Kinetic resolution of (R,S)-pyrazolides containing substituents in the leaving pyrazole for increased lipase enantioselectivity

Article abstract of DOI:10.1016/j.molcatb.2010.04.003

With hydrolysis of (R,S)-azolides in water-saturated methyl tert-butyl ether (MTBE) via Candida antarctica lipase B (CALB) as the model system, (R,S)-pyrazolides containing a leaving 3-, 4- or 3,4-substituted-pyrazole moiety are selected as the best substrates for preparing various optically pure carboxylic acids containing an α-chiral center. Great improvements of enzyme activity for the (R)-enantiomers with excellent enantioselectivity (V_R/V_S > 100) are obtainable, if (R,S)-pyrazolides containing a leaving 3- or 3,4-substituted-pyrazole moiety are employed for the hydrolysis or alcoholysis by methanol in anhydrous MTBE. A detailed kinetic analysis for (R,S)-N-2-phenylpropionylpyrazoles indicates that a bulky 3-substituent such as 3-(3-bromophenyl) or 3-(2-pyridyl) in the leaving pyrazole moiety has profound effects on decreasing the nucleophilic attack and proton transfer of catalytic serine for the slow-reacting enantiomer in anhydrous MTBE, as well as that and substrate affinity for both enantiomers in water-saturated MTBE. The resolution platform is also successfully applied to the hydrolysis of (R,S)-pyrazolides in water-saturated cyclohexane via Candida rugosa lipase (Lipase MY) having opposite enantioselectivity to CALB.

Full text of DOI:10.1016/j.molcatb.2010.04.003

Journal of Molecular Catalysis B: Enzymatic 66 (2010) 113–119
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Kinetic resolution of (R,S)-pyrazolides containing substituents in the leaving
pyrazole for increased lipase enantioselectivity
Pei-Yun Wang, Chia-Hui Wu, Jyun-Fen Ciou, An-Chi Wu, Shau-Wei Tsai∗
Institute of Biochemical and Biomedical Engineering, Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-Shan Tao-Yuan 33302, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
With hydrolysis of (R,S)-azolides in water-saturated methyl tert-butyl ether (MTBE) via Candida antarctica
lipase B (CALB) as the model system, (R,S)-pyrazolides containing a leaving 3-, 4- or 3,4-substituted-
pyrazole moiety are selected as the best substrates for preparing various optically pure carboxylic acids
containing an ␣-chiral center. Great improvements of enzyme activity for the (R)-enantiomers with
excellent enantioselectivity (VR/VS > 100) are obtainable, if (R,S)-pyrazolides containing a leaving 3- or
3,4-substituted-pyrazole moiety are employed for the hydrolysis or alcoholysis by methanol in anhy-
drous MTBE. A detailed kinetic analysis for (R,S)-N-2-phenylpropionylpyrazoles indicates that a bulky
3-substituent such as 3-(3-bromophenyl) or 3-(2-pyridyl) in the leaving pyrazole moiety has profound
effects on decreasing the nucleophilic attack and proton transfer of catalytic serine for the slow-reacting
enantiomer in anhydrous MTBE, as well as that and substrate affinity for both enantiomers in water-
saturated MTBE. The resolution platform is also successfully applied to the hydrolysis of (R,S)-pyrazolides
in water-saturated cyclohexane via Candida rugosa lipase (Lipase MY) having opposite enantioselectivity
to CALB.
Received 11 March 2010
Received in revised form 6 April 2010
Accepted 7 April 2010
Available online 13 April 2010
Keywords:
(R,S)-Pyrazolides
Lipases
Hydrolysis
Kinetic analysis
Substituent effects
Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved.
1. Introduction
attack, especially when accepting an proton from the imidazolium
of catalytic histidine and increasing the number of nitrogens of the
Lipases are widely employed as versatile biocatalysts for prepar-
ing a variety of chiral alcohols, amines, carboxylic acids, or their
derivatives for synthesizing pharmaceuticals and fine chemicals
[1,2]. In comparison with the resolution of secondary alcohols
and amines, the enzyme enantioselectivity toward carboxylic acids
is usually low to modest [3–6]. Therefore, various approaches of
using substrate engineering [7–10], medium engineering [11,12],
enzyme engineering [5,13–15], or their combinations have been
proposed for the fine-tuning of enzyme active-site structure to
enhance the enantiomer discrimination toward targeted race-
mates. Recently, anewresolutionprocessofusing (R,S)-azolides, i.e.
(R,S)-N-acylazoles, but not their corresponding ester, thioester, or
normal amide analogs as the substrate via lipase-catalyzed hydrol-
ysis or alcoholysis in organic solvents was reported [16,17]. The
results indicate that by altering the simple leaving azole moiety
from imidazole, pyrazole to 1,2,4-triazole, the enzyme activity and
enantioselectivity can be improved. One possible explanation is due
to the unique azolide structure that contains an unshared electron
pairs on the acyl-substituted nitrogen N(1), making the carbonyl
carbon atom more electrophilic and susceptible to nucleophilic
leaving azole [18–21].
If the rationale of obtaining the improved enzyme performance
is valid for the (R,S)-azolides containing a simple leaving azole moi-
ety, the question is then how one can further increase the substrate
affinity to the enzyme to enhance the reactivity, stabilize the tran-
sition state for formation of tetrahedral adduct of the fast-reacting
enantiomer, destabilize that of the slow-reacting enantiomer, or
use their combinations for enhancing the enantioselectivity. Appar-
ently, the substrate engineering approach of using (R,S)-azolides
with a leaving azole moiety containing substituents is a possible
and easy way to obtain the answer. This implies that the substituent
may exert non-covalent bonding to the amino acid residues in the
active site, leading to good substrate affinity, enantiomer discrim-
ination, and/or higher electrophilicity of the carbonyl carbon atom
for better nucleophilic attack. It is therefore aimed to investigate
the substituent effects on affecting the enzyme activity and enan-
tioselectivity by using (R,S)-azolides containing substituents in the
leaving moiety as the substrate in organic solvents.
Although N-acetylimidazole is 22 times more reactive than
the corresponding N-acetylpyrazole in water [20], this is not the
case for lipase-catalyzed hydrolysis or alcoholysis of N-acylazoles
[16,17]. By considering the variety of substituent allocation in the
leaving azole moiety, background stability, enzyme activity and
enantioselectivity, we mainly focus on the kinetic resolution of
∗
Corresponding author. Tel.: +886 3 2118800x3415; fax: +886 3 2118668.
E-mail address: tsai@mail.cgu.edu.tw (S.-W. Tsai).
1381-1177/$ – see front matter. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2010.04.003

114
P.-Y. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 113–119
1.25 ml of a benzene solution containing 3.25 mmol thionyl chlo-
Nomenclature
ride were added dropwise a mixture consisting of 6.0 ml benzene,
2.50 mmol 4-bromo-1H-pyrazole, 3.25 mmol racemic acid, and
10 mmol triethylamine. The resultant solution was stirred for 2 h at
room temperature. After being quenched in succession with 0.1 M
HCl solution (3 × 10 ml), 0.1 M NaOH solution (3 × 10 ml) and 0.1 M
NaCl solution (3 × 10 ml), the organic phase was separated, dried
over anhydrous MgSO4, filtered and concentrated under reduced
pressure, giving the desired (R,S)-N-acyl-4-bromopyrazoles (11, 18,
21, 23–25). With 4-bromo-1H-pyrazole replaced by another azole,
similar procedures were carried out to prepare other (R,S)-azolides
except for (R,S)-N-phenylpropionyl-3-aminopyrazole (3).
To 2.0 ml pyridine was added 1.0 mmol (R,S)-phenylpropionic
acid, 3.0 mmol triethylamine, and 1.0 mmol thionyl chloride with
stirring for 1 h at room temperature. The resultant mixture was then
added to 0.5 ml pyridine containing 1.0 mmol 3-aminopyrazole and
stirred for 2 h. After removing pyridine under reduced pressure,
the residue was dissolved in 20 ml benzene, washed in succession
with 0.1 M HCl solution (3 × 20 ml) and deionized water (20 ml).
The organic phase was separated, dried over anhydrous MgSO4, fil-
tered and concentrated under reduced pressure, giving the desired
substrate (3). All the synthesized substrates were confirmed from
the retention time in HPLC analysis and 1H NMR spectra recorded
at 400 MHz on Brucker AC-400 spectrometer in DMSO-d6 solution
with TMS as an internal standard.
ees
E
(Et)
k2i
enantiomeric excesses for the substrate
enantiomeric ratio, defined as k2RKmS/k2SKmR
enzyme concentration (mg/ml)
kinetic constants, i = R or S for (R)- or (S)-enantiomer
(mmol/g h)
Kmi
(Si)
(Si)0
Vi
,
kinetic constants, i = R or S for (R)- or (S)-enantiomer
(mM)
substrate concentration, i = R or S for (R)- or (S)-
enantiomer (mM)
initial concentration, i = R or S for (R)- or (S)-
enantiomer (mM)
initial rate, i = R or S for (R)- or (S)-enantiomer
(mM/h)
Xi, Xt
enantiomer conversion defined as [1 − (Si)/(Si)0],
i = R or S; racemate conversion defined as [XR + XS]/2
(R,S)-pyrazolides but not (R,S)-imidazolides and (R,S)-triazolides.
The CALB-catalyzed hydrolysis of (R,S)-N-2-phenylpropionylazoles
in anhydrous and water-saturated MTBE is first selected as the
model system for studying the substrate structure and water
content on the enzyme performance (Scheme 1). The kinetic con-
stants for different (R,S)-N-2-phenylpropionylpyrazoles are then
estimated and compared. Moreover, the resolution for other (R,S)-
pyrazolides via CALB and Lipase MY is addressed, showing the
promise of present resolution platform for preparing various (R)-
or (S)-carboxylic acids containing an ␣-chiral center.
2.3. Analysis
Hydrolysis or alcoholysis of (R,S)-azolides was monitored by
HPLC using a chiral column from Daicel (OD-H or OJ-H; Tokyo,
Japan) or Regis ((S,S)-whelk-01; Morton Grove, IL) that is capable
of separating the internal standard of benzene, 2-nitrotoluene, or
p-nitrophenol. UV detection at 220 or 270 nm was employed for
quantification at the room temperature. Detailed analytical condi-
tions are represented in Table S1 of the Supporting Information.
2. Materials and methods
2.1. Materials
Lipase MY from Candida rugosa and Novozym 435 from Candida
antartica lipase B were provided by Meito Sangyo (Tokyo, Japan)
and Novo Nordisk (Bagsvaerd, Denmark), respectively. Dimethyl
sulfoxide-d6 (DMSO-d6) containing 1% v/v tetramethylsilane (TMS)
for 1H NMR analysis was from Cambridge Isotope Laborato-
ries (Andover, MA). Other chemicals of analytical grade were
commercially available: 3,5-dimethyl-1H-pyrazole, 4-methyl-
1H-pyrazole and pyrazole from Acros (Geel, Belgium); (R,S)-
␣-bromophenylacetic acid, 1,1ꢀ-carbonylbis(2-methylimidazole)
and 4-bromo-1H-pyrazole from Aldrich (Milwaukee, WI); 3-
amino-1H-pyrazole, 3-(3-bromophenyl)-1H-pyraozle, 3-methyl-
1H-pyrazole, 3-(2-pyridyl)-1H-pyraozle, and 3-methyl-4-bromo-
1H-pyrazole from Alfa Aesar (Ward Hill, MA); 1H-benzotriazole,
N,Nꢀ-carbonyldi-1,2,4-triazole (CDT) and (R,S)-2-phenylpropionic
acid from Fluck (Buchs, Switzerland); 4-nitro-1H-pyrazole from
Matrix Scientific (Columbia, SC); (R,S)-2-ethyl-phenylacetic acid,
(R,S)-ketoprofen (i.e. (R,S)-2-(3-benzoylphenyl)-propionic acid),
(R,S)-␣-methoxyphenylacetic acid and (R,S)-naproxen (i.e. (R,S)-2-
(6-methoxy-2-naphthyl)-propionic acid) from TCI (Tokyo, Japan);
(R,S)-flurbiprofen (i.e. (R,S)-2-fluoro-␣-methyl-4-biphenylacetic
acid) from Sigma (St. Louis, MO); cyclohexane, isopropanol (IPA),
hexane, methanol and MTBE from Tedia (Fairfield, OH). Anhydrous
MTBE was prepared by adding calcium hydride from Riedel-de
Haen (Seelze, Germany) to MTBE for 24 h.
2.4. Effects of substrate structure
To 10 ml water-saturated MTBE containing 3 mM of different
(R,S)-azolide was added a specific amount of CALB for perform-
ing the hydrolytic resolution. The resultant solution was stirred
with a magnetic stirrer at 45 ◦C. Samples were removed from
the reaction medium at different time intervals for HPLC anal-
ysis, from which the time-course conversions XR and XS, initial
rates for both enantiomers VR and VS, racemate conversion Xt, and
enantiomeric excess for the substrate ees were determined. Simi-
lar experiments were performed in anhydrous MTBE via CALB, as
well as in water-saturated cyclohexane via Lipase MY having oppo-
site enantioselectivity to CALB and low tolerance to polar organic
solvents. The alcoholytic resolution was also carried out via CALB,
except that 100 mM methanol was further added to 10 ml anhy-
drous MTBE containing 3 mM (R,S)-azolide.
In order to compare the kinetic behaviors at 45 ◦C, the hydrolytic
reactions by varying the concentration of 7, 8, 10, and 11 in
anhydrous or water-saturated MTBE were carried out. Details for
estimating the kinetic constants KmS and k2S, KmR and k2R, and hence
E value (i.e. k2RKmS/k2SKmR) were described in the Supporting Infor-
mation.
3. Results and discussion
2.2. General procedures for substrates synthesis
3.1. Hydrolysis in water-saturated MTBE
To 5 ml benzene was added 1 mmol racemic acid and 1.5 mmol
CDT and the mixture was stirred at 55 ◦C for 2 h. The resultant mix-
ture was filtered and evaporated under reduced pressure, giving
the desired (R,S)-N-acyl-1,2,4-triazole (13, 16, 19, 22). Moreover to
Table 1 demonstrates effects of leaving azole moiety and acyl
part of (R,S)-azolides on changing the initial specific activities
VR/(Et) and VS/(Et), enantioselectivity in terms of VR/VS, racemate

P.-Y. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 113–119
115
Scheme 1. CALB-catalyzed hydrolysis or alcoholysis of (R,S)-azolides in organic solvents.
conversion Xt, and enantiomeric excess for the substrate ees at a
specified reaction time in water-saturated MTBE. In comparison
with 2, an introduction of 2-methyl substituent to the leaving imi-
dazole moiety of 1 mainly decreases the reactivity of fast-reacting
(R)-enantiomer and hence the enantioselectivity. By comparing
with 13, a benzene group fused into the leaving 1,2,3-triazole
moiety of 14 also greatly decreases the enzyme activity and enan-
tioselectivity. One may attribute the former to the high pKa of
2-methylimidazole on decreasing the electrophilicity of carbonyl
carbon atom for nucleophilic attack, and the latter to the steric
hindrance of leaving benzotriazole moiety for substrate affinity to
the active site. However for 6, 11, or 12 containing a 4-methyl, 4-
bromo, or 4-nitro substituent in the leaving pyrazole moiety, the
enantioselectivity increases with the penalty of slight decreasing of
VR/(Et) in comparison with 7. The strongest electro-withdrawing
ability of 4-nitro substituent of 12, giving the lowest pKa of the
resultant 4-nitropyrazole, should have benefits for the nucleophilic
attack of catalytic serine for formation of tetrahedral adduct and
then breakdown to release 4-nitropyrazole. Yet, only 3 times dif-
ference of VR/(Et) with 5.04 units pKa change of the leaving pyrazole
between (R)-6 and (R)-12 is shown, implying that there must exist
additional attractions or repulsions between the 4-substituent and
amino acid residues of the active site. These interactions may yield
minute distortion of the substrate orientation in the active site,
such that the distance between and carbonyl carbon atom and cat-
alytic serine increases to relax the electro-withdrawing ability of

116
P.-Y. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 113–119
Table 1
Effect of substrate structure on pKa of leaving azole, specific initial rates, VR/VS, Xt and ees for CALB-catalyzed hydrolysis in water-saturated MTBE.
Entry
pKa
VR/(Et) (mmol/h g)
VS/(Et) (mmol/h g)
VR/VS
(Et) (mg/ml)
Time (h)
Xt (%)
ees (%)
1
2
3
4
5
6
7
8
9
7.85a
7.00a
6.00b
4.06
3.77E−3
2.88E−2
6.57E−2
N
9.05E−2
5.28E−1
2.62
3.62E−3
1.32E−2
1.27E−2
5.56E−1
1.61
3.70E−3
4.76E−3
3.35E−4
N
1.0
6.1
196
N
161
111
81
291
264
482
156
243
95
1.6
N
188
342
9.9
7.4
247
23
30.0
20.0
20.0
60.0
20.0
20.0
2.0
40.0
60.0
40.0
12.0
20.0
2.0
20.0
40.0
10.0
20.0
20.0
2.0
20.0
20.0
2.0
20.0
4.3
5.0
118.0
4.0
1.0
29.9
51.8
51.4
N
1.0
58.8
100.0
N
3.27
3.04
5.62E−4
4.73E−3
3.23E−2
1.76E−5
5.00E−5
2.63E−5
3.55E−3
6.62E−3
4.58E−2
1.35E−5
N
2.25E−3
1.73E−5
1.41E−2
2.54E−1
9.75E−5
2.63E−3
9.05E−2
8.63E−5
8.25E−4
2.10E−4
51.5
50.5
53.5
51.1
51.8
50.7
50.8
50.1
55.6
9.5
48.5
51.5
51.8
50.5
68.3
50.8
50.3
64.4
54.7
50.5
57.5
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
1.7
2.52a
1.84b
1.43
1.0
48.0
18.0
24.0
2.0
0.3
0.7
72.0
147.5
2.0
48.0
0.8
1.3
9.5
12.0
3.0
24.0
2.0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
1.33b
0.63
−2.00
2.19a
1.60
4.38
2.10E−4
8.85E−4
4.25E−1
5.92E−3
1.39E−1
1.88
2.41E−2
6.04E−2
1.15
1.33b
2.19
94.2
100.0
100.0
68.3
100.0
100.0
78.9
100.0
100.0
100.0
100.0
1.33b
0.63
2.19
3.27
0.63
2.19
0.63
0.63
0.63
12
1.79E−2
2.15E−1
2.31E−2
207
260
110
40.0
20.0
40.0
25c
19.5
a
From Wang et al. [16].
b
Estimated by pKa = 2.289 − 5.642ꢀp (R2 = 0.988) correlated from known pKa and ꢀp of 5, 7, 11 and 12; Hammett para-substituent constants ꢀp for 3-amino, 3-(2-pyridyl),
and 3-(3-bromophenyl) as −0.66, 0.08 and 0.17, respectively, from Hansch et al. [22].
c
From Wu et al. [17]. Reaction conditions: 10 ml water-saturated MTBE containing 3 mM racemate at 45 ◦C and 400 rpm. Symbol E−1 as 10−1; notation N as too low to be
determined.
4-bromo substituent in (R)-11 and 4-nitro substituent in (R)-12,
and hence decreases the enzyme activity as elucidated from the
kinetic analysis.
tivity for each enantiomer. However no reactivity of 4 containing
a leaving 3,5-dimethylpyrazole moiety was detected at 118 h by
employing 60 mg/ml of CALB, indicating that the lipase did not
accommodate (R)- and (S)-azolides of 4 and 14 containing a 5-
substituent in the leaving azole moiety.
In order to demonstrate the merit of using (R,S)-azolides for
preparing various carboxylic acids containing an ␣-chiral center,
the ␣-methyl moiety of 1–14 was replaced by ␣-ethyl moi-
ety of 15 and 16. In comparison with 480-fold enhancement of
VR/(Et) with excellent enantioselectivity for 16 containing a leav-
ing 1,2,4-triazole moiety, very low reactivity of (R)-15 without
detecting (S)-15 reactivity at 147.5 h was found. Apparently, the
␣-ethyl moiety might impede the substrate affinity to the active
More improvements in enantioselectivity are perceived for 3,
5, 8, and 10 containing a 3-amino, 3-methyl, 3-(3-bromophenyl),
or 3-(2-pyridyl) substituent in the leaving pyrazole moiety. Yet,
no correlations between log(VR/(Et)) or log(VS/(Et)) and pKa of the
leaving 3-substitited-pyrazole were found, indicating that more
complicated interactions among the 3-substituent, amino acid
residues of the enzyme, and water adsorbed in the active site
should occur. In comparison with 5 and 11, the introduction of a
3-methyl-4-bromo substituent to the leaving pyrazole moiety of 9
can further enhance the enantioselectivity but decreases the reac-
Table 2
Effect of substrate structure on pKa of leaving azole, specific initial rates, VR/VS, Xt and ees for CALB-catalyzed hydrolysis in anhydrous MTBE.
Entry
pKa
VR/(Et) (mmol/h g)
VS/(Et) (mmol/h g)
VR/VS
(Et) (mg/ml)
Time (h)
Xt (%)
ees (%)
3
4
5
6
7
6.00
4.06
3.27
3.04
2.52
1.84
1.43
1.33
0.63
−2.00
2.19
1.60
1.33
2.19
1.33
0.63
2.19
3.27
2.19
0.63
2.51
1.92E−2
2.50E−5
1.91E−2
3.88E−2
1.82E−1
5.89E−3
7.73E−3
1.84E−2
7.91E−2
1.31E−1
7.23E−1
1.47E−4
1.10E−3
1.45E−2
4.43E−3
1.14E−1
1.61
130
3.8
259
52
2.0
60.0
2.0
6.0
2.0
2.0
2.0
2.0
2.0
6.0
2.0
20.0
4.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
0.9
118.0
1.0
0.7
5.0
1.3
5.0
2.0
0.8
0.7
0.2
72.0
2.0
0.8
1.0
0.8
0.2
3.5
6.0
23.2
53.8
54.4
54.2
50.6
52.1
52.5
53.4
63.5
58.0
34.2
50.6
50.7
52.5
55.0
51.4
50.3
58.7
50.2
100.0
14.4
9.50E−5
4.96
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
34.0
100.0
100.0
100.0
100.0
46.8
2.04
5.60
3.43
3.20
7.13
4.24
4.27
1.08E+1
1.08E−3
8.28E−1
3.91
2.83
5.63
7.05
1.09
6.06
3.70
30
8
9
582
414
387
53
32
14
7.3
752
269
638
49
4.4
322
19
10
11
12
13
14
15
16
17
18
19
20
22
24
3.38E−3
3.08E−1
2.95E−2
100.0
63.2
100.0
0.3
1.0
125
Reaction conditions: 10 ml anhydrous MTBE containing 3 mM racemate at 45 ◦C and 400 rpm. Symbol E−1 as 10−1
.

P.-Y. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 113–119
117
Table 3
site [23], when comparing VR/(Et) or VS/(Et) for 13 and 16, and
especially those for 10 and 15 containing a bulky 3-(2-pyridyl)
substituent in the leaving pyrazole moiety. When the ␣-methyl
moiety was changed to ␣-methoxy of 17–19 containing a leaving
1,2,4-triazole, 4-bromopyrazole, or 3-(2-pyridyl)pyrazole moiety,
similar enzyme performances as those for 10, 11, and 13 were
observed, except for giving very low enantioselectivity for 18 and
19. This really indicates the complicated interactions among the
substituent, ␣-methoxy moiety, and amino acid residues mainly
affecting VS/(Et) of slow-reacting substrates. If the ␣-methyl moiety
was further replaced by ␣-bromo of 20–22 containing a leaving 3-
methylpyrazole, 4-bromopyrazole, or 1,2,4-triazole moiety, only 20
gave excellent enantioselectivity. However, when employing (R,S)-
N-profenyl-4-bromopyrazoles of 23–25 as the substrate, increased
enantioselectivity with the penalty of slight deceasing of VR/(Et)
was perceived when comparing with the previous results of using
(R,S)-N-profenyl-1,2,4-triazoles as the substrate [16].
All the results in Table 1 demonstrate that a minute change of the
␣-substituent of acyl part or leaving pyrazole moiety may greatly
affect CALB activity and enantioselectivity. However, a guarantee of
giving excellent enantioselectivity may be made if (R,S)-pyrazolides
containing a leaving 3-, 4- or 3,4-substituted-pyrazole moiety are
selected as the substrate. This is especially true for 8, 10, and 17 con-
taining a bulky 3-(2-pyridyl) or 3-(3-bromophenyl) substituent. In
order to improve the reactivity of (R)-N-acylpyrazole containing a
leaving 3, 4- or 3,4-substituented-pyrazole, the hydrolytic resolu-
tion with water originally adsorbed on CALB as the acyl acceptor
was carried out in anhydrous MTBE.
Effects of leaving azole on kinetic constants and E values for CALB-catalyzed hydrol-
ysis in anhydrous and water-saturated MTBE.
Entry
13a
7
11
8
10
Anhydrous MTBE
k2R (mmol/h g)
KmR (mM)
k2R/KmR (l/h g)
k2S (mmol/h g)
KmS (mM)
2.01E+2
2.54E+1
7.91
2.07E+1
3.42E+1
6.05E−1
13.1
1.37E+2
2.28E+1
6.00
9.47E+1
1.62E+1
5.85
9.44E−1
1.06E+1
8.91E−2
65.6
4.82E+1
1.78E+1
2.71
6.40E−2
1.56E+1
4.10E−3
661
1.00E+2
1.72E+1
5.81
2.19E−1
1.41E+1
1.55E−2
375
3.11
2.16E+1
1.44E−1
41.7
k2S/KmS (l/h g)
E
Water-saturated MTBE
k2R (mmol/h g)
KmR (mM)
k2R/KmR (l/h g)
k2S (mmol/h g)
KmS (mM)
k2S/KmS (l/h g)
E
1.33E+3
4.59E+2
2.90
1.55E+1
5.31E+2
2.92E−2
99.3
1.14E+3
5.34E+2
2.13
1.25E+1
5.29E+2
2.36E−2
90.2
1.35E+2
4.17E+2
3.24E−1
9.44E−1
4.76E+2
1.98E−3
164
2.33E−3
5.54E−3
1.11E−5
1.58E−5
210
351
a
From Wang et al. [16]. Reaction conditions: 10 ml MTBE at 45 ◦C and 400 rpm.
Symbol E−1 as 10−1
.
which the kinetic constants represented in Table 3 are then esti-
mated and compared. In anhydrous MTBE, all Michaelis constants
KmR and KmS have the same order-of-magnitude. This is also per-
ceived for all kinetic constants k2R but not k2S, indicating that the
leaving 1,2,4-triazole or pyrazole moiety with or without contain-
ing a substituent has minor influences on varying the substrate
affinity and nucleophilic attack to the carbonyl carbon atom of
(R)-enantiomers but not their antipodes. Therefore, one obtains
the same order-of-magnitude of k2R/KmR but not k2S/KmS, lead-
ing to excellent enantioselectivity for 8 and 10, modest for 7 and
11, but low for 13. A detailed analysis of k2S/KmS gives the free
energy difference between the ground and transient states for
(S)-8 to be 9.4 kJ/mol higher than that for (S)-7. Similarly, only
5.9 kJ/mol difference between those of (S)-10 and (S)-7, as well
as 1.3 kJ/mol between those of (S)-11 and (S)-7, are estimated.
Therefore, more interactions between the 3-(3-bromophenyl) sub-
stituent and amino acid residues of the active site, modest for the
3-(2-pyridyl) substituent, and low for the 4-bromo substituent, on
impeding the nucleophilic attack and proton transfer for formation
of (S)-tetrahedral adduct can be deduced.
Table 3 also demonstrates nearly the same KmR and KmS for 7, 11
and 13 in water-saturated MTBE, which are an order-of-magnitude
higher than those in anhydrous MTBE. Very similar kinetic behav-
iors giving almost the same k2R/KmR or k2S/KmS and hence E values
for 7 and 13 are shown. In comparison with 7, about an order-
of-magnitude lower of k2R and k2R/KmR (or k2S and k2S/KmS), and
then higher E value (i.e. k2RKmS/k2SKmR), for 11 containing a leaving
4-bromopyrazole moiety are obtainable. This indicates that water
adsorbed in the active site has decreased the nucleophilic attack
and proton transfer of catalytic serine but not the substrate affinity,
and hence the reactivity of (R)- and (S)-11. For 8 and 10 containing
a bulky 3-substitutent in the leaving pyrazole moiety, liner rela-
tionships between the initial rates and substrate concentrations
are illustrated (Fig. S2). This indicates that the Michaelis con-
stants KmR and KmS are too large to be determined, giving a strong
support for the adsorbed water allocated in the active site near
the 3-substituent on impeding the substrate affinity. In compari-
son with 7, more than two order-of-magnitudes lower of k2R/KmR
and k2S/KmS but with excellent enantioselectivity for 8 and 10 are
shown, implying that the adsorbed water might also decrease the
nucleophilic attack and proton transfer of the catalytic serine.
It is reasonable to assume that KmR and KmS for all substrates in
Tables 1 and 2 are at least an order-of-magnitude higher than the
employed substrate concentration. As an approximation for esti-
3.2. Hydrolysis in anhydrous MTBE
In comparison with the reactivity of 13 in Table 1, about 2.5-
and 15.8-fold enhancements of VR/(Et) and VS/(Et), respectively,
are demonstrated, leading to lower enantioselectivity in anhy-
drous MTBE (Table 2). This indicates that water adsorbed in the
active site might affect the substrate affinity and/or nucleophilic
attack and proton transfer of catalytic serine for each enantiomer
[16]. The very low CALB activity and enantioselectivity of 4 and
14 (Tables 1 and 2) really reflects the difficult substrate affinity in
anhydrous or water-saturated MTBE. On the contrary, more than an
order-of-magnitude higher of VR/(Et) for (R)-6, (R)-11, and (R)-12 as
well as two order-of-magnitudes higher of VR/(Et) for (R)-5, (R)-8,
(R)-9, and (R)-10 are shown when comparing with those in Table 1.
This indicates that decreasing of water content in the reaction
medium hasprofoundeffectsonaffecting theenzymeperformance.
The high reactivity associated with excellent enantioselectivity for
3, 5, and 8–10 containing a leaving 3- or 3,4-substituted-pyrazole
in anhydrous MTBE may provide an indirect evidence that water
adsorbed in the active site should allocate in the region near the
3-substituent in water-saturated MTBE.
Similar arguments for the improvement of enzyme activity and
enantioselectivity can be applied to other (R)-azolides, especially
15–17, 20, and 24. Therefore, the best strategy for increasing the
(R)-enantiomer productivity with high optical purity is to employ
(R,S)-N-acyl-3-substituted-pyrazole as the substrate in the reaction
medium controlled at low water content in which water as the acyl
acceptor can be added in a feed-batch mode.
3.3. Kinetic analysis
In order to shed insights into effects of water content and
substrate structure on the hydrolytic resolution, the kinetic anal-
ysis based on Michaelis–Menten mechanism was carried out.
Figs. S1 and S2 of the Supplementary Information illustrate the
initial rates VR and VS varied with substrate concentrations for
7, 8, 10, and 11 in anhydrous and water-saturated MTBE, with

118
P.-Y. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 113–119
Good linear relationships for 2, 7 and 13, modest for 6, 11 and 12, but
bad for 3, 5 and 8–10 are shown. The unique azolide structure on the
reactivity is nicely reflected from the Brønsted slope of −0.45 for the
fast-reacting (R)-2, (R)-7, and (R)-13, and −0.20 of their antipodes
[16]. However, the slopes drop to −0.09 and −0.03 for (R)- and (S)-
enantiomers of 6, 11 and 12, respectively. This might provide an
indirect evidence for the existence of interactions among the 4-
substitent moiety, amino acid residues, and adsorbed water, such
that the electro-withdrawing effect exerted by 4-bromo or 4-nitro
substituent on enhancing the nucleophilic attack of catalytic ser-
ine and hence the enzyme activity is relaxed. No correlations for
(R)- and (S)-enantiomers of 3, 5 and 8–10 are perceived, reflecting
the more complicated interactions of the 3-substituent moiety in
the active site. As illustrated in Figs. S3 and S4 of the Supplemen-
tary Information, the above arguments in Fig. 1 can be applied to
the hydrolysis and alcoholysis of (R,S)-N-2-phenylpropionylazoles
in anhydrous MTBE, where the effect of adsorbed water on the
non-covalent interactions in the active site is minimized.
3.4. Alcoholysis by methanol in anhydrous MTBE
In order to demonstrate the novelty of using (R,S)-pyrazolides
containing substituents in the leaving pyrazole moiety for lipase-
catalyzed resolution in anhydrous organic solvents, the alcoholysis
of (R,S)-N-acylazoles by 100 mM methanol in anhydrous MTBE is
represented in Table 4. Similar enzyme performances as those in
Table 2 but with lower reactivity for each substrate are perceived,
yet still leading to excellent enantioselectivity for (R,S)-pyrazolides
containing a leaving 3- or 3,4-substitued-pyrazole moiety. The
Fig. 1. Variations of log(k2R/KmR) (᭹), log(k2S/KmS) (ꢁ), and log(VR/VS) (ꢀ) with pKa of
leaving azole for CALB-catalyzed hydrolysis in water-saturated MTBE. (—) Best-fit
results for (R)- or (S)-enantiomers of 6, 11 and 12, and those of 2, 7 and 13 from
Wang et al. [16].
mating k2R/KmR from VR/(Et)/(SR) and k2S/KmS from VS/(Et)/(SS) in
the hydrolysis, the structure-reactivity correlations in terms of pKa
of leaving azole in water-saturated MTBE are illustrated in Fig. 1.
Table 4
Effect of substrate structure on pKa of leaving azole, specific initial rates, VR/VS, Xt and ees for CALB-catalyzed alcoholysis in anhydrous MTBE.
Entry
pKa
VR/(Et) (mmol/h g)
VS/(Et) (mmol/h g)
VR/VS
(Et) (mg/ml)
Time (h)
Xt (%)
ees (%)
3
5
8
6.00
3.27
2.19
1.43
1.33
0.63
2.19
1.33
2.19
1.33
0.63
2.19
3.27
2.19
0.63
1.06
3.05E−3
3.63E−3
1.08E−3
1.82E−3
2.40E−3
3.79E−2
2.15E−1
1.40E−4
1.43E−2
2.76E−3
6.62E−2
1.09
347
266
739
278
829
78
2.0
10.0
2.0
10.0
2.0
10.0
2.0
20.0
2.0
2.0
2.0
2.0
10.0
2.0
3.0
1.0
20.0
6.0
3.4
0.3
0.6
2.0
3.5
3.1
1.3
0.3
3.5
1.0
1.5
50.7
50.6
51.1
50.4
50.8
50.4
60.0
50.2
50.1
51.4
54.4
64.8
50.6
54.6
52.1
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
72.1
9.69E−1
7.99E−1
5.07E−1
1.99
2.98
9.46
9
10
11
13
15
16
17
18
19
20
22
23
43
1.56E−1
111
114
247
35
5.1
133
31
1.63
6.82E−1
2.36
5.60
3.07E−1
2.30E−3
1.13E−1
4.58E−3
100.0
100.0
100.0
3.60
6.01E−1
131
6.0
Reaction conditions: 10 ml anhydrous MTBE containing 3 mM racemate and 100 mM methanol at 45 ◦C and 400 rpm. Symbol E−1 as 10−1
.
Table 5
Effect of lipase sources on pKa of leaving azole, specific initial rates, VS/VR, Xt and ees for Lipase MY-catalyzed hydrolysis in water-saturated cyclohexane.
Entry
pKa
6.00
VR/(Et) (mmol/h g)
VS/(Et) (mmol/h g)
VS/VR
(Et) (mg/ml)
Time (h)
Xt (%)
ees (%)
3
5
6
5.14E−4
1.16E−5
6.78E−4
3.45E−5
8.10E−4
8.95E−3
2.92E−2
N
2.40E−4
7.33E−2
5.30E−2
1.89E−3
1.69E−3
1.16E−3
2.37E−3
3.44E−2
4.79E−3
8.47E−2
6.68E−3
9.86E−2
4.61E−1
3.94E−1
N
66
41
124
193
121
51
13
N
37
14
3.8
58
52
62
31
40.0
40.0
20.0
40.0
20.0
20.0
10.0
40.0
60.0
10.0
2.0
5.0
72.0
5.0
47.0
5.0
1.0
2.0
115.0
9.0
0.6
15.4
2.0
4.0
20.0
22.5
53.2
42.8
52.8
46.1
54.8
53.1
54.6
N
51.5
58.1
44.2
53.0
53.4
59.5
62.6
100.0
68.0
100.0
82.9
100.0
100.0
82.9
N
89.2
96.2
31.5
100.0
100.0
100.0
87.5
3.27
2.19
1.43
0.63
−2.00
2.19
1.60
1.33
0.63
2.19
3.27
0.63
0.63
0.63
9
11
12
13
14
17
18
19
20
21
23
25
8.92E−3
1.08
2.02E−1
1.11E−1
8.90E−2
7.24E−2
7.56E−2
40.0
20.0
20.0
10.0
Reaction conditions: 10 ml water-saturated cyclohexane containing 3 mM racemate at 45 ◦C and 400 rpm. Symbol E−1 as 10−1; notation N as too low to be determined.

P.-Y. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 113–119
119
competitive enzyme inhibition by methanol has been proposed for
elucidating the lower enzyme activity [17]. Based on the hydrolysis
of (R)-N-naproxenyl-1,2,4-triazole in water-saturated MTBE, 8.5-
and 3.7-fold increases of VR/(Et) for the hydrolysis and alcoholysis,
respectively, in anhydrous MTBE were estimated. These enhance-
ments increase to 561 and 156 folds for (R)-10, 935 and 176 folds
for (R)-15, and 478 and 115 folds for (R)-17, implying that water
adsorbed in the active site does exert strong interactions to the
amino acid residues of the active site and 3-(2-pyridyl) substituent.
Therefore, the best strategy for increasing the (R)-ester productivity
and optical purity is to employ (R,S)-N-acyl-3-substituted-pyrazole
as the substrate in the reaction medium controlled at low methanol
concentration in which methanol as the acyl acceptor can be added
in a feed-batch mode.
MTBE. The good linear structure-reactivity correlations in water-
saturated MTBE for 2, 7 and 13, modest for 6, 11 and 12, and bad
for 3, 5 and 8–10, are regarded as indirect evidences for the strong
interactions of 3-substituent (but modest for the 4-substitutent)
with amino acid residues and adsorbed water in the active site.
The resolution platform has also been extended to the hydrolysis
of (R,S)-N-acylpyrazoles in water-saturated cyclohexane via Lipase
MY having opposite enantioselectivity to CALB.
Supplementary information: Information on product character-
ization specifications (1H NMR spectra and retention time in
HPLC analysis), initial rates varied with substrate concentrations
for CALB-catalyzed hydrolysis in anhydrous and water-saturated
MTBE, and variations of log(k2R/KmR), log(k2S/KmS), and log(VR/VS)
with pKa of leaving azole in anhydrous MTBE is available free of
charge via the Internet at http://www.sciencedirect.com.
3.5. Hydrolysis in water-saturated cyclohexane via Lipase MY
Acknowledgements
In order to test if the resolution strategy can be extended
to lipases having opposite enantioselectivity to CALB, Lipase
MY-catalyzed hydrolysis of (R,S)-azolides in water-saturated cyclo-
hexane but not MTBE owing to the low enantioselectivity and
stability in polar organic solvents was performed (Table 5). In com-
parison with 13 and 19 containing a leaving 1,2,4-triazole moiety,
(R,S)-pyrazolides containing a substituent, especially a 4- or 3,4-
substituent, in the leaving pyrazole moiety might yield good to
excellent enantioselectivity. By considering the enzyme perfor-
mance in the hydrolytic resolution, CALB is superior to Lipase MY
regardless of the different optical preferences. All the results rep-
resented in Tables 1–5 really indicate that one may employ the
present hydrolytic or alcoholytic resolution platform for preparing
(R)- and (S)-carboxylic acid containing an ␣-chiral center.
Financial supports from National Science Council (Grant No. NSC
97-2221-E-182-018-MY3) are appreciated.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.molcatb.2010.04.003.
References
[1] A. Ghanem, Tetrahedron 63 (2007) 1721–1754.
[2] A. Kamal, M.A. Azhar, T. Krishnaji, M.S. Malik, S. Azeeza, Coord. Chem. Rev. 252
(2008) 569–592.
[3] R. Chenevert, N. Pelchat, F. Jacques, Curr. Org. Chem. 10 (2006) 1067–1094.
[4] V. Gotor-Fernández, V. Gotor, Curr. Org. Chem. 10 (2006) 1125–1143.
[5] E. Santaniello, S. Casati, P. Ciuffreda, Curr. Org. Chem. 10 (2006) 1095–1123.
[6] U.T. Bornscheuer, R.J. Kazlauskas, Hydrolases in Organic Synthesis: Regio- and
Stereoselective Biotransformations, 2nd Ed., Wiley-VCH, Weinheim, 2006, pp.
37–275.
4. Conclusions
With CALB-catalyzed hydrolytic resolution of (R,S)-N-2-
phenylpropionylazoles (1–14) and other (R,S)-N-acylazoles
(15–24) in water-saturated MTBE as the model system, (R,S)-
pyrazolides containing a leaving 3-, 4- or 3,4-substituted pyrazole
moiety are exploited as the best substrates, when comparing with
13 containing a leaving 1,2,4-triazole moiety. Great improvements
of enzyme activity with excellent enantioselectivity (VR/VS > 100)
for the fast-reacting enantiomers are also obtainable, if (R,S)-
pyrazolides containing a leaving 3- or 3,4-substituted-pyrazole
moiety are employed for the hydrolysis or alcoholysis by methanol
in anhydrous MTBE. Therefore, the best strategy for carrying out
the present resolution process is to use (R,S)-pyrazolides contain-
ing a leaving 3-substituted pyrazole moiety as the substrate, and
keeps the reaction medium at low water (or methanol) contents
by adding the acyl acceptor in a fed-batch mode.
[7] K. Faber, S. Riva, Synthesis 10 (1992) 895–910.
[8] U. Hanefeld, Org. Biomol. Chem. 1 (2003) 2405–2415.
[9] S.W. Tsai, C.C. Chen, H.S. Yang, I.S. Ng, T.L. Chen, BBA Protein Proteomics 1764
(2006) 1424–1428.
[10] P.Y. Wang, S.W. Tsai, J. Mol. Catal. B: Enzym. 57 (2009) 158–163.
[11] F. Theil, Tetrahedron 56 (2000) 2905–2919.
[12] E.P. Hudson, R.K. Eppler, D.S. Clark, Curr. Opin. Biotechnol. 16 (2005) 637–
643.
[13] M.T. Reetz, Adv. Catal. 49 (2006) 1–69.
[14] M.D. Toscano, K.J. Woycechowsky, D. Hilvert, Angew. Chem. Int. Ed. 46 (2007)
3212–3236.
[15] P.Y. Wang, T.L. Chen, S.W. Tsai, Biotechnol. Bioeng. 101 (2008) 460–469.
[16] P.Y. Wang, Y.J. Chen, A.C. Wu, Y.S. Lin, M.F. Kao, J.R. Chen, J.F. Ciou, S.W. Tsai,
Adv. Synth. Catal. 351 (2009) 2333–2341.
[17] A.C. Wu, P.Y. Wang, Y.S. Lin, M.F. Kao, J.R. Chen, J.F. Ciou, S.W. Tsai, J. Mol. Catal.
B: Enzym. 62 (2010) 235–241.
[18] L.P. Lee, R. Bembi, T.H. Eife, J. Org. Chem. 62 (1997) 2827–2876.
[19] T.H. Eife, M.H. Werner, Bioorg. Chem. 26 (1998) 119–130.
[20] H.A. Staab, H. Bauer, K.M. Schneider, Azolides in Organic Synthesis and Bio-
chemistry, Wiley-VCH, Weinheim, 1998.
A
thorough
kinetic
analysis
for
(R,S)-N-2-
[21] J. Elguero, C. Foces-Foces, D. Sanz, R.M. Claramunt, Azolides: Structural Aspects
Advances in Nitrogen Heterocycles, vol. 4, Jai Press, Stamford, 2000, pp.
295–367.
[22] C. Hansch, L. Leo, R.W. Taft, Chem. Rev. 91 (1991) 165–195.
[23] R.T. Otto, H. Scheib, U.T. Bornscheuer, J. Pleiss, C. Syldatk, R.D. Schmid, J. Mol.
Catal. B: Enzym. 8 (2000) 201–211.
phenylpropionylpyrazoles indicates that a bulky 3-substituent in
the leaving pyrazole moiety has profound effects on decreasing
the nucleophilic attack and proton transfer of catalytic serine for
the slow-reacting enantiomer in anhydrous MTBE, as well as that
and substrate affinity for both enantiomers in water-saturated