A molecular modelling approach to rationalize the stereochemical outcome of the Burkholderia cepacia lipase-catalyzed transesterification of aromatic primary alcohols with vinyl esters with different chain lengths in chloroform

Article abstract of DOI:10.1016/j.tetasy.2009.07.024

The Burkholderia cepacia lipase-catalyzed transesterification of 2-methyl-3-phenyl-1-propanol with vinyl esters proceeds with high enantioselectivity independently of the acyl chain length and the low enantioselectivity of the same reaction with 2-phenyl-1-propanol is not affected by chain length of the vinyl esters. A molecular modelling approach has been developed in order to rationalize the enzymatic results.

Full text of DOI:10.1016/j.tetasy.2009.07.024

Tetrahedron: Asymmetry 20 (2009) 1833–1836
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
A molecular modelling approach to rationalize the stereochemical outcome
of the Burkholderia cepacia lipase-catalyzed transesterification of aromatic
primary alcohols with vinyl esters with different chain lengths in chloroform
b
b
a
Enzo Santaniello a, , Silvana Casati , Pierangela Ciuffreda , Giuseppe Meroni ,
*
Alessandro Pedretti c, Giulio Vistoli c
a Laboratory of Medical Chemistry, Department of Medicine, Surgery and Dentistry, Faculty of Medicine, Università degli Studi di Milano, Italy
b Department of Preclinical Sciences LITA Vialba, Faculty of Medicine, Università degli Studi di Milano, Italy
c Department of Pharmaceutical Sciences ‘Pietro Pratesi’, Faculty of Pharmacy, Università degli Studi di Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The Burkholderia cepacia lipase-catalyzed transesterification of 2-methyl-3-phenyl-1-propanol with vinyl
esters proceeds with high enantioselectivity independently of the acyl chain length and the low enanti-
oselectivity of the same reaction with 2-phenyl-1-propanol is not affected by chain length of the vinyl
esters. A molecular modelling approach has been developed in order to rationalize the enzymatic results.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 29 June 2009
Accepted 7 July 2009
Available online 3 September 2009
1. Introduction
This relevant difference in the stereoselective outcome of the enzy-
matic reaction for the alcohol 2 is most probably related to the subtle
The Burkholderia cepacia lipase (BCL), formerly known as Pseudo-
monas cepacia lipase (PCL), is a highly selective catalyst for a broad
range of substrates, catalyzing various reactions in water and non-
polar solvents under mild conditions.1 This includes the kinetic res-
olution of racemic secondary alcohols by hydrolysis in water or
esterification in organic solvents.2–4 The stereochemical outcome
of the lipase-catalyzed reactionscan be interpretedaccording to var-
ious models of the enzyme active site.5 In particular, the enantio-
specificity of the lipase from P. cepacia (PCL)6 has been extensively
investigated and a model can be adopted as a predictive rule for sec-
ondary alcohols.7 This model has been extended to primary alcohols
with a lower degree of confidence, since, in this case, it is more diffi-
cultto predictthestereopreference of theenzymaticreaction.8 Inthe
case of a primary alcohol, the enantioselectivity is frequently from
low to moderate and can be increased by optimizing the reaction
conditions. For instance, we were able to achieve a high enantiose-
lectivity in the acetylation of the primary alcohol 2-methyl-3-phe-
nyl-1-propanol 1.9 The reaction was stopped at approximately 40%
conversion and the (S)-acetate was obtained in enantiomerically
pure form. The unreacted alcohol could be recovered at the highest
enantiomeric excess (ee) when the reaction was carried out at 60%
conversion.10 The alcohol 1 has frequently been used as a model
for studies on the mechanism and stereochemistry of PCL-catalyzed
hydrolysis of esters of primary alcohols.11,12 From a stereochemical
point of view, the same enzymatic reaction proceeds in an unsatis-
factory manner with 2-phenyl-1-propanol (compound 2, Fig. 1).13
structural difference that exists between the two homologous alco-
hols 1 and 2.
OH
OH
2
1
Figure 1. Structure of the two aromatic primary alcohols 2-methyl-3-phenyl-1-
propanol 1 and 2-phenyl-1-propanol 2.
Recently, we have shown that the lipase-catalyzed resolution of
the esters (R,S)-3a–d affords (S)-1, with the ee depending on the
ester chain length.14 In fact, the excellent enantioselectivity of
the PCL-catalyzed hydrolysis of esters 3a and 3b dramatically fell
when the chain of the fatty acid was lengthened to esters 3c and
3d (Scheme 1).
Using a two-step MM-based modelling approach, it was pro-
posed that the drop in enantioselectivity was determined by an
overflowing conformational rearrangement due to the refolding
of the fatty acid chain inside the hydrophobic binding pocket. Con-
sequently, the compulsory increase in the activation energy of the
rate-determining step leading to the first tetrahedral intermediate
becomes a relevant factor. This effect was confirmed by calculating
the free energy differences between the complexes of PCL with the
(R)- and (S)-enantiomers.14
The foregoing results prompted us to undertake research aimed
at verifying whether similar conformational effects could be evi-
denced in an organic solvent. Therefore, we have extended the
* Corresponding author. Tel.: +39 025 031 9691; fax: +39 025 031 9631.
E-mail address: enzo.santaniello@unimi.it (E. Santaniello).
0957-4166/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2009.07.024

1834
E. Santaniello et al. / Tetrahedron: Asymmetry 20 (2009) 1833–1836
3. Molecular modelling results and discussion
H2O, PCL
OCOR
OH
Molecular modelling studies involved a preliminary MD simula-
tion of the enzyme in chloroform with a view to revealing possible
conformational shifts induced by the apolar medium. The so-ob-
tained BCL structure was then used in docking analyses to account
for the different enantioselectivity in the transesterification of
most significant esters. In order to rationalize the transesterifica-
tion data in silico, docking simulations for the aromatic primary
alcohols 1 and 2 were performed considering the Ser87 residue
covalently bound to the acyl group that should lead to the forma-
tion of the esters 3a–d and 5a–d. The resolved structure of BCL was
retrieved from PDB (Pdb Id: 3LIP).26 The structure was completed
by adding the hydrogen atoms. According to the physiological
pH, glutamate, aspartate, lysine, and arginine residues were con-
sidered ionized while histidine and cysteine residues were simu-
lated as neutral by default. The calcium ion which plays a
structural role in BCL was maintained in the simulation, while
the crystallization water molecules were removed to better en-
lighten the effects of apolar medium. The structure underwent a
minimization keeping the backbone atoms fixed to preserve the
experimental folding, and then it was inserted in a 70 Å side cubic
box of chloroform solvents.27 The simulations were carried out in
two phases: an initial period of heating from 0 K to 300 K over
6000 iterations (6 ps, i.e., 1 K/20 iterations), and a monitored phase
of simulation of 1 ns. Only the frames memorized during this last
phase were considered. The duration of an MD run was suitably
chosen to reveal significant conformational shifts induced by an
apolar solvent without affecting the folding stability. The final
structure was then minimized and used to model the covalently
modified enzyme after deleting the solvent molecules. In particu-
lar, Ser87 was acylated to simulate the first step of the transesteri-
fication process. Among the acyl groups, calculations were limited
to the acetyl group that should produce the acetates 3a and 5a in
the classical procedure that uses vinyl acetate as an acyl transfer
agent. The octanoyl group was selected as a typical medium-long
chain example of an acyl group that yields the octanoates 3c and
5a. The two covalently modified enzyme structures were finally
minimized and exploited in docking calculations. The simulated
aromatic primary alcohols (1 and 2, considering for both substrates
the two possible enantiomers) were built by VEGAZZ28 and opti-
mized by MOPAC2009. The modified structures of BCL were pre-
pared by AutoDock Tools as implemented in VEGAZZ software.
The 3D affinity grid box was designed to include the catalytic pock-
et around the modified Ser87 residue. Docking searches were per-
formed by AutoDock version 4 with the Lamarckian genetic
algorithm.29 The lowest energy pose was further minimized to
optimize the mutual complementarity between the enzyme and
substrate. All minimizations as well as the described MD run were
carried out by NAMD 2.6.30
(S)-1
3a-d
a R = CH3
bR = (CH2)2CH3
c R = (CH2)6CH3
dR = (CH2)10CH3
Scheme 1. The stereochemical outcome of the PCL-catalyzed hydrolysis of esters
3a–d.
study of the chain length effect to the BCL-catalyzed transesterifi-
cation of alcohols 1 and 2 using vinyl esters of different chain
lengths.
2. Results and discussion of the enzymatic results
The transesterification reactions of alcohols 1 and 2 were car-
ried out at room temperature in chloroform with commercially
available vinyl esters 4a–d (see Scheme 2).15
BCL in CHCl3
OH
OCOR
(
)
( )n
n
OCOR
3 n=1
5 n=0
1 n=1
2 n=0
4
a R = CH3
b R = (CH2)2CH3
c R = (CH2)6CH3
d R = (CH2)10CH3
Scheme 2. BCL-catalyzed transesterification of alcohols 1 and 2 with vinyl esters
4a–d.
We have found that, independent of the chain length of the vi-
nyl ester used, the enantioselectivity of the reaction with the alco-
hol 1 at about 30% conversion (expressed as enantiomeric ratio E16
)
remained considerably high. The enzymatic transesterification of
2-phenyl-1-propanol 2 with vinyl esters 4a–d was slower than that
of the alcohol 1 (20–30 h for 25–35% conversion) and low enantio-
meric ratio values (ca. 2) were obtained, irrespective of the chain
length of vinyl esters. All the results obtained from the transesteri-
fications are collected in Table 1.
Table 1
BCL-catalyzed transesterification of alcohols 1 and 2 with vinyl esters 4a–d
Product
Acyl donor
Time (h)
Conversiona (%)
eeb
Ec
3a
3b
3c
3d
5a
5b
5c
5d
4a
4b
4c
4d
4a
4b
4c
4d
3
4
3
31
33
32
31
40
21
33
35
>98
>98
>98
>98
39
35
15
22
>100
>100
>100
>100
2.9
2.3
1.6
1.4
5
24
20
20
30
A detailed description of the conformational changes that the
BCL enzyme experiences during the MD run in chloroform goes be-
yond the objectives of this study, but the analysis of the trajectory
produced allows some relevant considerations. Firstly, the per-
formed simulation does not vastly alter the experimental folding
as assessed by the percentage of residues falling in the allowed re-
gions of the Ramachandran plot, which remained constantly
around 75% during the MD run. Globally, such a result suggests
that the structural differences described below are mainly due to
the dynamic response of the enzyme to the apolar medium and
not to random structural distortions. Secondly, the analysis of sur-
face profiles discloses a divergent behavior since the SAS surface
slightly increases during the simulation suggesting a further open-
ing of the lid domain which exposes its apolar side chains, as
recently evidenced by longer simulations in apolar solvents.
Determined by GC analysis.15
a
Enantiomeric excess evaluated by 1H NMR analysis of the MTPA esters17 of
b
alcohols 1 and 2 obtained from the hydrolysis of the esters 3a–d and 4a–d.18
c
Enantiomeric ratio calculated according to Chen et al.16
Also the result obtained with the alcohol 2 is interesting, be-
cause in many other examples the change of the acyl group was
sufficient to increase the enantioselectivity of this reaction.19 How-
ever, it has also been observed that the enzymatic resolution of
alcohol 2 showed a negligible enantioselectivity (E <2) under sev-
eral conditions such as transesterification,20–22 alcoholysis23 or
acylation.24,25 In order to rationalize the above BCL-catalyzed enzy-
matic transesterification of alcohols 1 and 2, molecular modeling
studies were carried out.

E. Santaniello et al. / Tetrahedron: Asymmetry 20 (2009) 1833–1836
1835
Conversely, the polar surface shows a marked diminution suggest-
ing that the apolar solvent induces a hydrophilic collapse in which
the polar residues minimize their unfavorable contacts with the
medium. Lastly, such a hydrophilic collapse reflects on the catalytic
center which significantly decreases its wideness. This is probably
due to the approaching of polar residues as clearly assessed by the
volume of catalytic cavity which almost halves during the simula-
tion shifting from 1533 Å3 to 871 Å3 (as computed by CASTP ser-
ver). It should be noted that during the MD simulation the
catalytic cavity includes several solvent molecules that should pre-
vent an unrealistic collapse of the cavity despite the removal of
water molecules. These results are in line with those reported by
previous studies31,32 and suggest a contrasting situation since the
enzyme in chloroform should possess a more accessible, but less
wide, catalytic cavity.
As a preamble, it should be noted that the two acyl groups are
conveniently accommodated in the catalytic cavity. In both struc-
tures the carbonyl group interacts with Tyr23 and to a minor ex-
tent with Tyr29. The acetyl group realizes hydrophobic contacts
with Tyr29, His86, and Ile290, while the octanoyl chain is inserted
in a region lined by apolar residues (Ile33, His86, Ile110, Pro304,
Val305, Val307, and Ile308) with which it can stabilize many
hydrophobic interactions. Figure 2A depicts the putative complex
between (S)-1 and the BCL enzyme covalently bound to octanoyl
to yield (S)-3c ester. It reveals that the substrate can be conve-
niently accommodated in the catalytic center since the hydroxyl
group approaches Ser87 and interacts with Tyr23 and Thr18. The
methyl group is inserted in a suitable subpocket lined by aliphatic
residues (e.g., Leu17, Leu167, and Val266), while the phenyl ring
interacts with the apolar residues already mentioned for the
methyl group and stabilizes p-p stacking with Phe52 and Phe119
(not shown in Fig. 2 for clarity). Such a precise interaction pattern
is likewise also found with the acylated enzyme and can explain
well the enantioselectivity in the transesterification of (S)-1. In
fact, the (R)-1 alcohol can be harboured in the BCL catalytic cavity,
whereas the methyl group clashes against Leu17 moving the ester
function away from Ser87.
Docking results can also explain the lack of enantioselectivity
for the transesterification of the alcohol 2, since the methyl group
detrimentally clashes against catalytic residues in both isomers. In
particular, the methyl group bumps against the hydroxy function
of Thr18 in (R)-1 isomer (as seen in Fig. 2B) and approaches
Ser87 hampering the catalytic mechanism in the (S)-1 isomer. Fur-
thermore, both enantiomers of alcohol 2 lose the
p–p stacking
with Phe52 and Phe119 which characterizes the complexes of 1
isomers.
Table 2 shows the docking scores for the optimized complexes
as defined by electrostatic energies. Such energy values can well
rationalize the experimental data, since the electrostatic energies
for the two enantiomers of 1 are markedly different. This justifies
the reported enantioselectivity for this substrate, while the differ-
ence between the isomers of the alcohol 2 is clearly reduced. Fur-
thermore, the calculated energy scores are almost independent of
the length of the acyl chains. Although these substrates are sub-
stantially apolar molecules, the score functions were focused on
polar interactions since they play a key role in triggering the cata-
lytic mechanism as recently confirmed by us.33
4. Conclusions
Our results show that in the transesterification of primary alco-
hols 1 and 2 with various vinyl esters, the length of the acyl chain
does not influence the stereochemical outcome of the process. In
fact, the enantioselectivity remains high or low, depending signif-
icantly on the specific structure of the substrates 1 and 2 that pres-
ent a phenyl ring that is close to the stereocenter or part of it. Thus,
p–p interactions of the substrate with aromatic residues present in
the active site might become significant. Computational studies
were desired for an explanation of the reported observations. How-
ever, different to the lipase-catalyzed hydrolytic process of
esters,12,32 only a few molecular modeling studies have been pro-
posed to explain the results of transesterification of primary
alcohols catalyzed by a lipase in an organic solvent.34
The computational studies performed suggest that the BCL
structure, as derived by MD simulation in chloroform, can account
for the different enantioselectivity observed in the resolution of
Table 2
Electrostatic energies and differences between enantiomeric esters obtained by
docking simulations
Alcohol
Acyl
Ester
produced
Electrostatic energies
(kcal/mol)
Isomeric
difference
(S)-1
(R)-1
(S)-2
(R)-2
Acetyl
(S)-3a
(R)-3a
(S)-5a
(R)-5a
À21.28
À12.77
À14.03
À12.89
À18.29
À11.10
À12.33
À11.12
8.51
1.14
Figure 2. Putative complexes between octanoyl-Ser87 BCL and (S)-1 substrate (A)
as well as acetyl-Ser87 BCL and (S)-2 (B). The hydrophobic residues are represented
by gray surfaces. In both complexes the alcohol approaches the catalytic residues
and the main differences concern the hydrophobic contacts since (S)-1 suitably
inserts the methyl in the hydrophobic pocket lined by Leu 17, Leu167, and Val266,
while (S)-2 accommodates the phenyl ring into the apolar cavity losing the pivotal
stacking with Phe52 and bumping the methyl against Thr18.
(S)-1
(R)-1
(S)-2
(R)-2
Octanoyl
(S)-3c
(R)-3c
(S)-5c
(R)-5c
7.19
1.21

1836
E. Santaniello et al. / Tetrahedron: Asymmetry 20 (2009) 1833–1836
afforded the required derivative for 1H NMR analysis (500 MHz, Bruker AM
500). For racemic 1 the three multiplets corresponding to CH2–O (0.5, 1, and
0.5H) at 4.00–4.25 ppm were considered for establishing the ee; for racemic 2,
the signals of OCH3 corresponded to two singlets at 3.382 and 3.405 ppm and
were used for the same purpose.
substrates 1 and 2. In fact, using appropriate docking simulations,
an insightful rationalization of the enzymatic data is possible and
docking results are able to explain the experimental results in
terms of specific interactions of the substrates with aminoacid res-
idues of the catalytic site. Finally, our approach opens the opportu-
nity of exploiting such simple but successful modeling analyses to
predict the stereochemical outcome of the resolution of other
substrates.
18. The ee of the enzymatic transesterification was established by 1H NMR analysis
of the MTPA esters of alcohols
1 and 2 obtained by hydrolysis of the
enzymatically formed esters 3a–d and 5a–d. This hydrolysis was effected most
satisfactorily with lithium aluminum hydride, since after the work-up, the
alcohol was obtained practically pure and was used as such for specific rotation
measurement or evaluation of ee. Thus, to a solution of the ester (1 mmol) in
dry diethyl ether (2 mL), lithium aluminum hydride (0.38 g, 10 mmol) was
added and the mixture was stirred for the time required to complete
hydrolysis. To the above mixture, water (0.4 mL), 15% sodium hydroxide
(0.4 mL), and water (1.2 mL) were added sequentially. The solid was removed
by filtration on a Celite pad and washed with diethyl ether (2 Â 1 mL) and
evaporation of the solvent afforded the pure alcohol.
Acknowledgement
We thank the University of Milan for financial support.
19. (a) Goto, M.; Kawasaki, M.; Kometani, T. J. Mol. Catal. B: Enzym. 2000, 9, 245–
250; (b) Ottonson, J. J.; Hult, K. J. Mol. Catal. B: Enzym. 2001, 11, 1025–1108; (c)
Miyazawa, T.; Yukawa, T.; Koshiba, T.; Ueji, S.; Yamagihara, R.; Yamada, T.
Biotechnol. Lett. 2001, 23, 1547–1550; (d) Hirose, K.; Naka, H.; Yano, M.; Ohashi,
S.; Naemura, K.; Tobe, Y. Tetrahedron: Asymmetry 2000, 11, 1199–1210; (e)
Miyazawa, T.; Yukawa, T.; Koshiba, T.; Sakamoto, H.; Ueji, S.; Yamagihara, R.;
Yamada, T. Tetrahedron: Asymmetry 2001, 12, 1595–1602.
20. Kawasaki, M.; Goto, M.; Kawabata, S., Kodama, T.; Kometani, T. Tetrahedron
Lett. 1999, 40, 5223–5226. In this paper it has been shown that a highly
enantioselective resolution of the alcohol 2 was possible only using special
vinyl esters such as vinyl 3-(p-tolyl or 2-naphthyl)propanoates.
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