Enantioselective transesterification catalysis by nanosized serine protease subtilisin Carlsberg particles in tetrahydrofuran

Article abstract of DOI:10.1016/j.tet.2010.01.053

Enzyme catalysis in organic solvents is a powerful tool for stereo-selective synthesis but the enantioselectivity is still hard to predict. To overcome this obstacle, we employed a nanoparticulate formulation of subtilisin Carlsberg (SC) and designed a series of 14 structurally related racemic alcohols. They were employed in the model transesterification reaction with vinyl butyrate and the enantioselectivities were determined. In general, short alcohol side chains led to low enantioselectivties, while larger and bulky side chains caused better discrimination of the enantiomers by the enzyme. With several bulky substrates high enantioselectivities with E>100 were obtained. Computational modeling highlighted that key to high enantioselectivity is the discrimination of the R and S substrates by the sole hydrophobic binding pocket based on their size and bulkiness. While bulky S enantiomer side chains could be accommodated within the binding pocket, bulky R enantiomer side chains could not. However, when also the S enantiomer side chain becomes too large and does not fit into the binding pocket anymore, enantioselectivity accordingly drops.

Full text of DOI:10.1016/j.tet.2010.01.053

Tetrahedron 66 (2010) 2175–2180
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Enantioselective transesterification catalysis by nanosized serine protease
subtilisin Carlsberg particles in tetrahydrofuran
,
Betzaida Castillo a, Yamixa Delgado a, Gabriel Barletta b, Kai Griebenow a
*
a Department of Chemistry, University of Puerto Rico, R´ıo Piedras Campus, PO Box 23346, San Juan, Puerto Rico 00931-3346
b Department of Chemistry, University of Puerto Rico, College at Humacao, CUH Station, Humacao, Puerto Rico 00791
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 October 2009
Received in revised form 12 January 2010
Accepted 13 January 2010
Available online 25 January 2010
Enzyme catalysis in organic solvents is a powerful tool for stereo-selective synthesis but the enantio-
selectivity is still hard to predict. To overcome this obstacle, we employed a nanoparticulate formulation
of subtilisin Carlsberg (SC) and designed a series of 14 structurally related racemic alcohols. They were
employed in the model transesterification reaction with vinyl butyrate and the enantioselectivities were
determined. In general, short alcohol side chains led to low enantioselectivties, while larger and bulky
side chains caused better discrimination of the enantiomers by the enzyme. With several bulky sub-
strates high enantioselectivities with E>100 were obtained. Computational modeling highlighted that
key to high enantioselectivity is the discrimination of the R and S substrates by the sole hydrophobic
binding pocket based on their size and bulkiness. While bulky S enantiomer side chains could be ac-
commodated within the binding pocket, bulky R enantiomer side chains could not. However, when also
the S enantiomer side chain becomes too large and does not fit into the binding pocket anymore,
enantioselectivity accordingly drops.
Keywords:
Enantioselective catalysis
Non-aqueous enzymology
Enzyme
Secondary alcohol
Serine protease
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
changes.10 Generally, when the lyophilized enzyme catalysts are
suspended in an organic solvent, the enzyme is locked in a non-
The production of single enantiomers of chiral intermediates
has become increasingly important in the pharmaceutical in-
dustry.1 During the last few years, the synthesis of bio-relevant
compounds and their precursors using enzymes in aqueous and
non-aqueous media has been on the rise.2 One of the most
promising properties of enzymes is that they can be extremely
selective due to their three-dimensional substrate binding site and
active site structure. This selectivity can be utilized to introduce
chirality into organic molecules, resolve racemic mixtures, and
functionalize molecules regioselectively.2b,3 The usefulness of
enzymatic catalysis in organic solvents for producing chiral pre-
cursors of biologically relevant compounds, such as, anti-tuber-
native conformation resulting in a less active biocatalyst10c and
likely a less selective one as well.
Consequently, the development of formulations to improve
enzymes for use as catalysts in organic solvents has been attempted
frequently.2a,10c,11 Cyclodextrins are one of the most successful
groups of additives that activate enzymes in neat organic sol-
vents.11g Griebenow et al. reported that methyl-
(M
berg in organic solvents.11a The results suggest that M
b
-cyclodextrin
CD) dramatically activates the serine protease subtilisin Carls-
CD activates
b
b
this enzyme by two mechanisms: it acts as a molecular lubricant
enhancing enzyme dynamics and it reduces protein structural
perturbations during lyophilization.11a–c
MbCD also improved
cular drugs,4
b
-adrenergic blocking agents,5 anti epileptic agents,6
enantioselectivity in the model reaction of sec-phenethyl alcohol
potential anti-neoplastic agents,7 and drugs for the treatment of
typhoid fever and other infectious diseases,8 is well recognized.
However, the reduced activity and the lack of predictability of the
selectivity in organic solvents are still major drawbacks.9
and vinyl butyrate.11a
Enzyme enantioselectivity in organic solvents has been scruti-
nized many times in the literature, but fundamental problems and
arguments still remain. For example, the relationship between
enzyme flexibility (structural dynamics) and enantioselectivity is
being debated.12 Most models explaining differential enantiose-
lectivity in various solvents are typically based on differential
binding of R- and S-enantiomer substrates to the respective binding
pockets.12,13 It is, however, remarkable how much higher the
enantioselectivity of enzymes typically is under natural con-
ditions.12a One explanation for this is that structural and/or
One of the reasons for the lack of predictability of enantiose-
lectivity might be the fact that the necessary enzyme dehydration
step, frequently performed by lyophilization, causes structural
* Corresponding author. Tel.: þ787 764 0000x4781; fax: þ787 756 7717.
E-mail address: kai.griebenow@gmail.com (K. Griebenow).
0040-4020/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.01.053

2176
B. Castillo et al. / Tetrahedron 66 (2010) 2175–2180
Table 1
dynamical changes occurring upon removal of enzymes from
aqueous buffer and introduction into an organic solvent cause di-
minished enantioselectivity. There is ample evidence that enzyme
structural preservation during this process leads to improved
enantioselectivity.11a–c,14 We therefore argue that employing
a structurally improved formulation of an enzyme should result in
better experimental data on enantioselectivity when employing
various racemic substrates. Furthermore, it should be possible to
explain these data using computational modeling since the active
site structure should be more similar to the one obtained by X-ray
crystallography. Lastly, to remove as much experimental un-
certainty as possible from the data, such an approach should be
performed in one solvent only using one carefully selected enzyme
formulation.
Initial rates and enantioselectivities for the product formation in the trans-
esterification reaction of sec-alcohols with vinyl butyrate for different preparations
of subtilisin C
Enantioselectivityc,d
a,b,c
Substrates
Enzyme preparation
VS
1
14
93
126
Lyophilized M
Lyophilized M
b
CD
CD
OH
A
5
59
93
141
OH
b
B
C
7
228
82
104
OH
Lyophilized M
Lyophilized M
b
CD
CD
In this study, we selected subtilisin Carlsberg (SC) co-lyophilized
with Mb
CD as the model system based on our past experiences.11a–c
The particle size of the powder after suspension in organic solvents
is 110ꢀ20 nm and consequently there are no diffusional limitations
even at accelerated reaction conditions.15 A series of 14 racemic
secondary alcohols was employed as substrates in the trans-
esterification model reaction with vinyl butyrate to explore the
relationship between substrate structure and enantiomeric excess
in detail. Finally, computational modeling was carried out to ex-
plain the data obtained. As the result of the investigation, we are
able to present a consistent model of the enantioselectivity of the
protease in the organic solvent THF.
1
32
148
240
OH
b
D
a
Initial rates for the S enantiomer in nmol mgꢁ1 minꢁ1
Enzyme concentration 1 mg/mL in all experiments.
Experiments were performed in duplicate. Results are the average of both trials.
Measured by the ratio of [kcat/KM]S/[kcat/KM]R¼VS[R]/VR[S].
.
b
c
d
the substituents at the stereocenter (substrates 1–5, Table 2). In-
terestingly, the enzyme enantioselectivity increased with in-
creasing length of the linear carbon chain from 3 to 67 (Table 2).
A linear correlation was observed between the enzyme enan-
tioselectivity and the length of the linear aliphatic chain (Fig. 2).
Each methylene group increased the enzyme enantioselectivity by
about 17 units. The second group of secondary alcohols studied
differs from the first one in that a methyl group was added and
moved along the aliphatic chain from the beta to the epsilon car-
bons (substrates 6–9, Table 2). As with the previous group, we also
observed in this case a linear increase in enantioselectivity with an
increasing number of methylene groups in the aliphatic chain.
However, maximum enantioselectivity is reached with 5-methyl-2-
hexanol (substrate 8), decreasing sharply with the next substrate in
which an additional methylene is added and the methyl group
placed on the epsilon carbon (6-methyl-2-heptanol, substrate 9).
2. Results and discussion
Enzymes rarely have the combined properties necessary for
industrial chemical production of chiral compounds, such as, high
activity and selectivity toward non-natural substrates. They are
usually 102 to 106 times less active in organic solvents than in
water9 and show drastically reduced enantioselectivity.12a In this
study, SC was lyophilized from an aqueous buffer (pH 7.8) with or
without MbCD, which is a reported strategy designed to ameliorate
lyophilization- and organic solvent-induced partial denaturation or
structural changes.11a–c The preparations were suspended in THF
and the kinetics of the product formation in the transesterification
between different sec-alcohols and vinyl butyrate was followed by
gas chromatography (Fig. 1).
O
R
2
O
R
2
Enzyme
+
+
Acetaldehyde
O
*
Organic solvent
45°C
OH
R
1
*
O
R
1
140 mM
(a)
200 mM
(b)
Figure 1. Transesterification reaction between vinyl butyrate and the racemic secondary alcohols catalyzed by subtilisin Carlsberg.
First, in order to establish feasibility of our approach using the
CD containing subtilisin formulation, subtilisin prepared with-
out and with M CD was employed in four model reactions
employing racemic secondary alcohols and initial rates and enan-
tioselectivities were determined. In accordance with previous
studies using other substrates,11a–c,f our results show a 12- to 33-
fold increase in the SC activity as the result of co-lyophilization with
MbCD (see Table 1). In addition, enantioselectivity was improved
using this preparation for all substrates initially tested.
We next proceeded to employ a series of 14 structurally differ-
ent secondary alcohols in the model transesterification reaction
using the SC-MbCD formulation solely (Table 2). To better un-
The enzyme enantioselectivity toward the first three alcohol sub-
Mb
strates in this group increases about 65 units from one substrate to
the next, more sharply than what was observed with the previous
set of substrates discussed. This suggests that the added methyl
group imposes steric constrains, which averts the R enantiomer
from competing for the active site.
The third group of substrates was constructed by varying sub-
stituents at the gamma carbon (Table 2). In general, the enantio-
selectivity increased with increasing size and thus steric effect of
the substituent at the gamma carbon. For example, adding an ad-
ditional methyl group to the gamma carbon (as in 4,4-dimethyl-2-
pentanol, substrate 11) increases the enzyme enantioselectivity
substantially as compared to substrate 7 (4-methyl-2-pentanol).
The alcohol substrate for which the enzyme was found to be most
enantioselective was 4-methyl-4-phenyl-2-pentanol (substrate 12,
E¼240), in which the steric effect at the gamma carbon is increased
b
derstand the stereo and steric preferences of the enzyme, we
employed four groups of secondary alcohols in which one param-
eter was changed systematically. The first group was chosen to
systematically vary the length of the linear aliphatic chain of one of

B. Castillo et al. / Tetrahedron 66 (2010) 2175–2180
2177
Table 2
Initial rates and enantioselectivities for the product formation in the trans-
esterification reaction of different sec-alcohols with vinyl butyrate for subtilisin
Carlsberg co-lyophilized with M
bCD
Eb
Substrates
VS VR
Eb
a
a
a
a
Substrates
First group
VS VR
177 59
246 12
Second group
3
255 28
9
OH
OH
1
6
20
139 1.9 73
OH
OH
2
7
102 2.3
44
OH
59 0.4 141
OH
3
Figure 2. Effect of the length of the linear aliphatic chain of one of the substituents at
the stereocenter of the substrates 1–5 on enzyme enantioselectivity.
8
99 1.7
58
67
209 2.6 80
OH
OH
4
stereocenter caused higher enantioselectivity (Fig. 2). Second,
adding more bulky substituents increased the enantioselectivity of
the enzyme. However, in this case, enantioselectivity enhancement
depended on the distance of the bulky substituent from the
stereocenter.
9
OH 183 2.7
5
Third group
Fourth group
To understand in more detail the experimental results, molec-
ular modeling studies were conducted. This model included the
essential parts of the enzyme highlighted in the X-ray structure
shown in Figure 3, namely the active site residues, the residues
involved in the oxyanion hole, and residues contributing to the
hydrophobic binding pocket. All reactions discussed in the fol-
lowing were modeled as intermediates, which are structurally
similar to the transition state. All data obtained are available in
Supplementary data, but here we will showcase the most relevant
cases.
139 1.9
73
74 1.4 51
OH
OH
7
13
14
OH
228 2.2 104
49 24.5
2
OH
10
14 0.1 126c
OH
11
32 0.1 240c
OH
12
a
b
c
Initial rates for the S- and R-enantiomer in nmol mgꢁ1 minꢁ1
Measured by the ratio of [kcat/KM]S/[kcat/KM]R¼VS[R]/VR[S].
.
Enantioselectivity could not be determined accurately because of the very low
value of VR.
by the aromatic group. The latter is a derivative of 2-pentanol,
which is a key chiral intermediate required for synthesis of anti-
Alzheimer drugs that inhibit
b-amyloid peptide release and/or its
synthesis.16
Figure 3. Scheme of the structure of subtilisin Carlsberg created using the Protein Data
Bank file 1BFU. The active site residues (catalytic triad) are shown in blue, residues
contributing to the hydrophobic binding pocket in red.
Two additional secondary alcohols were tested: 1-phenyl-1-
propanol and 1-p-tolyl-1-propanol (substrates 13 and 14), and the
enzyme enantioselectivity was found to be lower than for most of
the other substrates tested.
Finally, we investigated whether the enantioselectivity and ac-
tivity changes were related. No correlation was evident when
plotting the initial rate for the S enantiomer (VS) versus enantio-
selectivity (R<0.5, data not shown). These results contrast the data
reported by Broos et al. where a correlation between enzyme
enantioselectivity and initial rates was found.12c
Computational modeling was able to explain the dependency
of the enantioselectivity on the length of the linear aliphatic chain
of one of the substituents at the stereocenter (1–5). For the
smallest substrate, 2-butanol, it was found that both, R and S
enantiomer fit comfortably in the binding pocket (Fig. 4). Con-
sequently, the discrimination of the enantiomers by the enzyme is
inefficient and the enantioselectivity is very low (Table 2). In-
creasing the length of the aliphatic chain leads to improved dis-
crimination of the enantiomers by the enzyme. This in particular
Our data show some remarkable trends. First, increasing the
length of the linear aliphatic chain of one of the substituents at the

2178
B. Castillo et al. / Tetrahedron 66 (2010) 2175–2180
Figure 4. Results of the computational modeling of the tetrahedral intermediates of (R)- (A) and (S)-2-butanol (B). It is apparent that both substrate side chains comfortably fit into
the substrate binding pocket (residues shown in green).
becomes apparent when investigating 2-heptanol and 2-octanol
(Fig. 5) where the S enantiomer side chain comfortably fits in the
binding pocket, while the R enantiomer simply does not.
anymore in the binding pocket and in consequence the enantio-
selectivity decreases.
Increasing the bulkiness of the side chain leads to even better
discrimination of the enantiomers by the enzyme (third group,
Table 2). For all the S enantiomers in this group it was found that
the side chain fitted well within the binding pocket, even for 12
(Fig. 6). In contrast, for all R enantiomers the side chains did not fit.
With increasing size of the side chain in this group and thus
increasing interactions between S enantiomer and binding pocket,
discrimination by the enzyme improved and enantioselectivity as
well.
Similar observations were made with the more bulky substrates
of the second group (see Supplementary data for details). For the
smallest substrate 6 the side chains of both enantiomers fit com-
fortably in the binding pocket. For 7 this is already not the
case anymore, the R enantiomer side chain simply does not fit in
the binding pocket anymore resulting in good discrimination of the
enantiomers by the enzyme. This trend continues in 8, but for 9 the
bulky side chain of now even the S enantiomer does not fit well
Figure 5. Results of the computational modeling of the tetrahedral intermediates of (R)- (A) and (S)-2-octanol (B). It is apparent that only the S enantiomer side chain comfortably
fits into the substrate binding pocket (residues shown in green). The R enantiomer side chain does not fit into the binding pocket.
Figure 6. Results of the computational modeling of the tetrahedral intermediates of (R)- (A) and (S)-4-methyl-4-phenyl-2-pentanol (B). It is apparent that only the S enantiomer
side chain comfortably fits into the substrate binding pocket (residues shown in green). The R enantiomer side chain does not fit into the binding pocket and in particular the
aromatic ring has no interactions with the binding pocket.

B. Castillo et al. / Tetrahedron 66 (2010) 2175–2180
2179
3. Conclusions
pure enantiomers (usually the ‘R’) synthesized from the corre-
sponding alcohol enantiomers.
In this study we employed an enhanced SC formulation to map
the active site with respect to enantioselectivity. The empirical
approach employed in this study, using a series of 14 substrates
with different structures was very useful in this context and the
active site of SC was systematically mapped for the first time. It was
demonstrated that the substrate structure is an important factor
that contributes to the enzyme enantioselectivity. Increasing the
size of substrates (in particular increasing the length of a linear
aliphatic chain or adding bulky groups with an optimum distance
from the stereocenter) caused SC to better discriminate between
the enantiomers. Computational modeling studies were conducted
to analyze in detail the structural basis for the experimental results.
These studies highlight that the improved discrimination of R and S
enantiomer is in fact based on the length and bulkiness of the side
chain. Furthermore, the experimental and computational results
agree with empirical rules developed.13d,e The 14 alcohols here
reported should serve as a guide for predicting the enantiose-
lectivity of SC toward similar substrates.
4.4. Synthesis of the ester products
Ester products were used to calibrate the GC instruments and
were prepared as previously described with some modifications.16b
To a solution of (R,S)-alcohol (0.01835 mol) in pyridine (5 mL),
butyric anhydride (8.65 mL) was added and the mixture kept at
room temperature for 16 h. The mixture was poured in ice cold
water and extracted with methyl tertiary butyl ether (MTBE,
3ꢃ20 mL). The MTBE extract was washed with water (2ꢃ10 mL),
1 N HCl (2ꢃ10 mL), and finally with water (3ꢃ10 mL), dried over
Na2SO4, filtered, and the solvent was removed on a water bath
maintained at 75–80 ꢂC. The product was purified by column
chromatography (silica gel 200–400 mesh, usually using 5:1 hex-
ane/ethyl acetate). After the solvent was removed, the ester prod-
ucts were characterized by NMR.
4.4.1. (R,S)-2-Pentyl butyrate (2b). 1H NMR (CDCl3/TMS, 400 MHz),
d
0.93–1.05 (m, 6H), 1.24 (d, J¼6.4 Hz, 3H), 1.28–1.52 (m, 4H), 1.61–
4. Experimental
4.1. Materials
1.77 (m, 2H), 2.29 (t, 2H), 4.96 (m, 1H).
4.4.2. (R,S)-4-Methyl-2-pentyl butyrate (7b). 1H NMR (CDCl3/TMS,
400 MHz),
d
0.88–1.06 (m, 6H),1.25 (d, J¼5.20 Hz, 2H),1.28–1.37 (m,
Subtilisin Carlsberg (SC), M
bCD, 2-butanol (1), 2-pentanol (2), 2-
3H), 1.61 (s, 3H), 1.64–1.78 (m, 2H), 1.90 (m, 1H), 2.30 (t, 1H), 2.49 (t,
1H), 3.79 (t, 1H).
hexanol (3), 2-heptanol (4), 2-octanol (5), 3-methyl-2-butanol (6),
4-methyl-2-pentanol (7), 5-methyl-2-hexanol (8), 6-methyl-2-
heptanol (9), 4-phenyl-2-butanol (10), 4,4-dimethyl-2-pentanol
(11), 4-methyl-4-phenyl-2-pentanol (12), 1-phenyl-1-propanol
(13), and 1-p-tolyl-1-propanol (14) were from Sigma–Aldrich (St.
Louis, MO). Anhydrous THF was purchased from Aldrich (water
content below 0.005%). Prior to use it was stored over activated
molecular sieves (3 Å). Vinyl butyrate was from TCI America
(Portland, OR).
4.4.3. (R,S)-5-Methyl-2-hexyl butyrate (8b). 1H NMR (CDCl3/TMS,
400 MHz),
d
0.92 (dd, J¼6.8 Hz, 6H), 0.99 (t, 3H), 1.18–1.30 (m, 2H),
1.24 (d, J¼6.0 Hz, 3H), 1.48–1.65 (m, 3H), 1.67 (q, 2H), 2.30 (t, 2H),
4.91–4.95 (m, 1H).
4.4.4. (R,S)-6-Methyl-2-heptyl butyrate (9b). 1H NMR (CDCl3/TMS,
400 MHz),
d
0.88 (d, J¼6.4 Hz, 6H), 0.96 (t, 3H), 1.15–1.22 (m, 2H),
1.2 (d, J¼6.4 Hz, 3H), 1.26–1.36 (m, 2H), 1.42–1.59 (m, 3H), 1.66 (q,
4.2. Enzyme preparation
2H), 2.26 (t, 2H), 4.92 (m, 1H).
SC powder was obtained by lyophilization of a 5 mg/ml sub-
tilisin solution in 20 mM phosphate buffer at pH 7.8 for 24 h.11a–c SC
4.4.5. (R,S)-4-Phenyl-2-butyl butyrate (10b). 1H NMR (CDCl3/TMS,
400 MHz),
d
1.03 (t, 3H), 1.31 (d, J¼6.0 Hz, 3H), 1.69–1.78 (m, 2H),
co-lyophilized with M
cept that cyclodextrin was co-dissolved with the enzyme at a 6:1 g/
g ratio of M CD-to-enzyme prior to lyophilization.
b
CD was prepared in the same manner ex-
1.83–1.91 (m, 1H), 1.96–2.03 (m, 1H), 2.33 (t, 2H), 2.64–2.77 (m, 2H),
5.00–5.05 (m, 1H), 7.22–7.26 (m, 3H), 7.3–7.36 (m, 2H).
b
4.4.6. (R,S)-4-Methyl-4-phenyl-2-pentyl butyrate (12b). 1H NMR
4.3. Kinetic measurements
(CDCl3/TMS, 400 MHz),
3H), 1.40 (s, 3H), 1.49–1.56 (m, 2H), 1.73–2.03 (m, 4H), 4.95–4.99 (m,
1H), 7.19–7.23 (m, 1H), 7.32–7.38 (m, 4H).
d
0.92 (t, 3H), 1.11 (d, J¼6.0 Hz, 3H), 1.39 (s,
Product formation was followed by gas chromatography (GC).
The GC instruments (Varian 3350, HP 6850, and HP 6890, with
Chirasil CB columns, FID detectors, He carrier gas) were cali-
brated with the chiral ester products. The enzyme powder (5 mg
for the lyophilized and 1 mg for the co-lyophilized preparations)
was placed in a 2-mL screw-cap scintillation vial fitted with
a mini-inert cap and placed in a vacuum desiccator for 4 h. The
organic solvent (1.0 mL) and the substrates (140 mM of the al-
cohol substrate and 200 mM vinyl butyrate) were then added
under nitrogen to initiate the reaction. The vial was sealed and
subjected to careful sonication using a sonication bath for 10 s to
homogenize the suspension. The mixture was then placed in
a controlled temperature shaker and agitated vigorously at
4.4.7. (R,S)-1-Phenyl-1-propyl butyrate (13b). 1H NMR (CDCl3/TMS,
400 MHz),
d 0.93–1.01 (m, 6H), 1.68–1.77 (m, 2H), 1.84–2.02 (m,
2H), 2.38 (td, 2H), 5.75 (t, 1H), 7.31–7.39 (m, 5H).
4.5. Computational methods
The computer modeling results were obtained using the pro-
gram MacroModel (Schro¨dinger Inc.) and the interface Maestro,17
frequently employed to model transition states of enzymes.18 All
calculations were conducted using the OPLS-AA force field. All
calculations were performed using the dielectric constant of THF
(7.6) as constant. The enzyme crystal structure used was PDB entry
1BFU.19 Transition states were modeled as the tetrahedral in-
termediate because of their structural similarity. The initial models
of the tetrahedral intermediates were sketched in the Maestro
program with the carbonyl oxygen oriented toward the oxyanion
hole. The system was divided in two subsets: moving residues and
frozen residues. The potential binding modes of the chiral products
300 rpm and 45 ꢂC. Periodically, 0.5
mL of the reacting solution
was withdrawn and analyzed by chiral-GC. Enzyme enantiose-
lectivity was determined by measuring the initial rates from plots
of the product concentration versus time for both enantiomers.
The enzyme enantioselectivity for either substrate is equal to the
ratio [kcat/KM]S/[kcat/KM]R¼VS[R]/VR[S].11a The retention times of
the ‘R’ and ‘S’ products were obtained by using samples of the

2180
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I.; Gotor, V. Chem. Rev. 2005, 105, 313–354.
4. Francalanci, F.; Cesti, P.; Cabri, W.; Bianchi, D.; Martinegro, T.; Foa, M. J. Org.
Chem. 1987, 52, 5079–5082.
5. Wang, Y. F.; Shen, S. T.; Liu, K. K. C.; Wong, C. H. Tetrahedron Lett. 1989, 30,
1917–1920.
6. Murata, M.; Terao, Y.; Achiwa, K.; Nishio, T.; Seto, K. Chem. Pharm. Bull. 1989, 37,
2670–2673.
were first determined by using the conformational search Monte
Carlo Multiple Minimum (MCMM) method with 7 moving residues
(Ser221, His64, Asp32, Ser125, Asn155, Gly127, Leu126). The lowest en-
ergy structures that were selected had hydrogen bonds between
the negatively charged oxygen of Ser221 and the backbone NH
group of Ser221 and with the side chain NH2 group of Asn155. The
moving residues subset later was expand to 10 Å away from the
Ser221, but the energy minimized structures obtained did not show
significant changes.
The parameters were set according to the following: the energy
window for saving structures was 50 kJ molꢁ1; the maximum it-
erations for minimizing the molecules were 5000 using the Polak–
Ribiere Conjugate Gradient (PRCG) method; the convergence
threshold was 0.001; and the other parameters were adopted de-
fault values.
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Acknowledgements
This publication was made possible by grant number SC1
GM086240 (PI K.G.) from the National Institute for General Medical
Sciences (NIGMS) at the National Institutes of Health (NIH) through
the Support of Competitive Research (SCORE) Program. B.C. was
recipient of an NIH-RISE Program doctoral fellowship (2R25
GM061151, PI Morales). Its contents are solely the responsibility of
the authors and do not necessarily represent the official views of
NIGMS.
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Supplementary data
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