(R,S)-2-chlorophenoxyl pyrazolides as novel substrates for improving lipase-catalyzed hydrolytic resolution

Article abstract of DOI:10.1002/chir.21024

The best reaction condition of Candida antartica lipase B as biocatalyst, 3-(2-pyridyl)pyrazole as leaving azole, and water-saturated methyl t-butyl ether as reaction medium at 45°C were first selected for performing the hydrolytic resolution of (R,S)-2-(4-chlorophenoxyl) azolides (1-4). In comparison with the kinetic resolution of (R,S)-2-phenylpropionyl 3-(2-pyridyl)pyrazolide or (R,S)-α-methoxyphenylacetyl 3-(2-pyridyl)pyrazolide at the same reaction condition, excellent enantioselectivity with more than two order-of-magnitudes higher activity for each enantiomer was obtained. The resolution was then extended to other (R,S)-3-(2-pyridyl)pyrazolides (5-7) containing 2-chloro, 3-chloro, or 2,4-dichloro substituent, giving good (E > 48) to excellent (E > 100) enantioselectivity. The thermodynamic analysis for 1, 2, and 4-7 demonstrates profound effects of the acyl or leaving moiety on varying enthalpic and entropic contributions to the difference of Gibbs free energies. A thorough kinetic analysis further indicates that on the basis of 6, the excellent enantiomeric ratio for 4 and 7 is due to the higher reactivity of (S)-4 and lower reactivity of (R)-7, respectively.

Full text of DOI:10.1002/chir.21024

CHIRALITY 24:60–66 (2012)
(R,S)-2-Chlorophenoxyl Pyrazolides as Novel Substrates for
Improving Lipase-Catalyzed Hydrolytic Resolution
*
MIN-FANG KAO, PEI-YU LU, JOU-YAN KAO, PEI-YUN WANG, AN-CHI WU, AND SHAU-WEI TSAI
Institute of Biochemical and Biomedical Engineering, Chang Gung University, Kwei-Shan Tao-Yuan, Taiwan
ABSTRACT
The best reaction condition of Candida antartica lipase B as biocatalyst, 3-(2-pyridyl)-
pyrazole as leaving azole, and water-saturated methyl t-butyl ether as reaction medium at 458C
were first selected for performing the hydrolytic resolution of (R,S)-2-(4-chlorophenoxyl) azo-
lides (1–4). In comparison with the kinetic resolution of (R,S)-2-phenylpropionyl 3-(2-pyridyl)-
pyrazolide or (R,S)-a-methoxyphenylacetyl 3-(2-pyridyl)pyrazolide at the same reaction
condition, excellent enantioselectivity with more than two order-of-magnitudes higher activity
for each enantiomer was obtained. The resolution was then extended to other (R,S)-3-(2-pyridyl)-
pyrazolides (5–7) containing 2-chloro, 3-chloro, or 2,4-dichloro substituent, giving good (E > 48)
to excellent (E > 100) enantioselectivity. The thermodynamic analysis for 1, 2, and 4–7
demonstrates profound effects of the acyl or leaving moiety on varying enthalpic and entropic
contributions to the difference of Gibbs free energies. A thorough kinetic analysis further
indicates that on the basis of 6, the excellent enantiomeric ratio for 4 and 7 is due to the
higher reactivity of (S)-4 and lower reactivity of (R)-7, respectively. Chirality 24:60–66,
C
VV
2012.
2011 Wiley Periodicals, Inc.
KEY WORDS: (R,S)-2-chlorophenoxyl 3-(2-pyridyl)pyrazolides; lipases; hydrolysis; kinetic and
thermodynamic analysis
INTRODUCTION
ness because of low reactivity have hindered such methods
practicably used in industry.
Lipases have proved the capability for the preparation of
optically pure alcohols, amines, and carboxylic acids for syn-
thesizing a variety of pharmaceuticals and agrochemicals.1
To enhance the enzyme performance, the substrate engi-
neering approach of using activated or irreversible acyl
donors or acceptors,2 medium engineering strategy of vary-
ing temperature, reaction media, or additives,3 as well as
enzyme engineering approach of using site-directed muta-
tions, evolutions, or chemical modifications4,5 have been
proposed for the fine-tuning of enzyme active-site structure.
Recently, an enzymatic resolution process using (R,S)-azo-
lides but not their corresponding ester, thioester, or normal
amide analogs as the substrate was reported for preparing
optically pure carboxylic acids containing a 2-aryl group to
the a-chiral center.6–8
(R,S)-2-Chlorophenoxypropionic acids (CPAs) and their
esters are widely used as herbicides with the biological
activity mainly residing on the (R)-isomers. In addition,
(R)-2-(4-chlorophenoxy)propionic acid can lower the level of
serum cholesterol to prevent platelet aggregation, whereas
the (S)-antipode inhibits the chloride channel in muscles.9
Optically pure CPAs have been prepared via lipase-catalyzed
hydrolysis or esterification of their racemates. In general,
low enantioselectivity for crude Candida rugosa lipases
(CRL), moderate to high enantioselectivity but with
low reactivity for Carica papaya lipase (CPL) were
reported.10–12 Many efforts have been made to improve
CRL activity and enantioselectivity, for example, use of
purified or isopropanol (IPA)-treated lipase,13–18 addition
of dimethyl sulfoxide (DMSO), carbon tetrachloride, or
benzene,17,19–21 ionic liquids as the reaction medium,22 and
organosilicon alcohols as the acyl acceptor.11,12,23 Neverthe-
less, considerations of solvent toxicity, biocatalyst recycle
and downstream separation capability, and cost effective-
It is, therefore, aimed to use the resolution platform of
using (R,S)-azolides as novel substrate for improving the
preparation of optically pure CPAs (Fig. 1). The best reaction
conditions for the hydrolysis of (R,S)-2-(4-chlorophenoxy)-
propionyl azolides (1–4) in water-saturated solvents is first
selected and then extend to other (R,S)-2-chlorophenoxypro-
pionyl 3-(2-pyridyl)pyrazolides (5–7). Moreover, the thermo-
dynamic and kinetic analysis is also performed for elucidat-
ing the favorable results.
EXPERIMENTAL
Materials
Lipase MY (30,000 U g21 using olive oil emulsion as substrate at 378C
and pH 7.0) from Candida rugosa, Novozym 435 as an immobilized lipase
(7000 PLU g21 using lauric acid and 1-propanol as substrates at 608C)
from Candida antartica lipase B (CALB), and partially purified CPL
(26,700 U g21 using olive oil emulsion as substrate at 408C and pH 8.5)
were provided by Meito Sangyo (Tokyo, Japan), Novo Nordisk (Bags-
vaerd, Denmark), and Challenge Bioproducts (Yun-Lin Hsien, Taiwan),
respectively. DMSO-d6 containing 1% (v/v) tetramethylsilane (TMS) for
1H NMR analysis was from Cambridge Isotope Laboratories (Andover,
MA). Other chemicals of analytical grade were commercially available:
(R,S)-2-(3-chlorophenoxy)propionic acid and (R,S)-2-(2,4-dichlorophenox-
y)propionic acid from TCI (Tokyo, Japan); (R,S)-2-(2-chlorophenoxy)
propionic acid and 4-methypyrazole from Acros (Geel, Belgium); 4-bro-
mopyrazole, (R,S)-2-(4-chlorophenoxy)propionic acid, and N,N0-carbon-
Contract grant sponsor: National Science Council; Contract grant number:
100-2221-E-182-026-MY2.
*Correspondence to: Shau-Wei Tsai, Institute of Biochemical and Biomedical
Engineering, Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-Shan
Tao-Yuan, Taiwan, 33302. E-mail: tsai@mail.cgu.edu.tw
Received for publication 14 December 2010; Accepted 10 August 2011
DOI: 10.1002/chir.21024
Published online 20 October 2011 in Wiley Online Library
(wileyonlinelibrary.com).
C
VV
2011 Wiley Periodicals, Inc.

CHLOROPHENOXYL PYRAZOLIDES AS NOVEL SUBSTRATES
61
Fig. 1. Lipase-catalyzed hydrolytic resolution of (R,S)-2-chlorophenoxypropionyl azolides.
yldi(1,2,4-triazole) (CDT) from Sigma-Aldrich (Milwaukee, WI); 3-(2-pyri-
dyl)-1H-pyraozle from Alfa Aesar (Ward Hill, MA); benzene, cyclohexane
(CYC), dipropylether (IPE), hexane, IPA, and methyl t-butyl ether
(MTBE) from Tedia (Fairfield, OH).
(1H, q), 7.02–7.04 (1H, m), 7.06–7.07 (1H, m), 7.22 (1H, d), 7.29–7.32
(1H, m), 7.46–7.48 (1H, m), 7.92–7.95 (1H, m), 8.09 (1H, d), 8.55 (1H,
d), 8.68–8.69 (1H, m).
(R,S)-2-(2-Chlorophenoxy)propionyl 3-(2-pyridine)pyrazolide
(6). 1H NMR (DMSO-d6/TMS) d: 1.77 (3H, d), 6.20–6.24 (1H, q), 6.95–
7.04 (2H, m), 7.22–7.27 (2H, m), 7.44–7.48 (2H, m), 7.90–7.94 (1H, m), 8.03
(1H, d), 8.55 (1H, d), 8.68–8.69 (1H, m).
Substrates Synthesis
To 2.5 ml of benzene containing 1.3 mmol CPA, 1.0 mmol pyrazole,
and 4 mmol triethylamine, a mixture containing 0.5 ml of benzene and
1.3 mmol thionyl chloride was added dropwise with stirring for 2 h at
08C. The resultant mixture was filtered, quenched in succession with
0.1 M HCl solution (3 3 10 ml), 0.1 M NaOH solution (3 3 10 ml), and
0.1 M NaCl solution (3 3 10 ml). The organic phase was separated,
dried over anhydrous MgSO4 for 12 h, filtered, and concentrated under
reduced pressure, giving the desired (R,S)-pyrazolides. Moreover, to
2.5 ml of benzene, 1 mmol (R,S)-2-(4-chlorophenoxy)propionic acid and
1.5 mmol CDT were added and stirred for 2 h at 558C. The resultant
mixture was filtered and evaporated under reduced pressure, giving the
desired (R,S)-2-(4-chlorophenoxy)propionyl-1,2,4-triazolide. All the
synthesized substrates were confirmed from the 1H NMR spectra
recorded at 500 MHz on Brucker Avance DRX 500 spectrometer in
DMSO-d6 solution with TMS as an internal standard (IS).
(R,S)-2-(2,4-Dichlorophenoxy)propionyl 3-(2-pyridine)pyrazo-
lide (7). 1H NMR (DMSO-d6/TMS) d: 1.78 (3H, d), 6.19–6.23 (1H, q),
7.01 (1H, d), 7.22 (1H, d), 7.34–7.37 (1H, m), 7.47–7.49 (1H, m), 7.60
(1H, d), 7.91–7.99 (1H, m), 8.00 (1H, d), 8.55 (1H, d), 8.69–8.70 (1H, q).
Analysis
The hydrolysis (R,S)-azolides was monitored by high performance liq-
uid chromatography (HPLC) using a chiral column from Daicel (OJ-H or
OD-H; Tokyo, Japan) or Regis [(S,S)-Whelk-O 1; Morten Grove, IL], that
is, capable of separating the IS and substrates. UV detection at 220, 240, or
270 nm was used for quantification at room temperature. Detailed condi-
tions, such as the mobile phase composition and retention time for each
compound, are given in Table 1. The optical rotation of (R)-2-(2,4-dichloro-
phenoxy)propionic acid dissolved in ethanol was determined at 589 nm on
a Atago AP-100 polarimeter.
(R,S)-2-(4-Chlorophenoxy)propionyl 1,2,4-triazolide (1). 1H
NMR (DMSO-d6/TMS) d: (DMSO-d6/TMS) d: 1.73 (3H, d), 5.95–6.00
(1H, q), 6.94–7.05 (2H, m), 7.22–7.29 (1H, m), 7.42–7.48 (1H, m), 8.46
(1H, s), 9.42 (1H, s). The abbreviations d, q, m, and s were the peak mul-
tiplicities of doublet, quartet, multiplet, and single, respectively.
Effects of Lipase Sources, Temperature,
and Substrate Structure
To 10 ml of water-saturated MTBE containing 3 mM of 1–4, a specific
amount of CALB was added for performing the hydrolysis in a batch
reactor at 458C. Samples were removed from the reaction medium at
different time intervals for HPLC analysis, from which the time-course
conversions XR (i.e., [1 2 (SR)/(SR0)], with (SR0) as the initial (R)-enan-
tiomer concentration) and XS (i.e., [1 2 (SS)/(SS0)], with (SS0) as the
initial (S)-enantiomer concentration), initial rates for both enantiomers
VR and VS based on several conversion determinations, racemate conver-
sion Xt [i.e., 0.5(XR 1 XS)], and enantiomeric excess for the substrate ees
were determined. Similar experiments were performed in water-satu-
rated CYC for CRL or CPL. To study the temperature effect on CALB
performances, the reaction was also performed in MTBE at 25 and 358C.
More experiments were performed for investigating the acyl moiety in
5–7 on enzyme activity and enantioselectivity. To compare the kinetic
behaviors, the hydrolysis of varying initial substrate concentrations (SR)
(R,S)-2-(4-Chlorophenoxy)propionyl 4-bromopyrazolide (2). 1H
NMR (DMSO-d6/TMS) d: 1.66 (3H, d), 6.04–6.09 (1H, q), 6.85–6.87 (1H,
m), 6.94–7.03 (2H, m), 7.27–7.31 (1H, m), 8.17 (1H, s), 8.76 (1H, s).
(R,S)-2-(4-Chlorophenoxy)propionyl 4-methylyrazolide (3). 1H
NMR (DMSO-d6/TMS) d: 1.66 (3H, d), 2.19 (3H, s), 5.99–6.04 (1H, q),
6.88–6.92 (2H, m), 7.29–7.36 (2H, m), 7.88 (1H, s), 8.24 (1H, s).
(R,S)-2-(4-Chlorophenoxy)propionyl 3-(2-pyridine)pyrazolide
(4). 1H NMR (DMSO-d6/TMS) d: 1.73 (3H, d), 6.16 (1H, d), 6.96–6.98
(2H, m), 7.23 (1H, d), 7.32–7.34 (2H, m), 7.47–7.49 (1H, q), 7.93–7.96
(1H, m), 8.10 (1H, d), 8.55 (1H, d), 8.70 (1H, d).
(R,S)-2-(3-Chlorophenoxy)propionyl 3-(2-pyridine)pyrazolide
(5). 1H NMR (DMSO-d6/TMS) d: 1.72 (3H, d), 6.20 (1H, d), 6.90–6.92
Chirality DOI 10.1002/chir

62
KAO ET AL.
TABLE 1. HPLC analytic conditions
Retention time (min)
Wave length
(nm)
Flow rate
Entry
Column
OD-H
OD-H
OD-H
OD-H
(S,S)-Whelk-O 1
OJ-H
(ml min21
)
Mobile phase
IS
(R)-Azolide
(S)-Azolide
1
2
3
4
5
6
7
220
240
270
270
270
270
570
2.0
1.5
2.0
2.0
2.0
2.0
2.0
90:10:0
93:7:0
90:10:0
99:1:0
90:10:0
90:10:0
98.5:1:0.5
3.1 (Nitrotoluene)
2.6 (Benzene)
2.0 (Benzene)
2.0 (Benzene)
2.5 (Nitrotoluene)
2.1 (benzene)
2.0 (benzene)
14.1
3.7
6.7
10.5
5.8
18.5
14.9
12.2
3.3
5.7
9.3
7.1
13.7
11.8
OD-H
Mobile phase composition in HEX:IPA:glacial acetic acid (v/v/v); IS as the internal standard.
and (SS) of 5–7 in MTBE at 458C was performed, with which the speci-
ficity constants k2R/KmR and k2S/KmS, and hence enantiomeric ratio (i.e.,
E 5 k2RKmS/k2SKmR) were estimated from the experimental data coupled
triads on decreasing the nucleophilic attack and proton trans-
fer (i.e., k2R and k2S) but not substrate affinity to the active
site (KmS and KmR), as previously reported for the hydrolysis
of (R,S)-2-phenylpropionyl azolides.8
with VR
5 k2R(SR)(Et)/{KmR 1 (SR)[1 1 KmR/KmS]} and VS 5
k2S(SS)(Et)/{KmS 1 (SS)[1 1 KmS/KmR]} at the initial stage. The kinetic
constants, k2R, k2S, KmR, and KmS, using Michaelis–Menten kinetics for
both enantiomers were previously defined.8
By further changing the substrate to 3 containing a leav-
ing 4-methylpyrazole, the enzyme activity for each enan-
tiomer increases, yielding a slight increase of initial VS/VR to
47.7. This again provides an indirect evidence of the nonco-
valent bonding between 4-methyl substituent and amino acid
residues, when considering the higher pKa of 3.04 for
4-methylpyazolium that is disadvantageous for the nucleo-
philic attack to the carbonyl carbon atom, and leaving of
4-methylpyrazole in nonenzymatic reactions. On the con-
trary, decreasing of temperature to 258C is a more effective
way to increase VS/VR to 73.2 for 2, yet with the penalty of
fourfold lower of VS/(Et) for the fast-reacting enantiomer.
It has been shown that, a bulky 3-(2-pyridyl) or 3-(3-bromo-
phenyl) substituent to the leaving pyrazole is beneficial for
giving excellent enantioselectivity for Novozym 435.8 Using
Products Separation
After terminating the hydrolysis and removing the lipase by filtration,
the resultant solution of Xt 42.5% for 7 was collected, added to 0.1 M
NaOH solution (2 3 10 ml) in succession, and stirred for 20 min. The
aqueous solutions were collected, acidified to pH 2 by adding 0.3 ml
of HCl solution (37%, w/w), and added in succession (2 3 10 ml) to
benzene with stirring for 20 min. The organic phase was collected,
dried over anhydrous MgSO4 for 12 h, concentrated under reduced
pressure, and gave the acid product. From the optical rotations of
20
[a]D 5 228.0 (c 0.5, EtOH) (Lit. [a]D20 5 228.6 (c 1.6, EtOH) for (S)-
7),17 the (S)-preference for all lipases was determined.
The organic phase after the extraction via the NaOH solution was
separated, added to 0.1 M HCl solution (20 ml), and stirred for 2 h. The
organic phase was collected, dried over anhydrous MgSO4 for 12 h, con-
centrated under reduced pressure, and gave the remaining substrate.
The aqueous phase was added to 10 ml of benzene containing 0.2 M
NaOH solution (0.2 ml), and stirred for 20 min. The organic phase was
separated, dried over anhydrous MgSO4 for 12 h, and concentrated
under reduced pressure to give 3-(2-pyridyl)pyrazole product.
4
containing a leaving 3-(2-pyridine)pyrazole as the
substrate, excellent enantioselectivity of VS/VR 5 220.0 with
VS/(Et) 5 3.96 mmol h21 g21 at 458C is obtainable. In com-
parison with the hydrolysis of (R,S)-2-phenylpropionyl 3-(2-
pyridyl)pyrazolide at the same reaction condition (i.e., VR/
(Et) 5 1.27 3 1022 mmol h21 g21, VS/(Et) 5 2.63 3 1025
mmol h21 g21, and VR/VS 5 482), more than 311- and
684-fold enhancements of specific activity for the fast- and
slow-reacting enantiomers, respectively, are estimated.
These corresponds to free energy contributions of about
15.2 and 17.2 kJ mol21 (i.e., RTln(311) and RTln(684),
respectively, at 458C) well within the range of a weak hydro-
gen bond between the 4-chlorophenoxy oxygen and amino
acid residues or adsorbed water in a hydrophobic environ-
ment. The inductive effect due to electronegative 4-chloro-
phenoxy moiety at the 2-position of acyl donor can be ruled
out, when considering the lowest reactivity of 7 containing a
more electronegative 2,4-dichlorophenoxy moiety.
Similar activity enhancements of CALB or Pseudomonas
cepacia lipase, for the resolution of (R,S)-amines in organic
solvents, have been reported using methyl methoxyacetate
as a highly active acyl donor.24,25 Apparently, an extra hydro-
gen bond between the methoxy oxygen and amine hydrogen
previous proposed does not apply to the present case. It is
also interesting to note the low CALB activity (i.e., VR/(Et) 5
5.29 3 1023 mmol h21 g21 and VS/(Et) 5 1.73 3 1025 mmol
h21 g21) for hydrolyzing (R,S)-a-methoxyphenylacetyl 3-(2-
pyridyl)pyrazolide in water-saturated MTBE.8 Although the
2-position of acyl part also contains methoxy oxygen, no
formation of an extra hydrogen bond is perceived. This may
RESULTS AND DISCUSSION
Effects of Lipase Sources, Solvent, Temperature, and
Substrate Structure
Table 2 demonstrates effects of lipase sources, solvent,
temperature, and substrate structure on the hydrolysis of
(R,S)-2-chlorophenoxypropionyl azolides on changing the ini-
tial specific activities VR/(Et) and VS/(Et), enantioselectivity
in terms of initial VR/VS (or VS/VR), Xt, and ees at a specified
reaction time. In comparison with the bad CPL performance
for 1, a moderate enantioselectivity with an opposite stereo-
preference for CRL and CALB is found. A decrease of tem-
perature results in lowering VR/(Et) and VS/(Et), but with a
slight increase of enantioselectivity for CALB. By replacing
the leaving 1,2,4-triazole with 4-bromopyrazole for 2, only
slight changes of VR/(Et) and VS/(Et), and hence VR/VS, for
CRL and CPL are shown. Yet for CALB at 458C, more than
fourfold and 11-fold decreases of VR/(Et) and VS/(Et),
respectively, lead to an enhancement of VS/VR from 17.9 to
40.4. Therefore, there should have been noncovalent bonds
between the 4-bromo substituent and amino acid residues,
when considering the pKa of 0.63 for 4-bromopyazolium
lower than 2.19 for 1,2,4-triazolium. This bonding may induce
a minute change of the orientations of substrate and catalytic
Chirality DOI 10.1002/chir

CHLOROPHENOXYL PYRAZOLIDES AS NOVEL SUBSTRATES
63
TABLE 2. Effect of lipase sources, solvent, temperature, and substrate on hydrolysis of 1–7
Entry
Lipase
Temp.
(8C)
VR/(Et)
VS/(Et)
VR/VS or
VS/VR
(Et)
Time
(h)
Solvent
(mmol h21 g21
)
(mmol h21 g21
)
(mg ml21
)
Xt (%)
ees (%)
1
CRL
CPL
CALB
CALB
CALB
2
CYC
CYC
MTBE
MTBE
MTBE
45
45
45
35
25
1.64
8.36 E 2
1.58 E 21
1.09 E 11
6.99
19.6
1.2
17.9
18.9
21.9
2
2
1
1
2
3.8
8.3
0.8
0.7
1.0
49.2
82.1
61.7
57.5
64.2
28.3
13.1
100.0
100.0
100.0
1.87 E 21
6.06 E 21
3.69 E 21
2.22 E 21
4.86
CRL
CPL
CALB
CALB
CALB
3
CYC
CYC
MTBE
MTBE
MTBE
45
45
45
35
25
9.48 E 21
2.95 E 22
5.50 E 22
3.65 E 22
7.65 E 23
4.22 E 22
1.58 E 22
2.22
1.52
5.60 E 21
22.5
1.9
40.4
41.6
73.2
2
2
2
2
2
4.5
10.5
3.5
4.0
26.0
37.1
51.3
63.0
57.0
60.9
20.9
12.3
100.0
100.0
100.0
CALB
4
MTBE
45
1.42 E 21
6.78
47.7
1
8.7
78.7
100.0
CALB
CALB
CALB
5
MTBE
MTBE
MTBE
45
35
25
1.80 E 22
9.30 E 23
4.20 E 23
3.96
2.39
1.19
220.0
257.0
283.3
2
2
6
2.5
9.0
3.1
52.3
56.4
50.2
100.0
100.0
100.0
CALB
CALB
CALB
6
MTBE
MTBE
MTBE
45
35
25
1.94 E 21
6.30 E 22
2.90 E 22
5.08
2.31
1.38
26.2
36.8
48.1
10
10
5
4.1
2.0
1.0
60.9
58.4
55.7
100.0
4.9
100.0
CALB
CALB
CALB
7
MTBE
MTBE
MTBE
45
35
25
2.73 E 22
1.22 E 22
4.44 E 23
1.13
7.03 E 21
3.29 E 21
41.4
57.6
74.1
12
12
12
0.5
1.0
3.5
53.2
53.2
56.6
100.0
83.2
100.0
CRL
CPL
CYC
CYC
CYC
IPE
MTBE
MTBE
MTBE
45
45
45
45
45
35
25
1.31 E 23
5.05 E 23
3.79 E 23
6.91 E 23
4.24 E 23
2.01 E 23
1.00 E 23
5.68 E 23
1.11 E 21
7.86 E 21
1.49
1.08
6.40 E 21
3.74 E 21
4.3
22.0
12
12
12
12
12
12
12
4.3
10.5
5.0
5.0
1.0
54.7
63.8
59.6
65.7
55.9
56.2
54.8
30.3
83.2
CALB
CALB
CALB
CALB
CALB
207.4
215.6
254.7
318.4
374.0
100.0
100.0
100.0
100.0
100.0
7.1
30.0
Reaction conditions: 10 ml solvent containing 3 mM racemate at 400 rpm. Symbol E-1 as 1021
.
be attributed to the largest pocket of active site originally
occupied by the methoxy oxygen of methyl methoxyacetate
or chlorophenoxy oxygen of 1–7, being now occupied by
2-phenyl moiety to lose the capability of forming a hydrogen
bond with the methoxy oxygen.
Table 2 further demonstrates the results for 5–7 contain-
ing a leaving 3-(2-pyridine)pyrazole moiety. More than 10-
fold increases of specific activity for the slow-reacting (R)-5,
but not (S)-5, at 458C were obtained and led to great decreas-
ing of the enantioselectivity. A decrease of temperature to
258C is advantageous to improve VS/VR from 26.2 to 48.1, yet
with the penalty of 3.6-fold decreasing of initial VS/(Et).
On the contrary, a decrease of specific activity for each enan-
tiomer yields a slight enhancement of VS/VR to 41.1 at 458C,
when using 6 containing a 2-chlorophenoxy moiety as
the substrate. This can be improved to 74.1 by lowering the
temperature to 258C.
Using 7 containing a 2,4-dichlorophenoxy moiety as the
substrate, it is interesting to find the change of stereoprefer-
ence of CRL and CPL, but not CALB. Similar kinetic behav-
iors of giving the (S)-preference for crude CRL but not the
IPA-treated preparation have been reported,17 and were
attributed to the isoenzymes having an opposite stereoprefer-
ence. The excellent enantioselectivity of CALB is shown in
Table 2, for the hydrolysis of 7 in MTBE at 458C. Moreover,
changing of the solvent to IPE or CYC has caused minor
influences on varying the enzyme activity and enantioselec-
tivity. Yet the enantioselectivity improves by decreasing
temperature to 258C. On the basis of above results, the best
condition of CALB as the biocatalyst, water-saturated MTBE
as the reaction medium, 458C for 4 and 7 and 258C for 5
and 6 is concluded. To shed insights into effects of substrate
structure on the hydrolytic resolution, the thermodynamic
and kinetic analysis for CALB was performed.
Thermodynamic Analysis
According to the transition theory, the specificity constant
can be expressed as ln(k2i/Kmi) 5 2DHi/RT 1 (A 1 DSi)/R,
i 5 R or S. A constant A 5 Rln(jkBT/h), with h, kB, R, T, j
as the Plank’s constant, Boltzmann’s constant, gas constant,
absolute temperature, and transmission coefficient, respec-
tively, can be assumed, when varying the temperature in a
narrow range. From the enthalpic and entropic differences
(i.e., DHi and DSi) between the transition and ground states
for each enantiomer, one obtains ln(E) 5 2DDG/RT 5
2DDH/RT 1 DDS/R, where 2DDG, 2DDH, and 2DDS rep-
resent the differences of Gibbs free energies (DGR and DGS),
enthalpies (DHR and DHS), and entropies (DSR and DSS)
between the transition states of both the enantiomers,
respectively.
Chirality DOI 10.1002/chir

64
KAO ET AL.
Fig. 4. Variations of ln(k2R/KmR
ln(E) (&, ~) with inverse of absolute temperature for 5 (*, !, &) and
6 (l, !, ~).
) (*, l), ln(k2S/KmS) (!, !), and
Fig. 2. Variations of ln(k2R/KmR
ln(E) (&, ~) with inverse of absolute temperature for 1 (*, !, &) and
2 (l, !, ~).
) (*, l), ln(k2S/KmS) (!, !), and
discrimination is found to be mainly enthalpic-driven for 1,
with compensation from the entropic gain for 2, and even
with favor from the entropic loss for 4.
Good liner relationships between ln(E) and ln(k2i/Kmi)
and the inverse of absolute temperature (Figs. 2–4) were
found, in which DHi, (A 1 DSi), 2DDH, and 2DDS were
estimated and represented in Table 3. The thermodynamic
parameter DHS for 1, 2, or 4 increases when the leaving
azole moiety contains a substituent, implying that the unfav-
orable enthalpic contribution due to desolvation of polar
water at the transition state cannot compensate from the
favorable enthalpic gain of the substituent with amino acid
residues or adsorbed water in the active site. This is
especially valid for DHR of slow-reacting enantiomer (R)-2
containing a 4-bromopyrazole moiety.
It is difficult to elucidate DSR and DSS varied with the sub-
stituent, owing to the complicated entropic contributions
from interactions between water, solvent, substrate, and
enzyme molecules on changing the number of degrees of
freedom. Yet the enthalpy–entropy compensation relation-
ships between DHR and (A 1 DSR), DHS and (A 1 DSS), and
hence 2DDH and 2DDS, still hold. Moreover, a comparison
of 2DDG and 2DDH at 458C indicates that the enantiomer
Table 3 also demonstrates DHR and DHS varied with the
acyl group for 4–7. Apparently, the 4-chloro substituent in 4
or 7 especially for the slow-reacting (R)-enantiomer, but not
the 3- or 2-chloro group in 5 or 6, is capable of compensating
the unfavorable enthalpic contribution due to desolvation of
polar water at the transition state. The good enthalpy–
entropy compensation relationships of DSR 5 2113.5 1
4.315DHR – A (r2 5 0.97) and DSS 5 240.41 1 4.074DHS – A
(r2 5 0.91), and hence DDS 5 61.43 1 4.487DDH (r2 5 0.98)
might be attributed to the similar acyl donors investigated
here. It is interesting to compare the thermodynamic param-
eters for 4, 6, and 7. The introduction of a 4-chloro substitu-
ent in 7 is more favorable on lowering DHR and (A 1 DSR),
leading to 2DDH of 15.11 kJ mol21 and 2DDS of 1.33 J
mol21 K21. Thus, the entropic compensation effect is nearly
negligible at 458C for obtaining an excellent CALB enantiose-
lectivity. On the contrary, the introduction of a 2-chloro
group in 4 mainly lowers DHS and (A 1 DSS), and leads to
the enthalpic gain just compensated from the entropic gain,
resulting in nearly the same 2DDG, and hence enzyme enan-
tioselectivity for 4 and 7.
It envisages that a weak hydrogen bond should exist
between the 4-chloro substituent of (R)-4 and (R)-7 and
amino acid residues. This can not only lower DHR but also
decreases the number of degrees of freedom of the transition
state, such as DSR < DSS to obtain a negative 2DDS for 4.
Moreover, the 2-chloro substituent of (S)-7 can, furthermore,
contribute a weaker hydrogen bond to lower DHS and (A 1
DSS) in comparison with those for (S)-4. To confirm the elu-
cidation, more experiments of varying the substituent to the
2-phenoxyl group or use of theoretical predictions via molec-
ular modeling are needed.
Kinetic Analysis
Figure 5 illustrates the specific activity varied with the ini-
tial enantiomer concentration (SR) or (SS) for 5–7, with
which the specificity constants k2R/KmR and k2S/KmS can be
estimated and represented in Table 4. It is also reasonable to
Fig. 3. Variations of ln(k2R/KmR
ln(E) (&, ~) with inverse of absolute temperature for 4 (*, !, &) and
7 (l, !, ~).
) (*, l), ln(k2S/KmS) (!, !), and
Chirality DOI 10.1002/chir

CHLOROPHENOXYL PYRAZOLIDES AS NOVEL SUBSTRATES
65
TABLE 3. Thermodynamic analysis for CALB-catalyzed hydrolysis of 1, 2, and 4–7 in water-saturated MTBE
DHR
DHS
A 1 DSR
A 1 DSS
DGR 2 AT
DGS – AT
2DDH
2DDS
2DDG
Entry (kJ mol21
)
(kJ mol21
)
(J mol21 K21
)
(J mol21 K21
)
(kJ mol21
)
(kJ mol21
)
(kJ mol21
)
(J mol21 K21
)
(kJ mol21
)
1
2
4
5
6
7
39.58
78.24
57.42
74.76
71.67
56.92
31.80
54.55
47.47
51.10
48.76
41.80
116.8
219.9
143.8
217.4
192.1
129.9
116.3
175.5
157.5
170.3
151.2
128.6
2.42
8.27
11.67
5.59
10.55
15.59
25.20
21.28
22.63
23.08
0.64
7.78
23.69
9.94
23.66
22.92
15.11
0.56
44.45
213.66
47.09
40.94
1.33
7.60
9.55
14.28
8.68
9.89
14.69
0.88
Reaction conditions as given in Tables 1–3; 2DGR, 2DGS and 2DDG calculated at 458C.
estimate the constants equal to VR/(Et)/(SR) and VS/(Et)/
(SS) for 4 at the low substrate concentration of 3 mM in Ta-
ble 2. Moreover, by further assuming the same enantiomer
affinity to the active site for each racemate, one obtains KmR
5 KmS 5 38.5 mM for 5, 29.7 mM for 6, and 120 mM for 7.
The highest Michaelis constants for 7 can be attributed to
the dichloro substituent on hindering the substrate affinity
to active site. In the hydrolysis of (R,S)-2-phenylpropionyl
pyrazolides containing a bulky leaving 3-(2-pyridyl)pyrazole
or 3-(3-bromophenyl)pyrazole,8 the Michaelis constants KmR
and KmS are too large to be determined, and were attributed
to the adsorbed water allocated in active site near the 3-sub-
stituted substituent on impeding the substrate affinity. Appa-
rently, this will relax if the 2-phenyl moiety is replaced by a
chlorophenoxyl group in 5–7. More data from experiments
or theoretical predictions via molecular modeling are needed
to elucidate the different behaviors from interactions
between chlorophenoxyl oxygen atom and amino acid resi-
dues/adsorbed water for fine-tuning the active site.
lytic serine to the carbonyl carbon atom and proton transfer
to the leaving 3-(2-pyridyl)pyrazole of (S)-5 or (S)-7 but not
(S)-6. On the contrary, a 2-chloro substituent in (R)-6 or (R)-
7 but not (R)-5 mainly decreases the nucleophilic attack and
proton transfer when considering an order-of-magnitude
higher k2R for the later. Therefore, on the basis of 6, the
excellent enantiomeric ratio is attributed to the high k2S/KmS
of fast-reacting enantiomer for 4 and lower k2R/KmR of slow-
reacting antipode for 7, as envisaged from changes of (DGR
– AT) and (DGS – AT) at 458C in Table 3.
CONCLUSION
The lipase-catalyzed hydrolytic resolution of (R,S)-2-chloro-
phenoxylpyrazolides was studied for preparing optically pure
CPAs containing a 2-, 3-, 4-chloro, or 2,4-dichloro substituent
to the 2-phenoxyl moiety. The best reaction condition of
CALB as the biocatalyst, water-saturated MTBE as the reac-
tion medium, 258C for 5 and 6 and 458C for 4 and 7 was
selected, leading to good (E > 48) to excellent (E > 100)
enantioselectivity and high reactivity for the fast-reacting
enantiomer, when comparing the results for CRL or CPL. A
detailed thermodynamic analysis for CALB-catalyzed hydro-
lysis of 1, 2, and 4–7 demonstrates profound influences of
the acyl or leaving group on varying enthalpic and entropic
contributions to the difference of Gibbs free energies DGR,
DGS, and 2DDG. The kinetic analysis also indicates that on
the basis of 6, the excellent enantiomeric ratio for 4 and 7 is
It is difficult to explain why the maximum specificity con-
stants occur for 5 but not 4 containing the least hindered 4-
chlorophenoxyl group for performing the nucleophilic attack
by catalytic serine. However, by considering the lowest k2S
for (S)-6, a 3- or 4-chloro substituent to the phenoxyl group
is advantageous to enhance the nucleophilic attack of cata-
TABLE 4. Effects of substrate structure on kinetic constants
for CALB-catalyzed hydrolysis at 458C
Entry
4
5
6
7
k2R/KmR
1.20 E 22 1.64 E 21 2.10 E 22 3.16 E 23
(l h21 g21
)
KmR (mM)
k2R
ND
ND
3.85 E 11 2.97 E 11 1.20 E 12
6.32
6.25 E 21 3.79 E 21
(mmol h21 g21
k2S/KmS
)
2.64
4.16
1.16 7.57 E 21
(l h21 g21
)
KmS (mM)
k2S
ND
ND
3.85 E 11 2.97 E 11 1.20 E 12
1.60 E 12 3.44 E 11 9.09 E 11
(mmol h21 g21
)
E
220.0
25.3
55.1
239.5
Fig. 5. Specific activity varied with initial enantiomer concentration (SR0
)
Reaction conditions: 10 ml water-saturated MTBE at 400 rpm. Symbol E 21
as 1021. ND as not determined due to shortage of (R,S)-2-(4-chlorophenoxy)-
propionic acid from Sigma–Aldrich.
or (SS0) for CALB-catalyzed hydrolysis in water-saturated MTBE at 458C: VS/
(Et) for (S)-5 (!), (S)-6 (&), and (S)-7 (*); 20VR/(Et) for (R)-5 (!),
100VR/(Et) for (R)-6 (~), and (R)-7 (l). (—) Best-fit results.
Chirality DOI 10.1002/chir

66
KAO ET AL.
14. Colton IJ, Ahmed SN, Kazlauskas RJ. A 2-propanol treatment increases
the enantioselectivity of Candida rugosa lipase toward esters of chiral
carboxylic acids. J Org Chem 1995;60:212–217.
attributed to the higher k2S/KmS of (S)-4 and lower k2R/KmR
of (R)-7.
LITERATURE CITED
15. Tsai SW, Dordick JS. Extraordinary enantiospecificity of lipase catalysis
in organic media induced by purification and catalyst engineering.
Biotechnol Bioeng 1996;52:296–300.
1. Bornscheuer UT, Kazlauskas RJ. Hydrolases in organic synthesis: regio-
and stereoselective biotransformations. 2nd ed. Weinheim: Wiley-VCH
Verlag; 2006. p 37–275.
16. Cipiciani A, Cittadini M, Fringuelli F. Improving the enantioselectivity
of Candida rugosa lipase in the kinetic resolution of racemic
methyl 2-(2,4,-dichlorophenoxy)propionate. Tetrahedron 1998;54:7883–
7890.
2. Hanefeld U. Reagents for (ir)reversible enzymatic acylations. Org Biomol
Chem 2003;1:2405–2415.
3. Hudson EP, Eppler RK, Clark DS. Biocatalysis in semi-aqueous and
nearly anhydrous conditions. Curr Opin Biotechnol 2005;16:637–643.
17. Cipiciani A, Bellezza F, Fringuelli F, Stillitano M. Enantioselectivity of
alcohol-treated Candida rugosa lipase in the kinetic resolution of racemic
methyl 2-aryloxypropionates in water and aqueous organic media.
Tetrahedron: Asymmetry 1999;10:4599–4605.
4. Toscano MD, Woycechowsky KJ, Hilvert D. Minimalist active-site redesign:
teaching old enzymes new tricks. Angew Chem Int Ed 2007;46: 3212–3236.
5. Wang PY, Chen TL, Tsai SW. Improvements of enzyme activity and enan-
tioselectivity via combined substrate engineering and covalent immobili-
zation. Biotechnol Bioeng 2008;101:460–469.
18. Ueji SI, Tanaka H, Hanaoka T, Ueda A, Watanabe K, Kaihatsu K, Ebara
Y. Effects of chemical modification of lipase on its enantioselectivity in
organic solvents. Chem Lett 2001;1:1066–1067.
6. Wang PY, Chen YJ, Wu AC, Lin YS, Kao MF, Chen JR, Ciou JF, Tsai SW,
(R,S)-Azolides as novel substrates for lipase-catalyzed hydrolytic resolu-
tion in organic solvents. Adv Synth Catal 2009;351:2333–2341.
19. Watanabe K, Ueji SI. Dimethyl sulfoxide as a co-solvent dramatically
enhances the enantioselectivity in lipase-catalyzed resolutions of 2-phe-
noxypropionic acyl derivatives. J Chem Soc Perkin Trans 1 2001;30:1386–
1390.
7. Wu AC, Wang PY, Lin YS, Kao MF, Chen JR, Ciou JF, Tsai SW, Improve-
ments of enzyme activity and enantioselectivity in lipase-catalyzed alco-
holysis of (R,S)-azolides. J Mol Catal B: Enzym 2010;62:235–241.
20. Ammazzalorso A, Amoroso R, Bettoni G, De Filippis B, Fantacuzzi M,
Giampietro L, Maccallini C, Tricca ML. Candida rugosa lipase-catalysed
kinetic resolution of 2-substituted-aryloxyacetic esters with dimethylsulf-
oxide and isopropanol as additives. Chirality 2008;20:115–118.
8. Wang PY, Wu CH, Ciou JF, Wu AC, Tsai SW. Kinetic resolution of (R,S)-
pyrazolides containing substituents in the leaving pyrazole for increased
lipase enantioselectivity. J Mol Catal B: Enzym 2010;66:113–119.
21. Ujang Z, Husain WH, Seng MC, Rashid AHA. The kinetic resolution of
2-(4-chlorophenoxy) propionic acid using Candida rugosa lipase. Proc
Biochem 2003;38:1483–1488.
9. Liantonio A, Accardi A, Carbonara G, Fracchiolla G, Loiodice F, Tortor-
ella P, Traverso S, Guida P, Pierno S, De Luca A, Camerino DC, Pusch
M. Molecular requisites for drug binding to muscle CLC-1 and renal
CLC-K channel revealed by the use of phenoxy-alkyl derivatives of 2-(p-
chlorophenoxy)propionic acid. Mol Pharmacol 2002;62:265–271.
22. Mohile SS, Potdar MK, Harjani JR, Nara SJ, Salunkhe MM. Ionic liquids:
efficient additives for Candida rugosa lipase-catalysed enantioselective
hydrolysis of butyl 2-(4-chlorophenoxy)propionate. J Mol Catal B: Enzym
2004;30:185–188.
10. Dernoncour R, Azerad R, Enantioselective hydrolysis of 2-(chlorophenoxy)
propionic esters by esterases. Tetrahedron Lett 1987;28:4661–4664.
23. Kawamoto T, Sonomoto K, Tanaka A, Efficient optical resolution of 2-(4-
chlorophenoxy)propanoic acid with lipase by use of organosilicon com-
pounds as substrate: the role of silicon atom in enzymatic recognition.
J Biotechnol 1991;18:85–92.
11. Cheng YC, Tsai SW. Enantioselective esterification of (R,S)-2-(4-chloro-
phenoxy)-propionic acid via Carica Papaya lipase in organic solvents.
Tetrahedron: Asymmetry 2004;15:2917–2920.
24. Cammenberg M, Hult K, Park S. Molecular basis for the enhanced
lipase-catalyzed N-acylation of 1-phenylethanamine with methoxyacetate.
ChemBioChem 2006;7:1745–1749.
12. Cheng YC, Tsai SW. Carica papaya lipase: an effective biocatalyst
for esterification resolution of (RS)-2-(chlorophenoxy)propionic acid.
Biochem Eng J 2007;35:318–324.
25. Sun JH, Dai RJ, Meng WW, Deng YL. Efficient enzymatic kinetic resolu-
tion of 2-heptylamine with a highly active acyl donor. Catal Commun
2010;11:987–991.
13. Wu SH, Guo ZW, Sih CJ. Enhancing the enantioselectivity of Candida
lipase catalyzed ester hydrolysis via noncovalent enzyme modification.
J Am Chem Soc 1990;112:1990–1995.
Chirality DOI 10.1002/chir