis was to calculate the
ECD spectra of each conformer found for 20 (conformers 1–10) con-
ducted with the B3LYP functional and TZVP basis set using the PCM
solvent continuum model with acetonitrile as the solvent. The pre-
dicted ECD spectrum, weighted on the basis of the Gibbs free energy,
is shown in Figure 5. As can be seen, the Boltzmann averaged ECD
spectrum of the (R)-enantiomer of compound 20 is in perfect agree-
ment with the experimental one. This allowed us to unequivocally
assign the absolute configuration to be (R). Based on the similarity
of the experimental spectra of all of the compounds obtained by
the enzymatic transesterification reaction, the same (R) absolute
configuration was assigned to the remaining compounds 30–50.
4.3.2. Analytical scale enzymatic hydrolysis of diesters 200, 300,
and 500
An enzymatic reaction was performed in a solution containing
diester 200, 300, or 500 (1 ꢁ 10ꢀ4 mol), BCL (100 mg), and TBME–water
80:20 v/v (1 mL). The reaction mixture was kept at a constant tem-
perature (50 °C). After the reaction, the enzyme was removed by
filtration and the resulting solution was concentrated. The enantio-
meric ratios were determined on an HPLC system using a chiral
column. The mobile phase consisted of hexane–propan-2-ol.
3. Conclusion
4.3.3. Preparative scale enzymatic acylation of diol 1
We have reported the desymmetrization of identical hydroxyl
groups of a prochiral diol in an enzymatic transesterification reac-
tion. Both the solvent and the acyl group donor employed in the
To a solution of 1 (1 ꢁ 10ꢀ3 mol) in TBME (20 mL), lipase BCL
(3.5 g) and acyl donor (1 ꢁ 10ꢀ3 mol) were added. The reaction

688
R. Kołodziejska et al. / Tetrahedron: Asymmetry 23 (2012) 683–689
Figure 4. Structures of the three lowest energy conformers of the (R)-enantiomer of compound 20 calculated at the B3LYP/6-31G(d) theory level.
4.3.3.3. 3-Hydroxy-2-((5-methyl-2,4-dioxo-3,4-dihydropyrimi-
din-1(2H)-yl)methoxy)propyl butyrate 30.
Isolated yield:
42%; 1H NMR (CDCl3):d (ppm) = 0.95 (t, J = 7.5 Hz, 3H), 1.65 (m,
2H), 1.94 (d, J= 1.2 Hz, 3H), 2.28 (t, J = 7.5 Hz, 2H), 3.64 (dd,
J = 6.0 Hz, J = 12.0 Hz, 1H), 3.72 (dd, J = 3.9 Hz, J = 12.0 Hz, 1H),
3.90 (m, 1H + OH), 4.10 (dd, J = 6.0 Hz, J = 12.0 Hz, 1H), 4.25 (dd,
J = 4.5 Hz, J = 12.0 Hz, 1H), 5.25 (m, 1H), 7.15 (q, J = 1.2 Hz, 1H),
8.30 (s, 1H); MS-CI (m/z) 301 (MH)+.
4.3.3.4.
2-((5-Methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)methoxy)propane-1,3-diyl dibutyrate 300.
Isolated yield:
11%; 1H NMR (CDCl3): d (ppm) = 0.95 (t, J = 7.5 Hz, 3H), 1.65 (m,
2H), 1.94 (d, J = 1.2 Hz, 3H), 2.28 (t, 7.2 Hz, 2H), 4.08 (m, 3H),
4.25 (m, 2H), 5.25 (s, 2H), 7.10 (q, J = 1.2 Hz, 1H), 8.20 (s, 1H);
MS-CI (m/z) 371 (MH)+.
4.3.3.5. 3-Hydroxy-2-((5-methyl-2,4-dioxo-3,4-dihydropyrimi-
Figure 5. Computed ECD spectra (
) at the B3LYP/TZVP/PCM (CH3CN) level of
din-1(2H)-yl)methoxy)propyl benzoate 40.
Isolated yield:
theory obtained as
a population-weighted sum of the calculated spectra of
39%; 1H NMR (CDCl3): d (ppm) = 1.80 (d, J = 1.2 Hz, 3H), 3.75 (dd,
J = 6.0 Hz, J = 12.0 Hz, 1H), 3.83 (dd, J = 4.2 Hz, J = 12.0 Hz, 1H),
4.10 (m, 1H + OH), 4.41 (dd, J = 5.7 Hz, J = 12.0 Hz, 1H), 4.46 (dd,
J = 4.5 Hz, J = 12.0 Hz, 1H), 5.27 (m, 2H), 7.10 (q, J = 1.2 Hz, 1H),
7.45 (m, 2H), 7.58 (m, 1H), 7.99 (m, 1H), 8.02 (m, 1H); MS-CI (m/
z) 335 (MH)+.
individual conformers of (R)-20 compared to experimental (
) in CH3CN.
Table 8
The retention times of the 1, mono- and di-derivatives
Compound
Retention time (min)
Compound
Retention time (min)
1
5.4a
9.8a
(S)-40
(R)-40
400
10.8a
11.9a
33.9a
29.1b
26.9b
31.9b
13.4b
(S)-20
(R)-20
200
4.3.3.6.
2-((5-Methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
1H NMR
13.9a
28.2a
7.3a
yl)methoxy)propane-1,3-diyl dibenzoate 400.
1
(CDCl3): d (ppm) = 1.68 (d, J = 1.2 Hz, 3H), 4.50 (m, 5H), 5.30 (s,
2H), 7.06 (q, J = 1.2 Hz, 1H), 7.45 (m, 4H), 7.59 (m, 2H), 8.01 (m,
4H); MS-CI (m/z) 439 (MH)+.
(S)-30
(R)-30
300
(S)-50
(R)-50
500
9.8a
14.7a
a
n-Hexane–propan-2-ol 60:40; 1 mL/min.
n-Hexane–propan-2-ol 80:20; 0.5 mL/min.
4.3.3.7. 3-Hydroxy-2-((5-methyl-2,4-dioxo-3,4-dihydropyrimi-
b
din-1(2H)-yl)methoxy)propyl decanoate 50.
Isolated yield:
19%; 1H NMR (CDCl3): d (ppm) = 0.95 (m, 3H), 1.25 (s, 14H), 1.60
(m, 2H), 1.95 (d, J = 1.2 Hz, 3H), 2.25 (m, 2H), 4.15 (m, 3H + OH),
4.22 (m, 2H), 5.23 (s, 2H), 7.15 (q, J = 1.2 Hz, 1H), 9.20 (s, 1H);
MS-CI (m/z) 413 (MH)+.
mixture was kept at a constant temperature (50 °C). The reaction
progress was monitored by TLC and HPLC. After 24 h, the reaction
was quenched by filtration and the filtrate was concentrated under
reduced pressure. The crude product was purified by PLC using
ethyl acetate as eluent.
4.3.3.8.
2-((5-Methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)methoxy)propane-1,3-diyl didecanoate 500.
Isolated yield:
5%; 1H NMR (CDCl3): d (ppm) = 0.95 (m, 6H), 1.28 (m, 32 H), 1.98
(d, J = 1.2 Hz, 3H), 2.25 (m, 4H), 4.13 (m, 3H), 4.21 (m, 2H), 5.24
(s, 2H), 7.20 (q, J = 1.2 Hz, 1H), 9.20 (s, 1H); MS-CI (m/z) 595 (MH)+.
4.3.3.1. 3-Hydroxy-2-((5-methyl-2,4-dioxo-3,4-dihydropyrimi-
din-1(2H)-yl)methoxy)propyl acetate 20.
Isolated yield:
30%; 1H NMR (D2O): d (ppm) = 1.80 (d, J = 1.2 Hz, 3H), 1.95 (s,
3H), 3.54 (dd, J = 5.9 Hz, J = 12.3 Hz, 1H), 3.62 (dd, J = 3.9 Hz,
J = 12.3 Hz, 1H), 3.89 (m, 1H), 4.00 (d, J = 6.7 Hz, 1H), 4.15 (dd,
J = 8.6 Hz, J = 11.5 Hz, 1H), 5.21 (s, 2H), 7.48 (d, J = 1.2 Hz, 1H),
11.00 (s, 1H); MS-CI (m/z) 273 (MH)+.
4.3.4. ECD spectroscopy and quantum chemical calculations
The electronic circular dichroism spectra were measured using
a Jasco J-815 CD Spectropolarimeter (Jasco Co., Japan). ECD spectra
were recorded between 180 and 450 nm at room temperature in
spectroscopic grade acetonitrile. Solutions with concentrations in
the range from 2 ꢁ 10ꢀ4 to 4.8 ꢁ 10ꢀ4 M were examined in a
quartz cell with a path length of 2–0.1 cm. All spectra were re-
corded using 100 nm/min scanning speed, a step size of 0.2 nm, a
bandwidth of 1 nm, a response time of 0.5 s, and an accumulation
of five scans. The spectra were background corrected.
4.3.3.2.
2-((5-Methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)methoxy)propane-1,3-diyl diacetate 200.
Isolated yield:
15%; 1H NMR (CDCl3): d (ppm) = 1.95 (d, J = 1.2 Hz, 3H), 2.03 (s,
6H), 4.04 (m, 3H), 4.20 (m, 2H), 5.23 (s, 2H), 7.14 (s, 1H), 9.48 (s,
1H); MS-CI (m/z) 315 (MH)+.

R. Kołodziejska et al. / Tetrahedron: Asymmetry 23 (2012) 683–689
689
The conformational search was carried out using a HyperChem
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Acknowledgements
The authors acknowledge the National Science Centre grant
entitled ‘Advanced techniques of circular dichroism as a valuable
tool in structural analysis of pharmacologically important com-
pounds’ for a financial support, and a Grant No. G34-15 for compu-
tational time at the Interdisciplinary Centre for Mathematical and
Computational Modeling (ICM) of University of Warsaw, Poland.