Lipase-catalyzed enantioselective resolution of (R,S)-N-2-methylalkanoyl-3- (2-pyridyl)pyrazoles in organic solvents

Article abstract of DOI:10.1016/j.molcatb.2010.11.012

The lipase-catalyzed resolution of (R,S)-pyrazolides containing a 2-aryl substituent to the α-chiral center has been successfully extended to (R,S)-N-2-methylalkanoyl-3-(2-pyridyl)pyrazoles (1-4) containing different alkanoyl-chain lengths. The best reaction condition for CALB-catalyzed hydrolysis of (R,S)-N-2-methylheptanoyl-3-(2-pyridyl)pyrazole (1) in water-saturated MTBE at 35 °C is selected, leading to an excellent enantioselectivity (V_R/V_S > 100) with improved initial specific activities in comparison with that of (R,S)-N-2-phenylpropionyl-3-(2- pyridyl)pyrazole. The thermodynamic analysis for the hydrolysis of 1 demonstrates great influences of water content and solvent hydrophobicity on varying the ehthalpic and entropic contributions in water-saturated and anhydrous MTBE and IPE, and leads to an excellent enthalpy-entropy compensation relationship ΔΔS = 3.113ΔΔH + 33.86 (r² = 0.999). Moreover, a thorough kinetic analysis for all substrates indicates that a critical valeroyl-chain length for obtaining the enantiomer discrimination and improved lipase activity for the fast-reacting (R)-pyrazolide is needed. Copyright

Full text of DOI:10.1016/j.molcatb.2010.11.012

Journal of Molecular Catalysis B: Enzymatic 68 (2011) 245–249
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Lipase-catalyzed enantioselective resolution of
(R,S)-N-2-methylalkanoyl-3-(2-pyridyl)pyrazoles in organic solvents
∗
Yi-Sheng Lin, Pei-Yun Wang, 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:
Received 2 September 2010
Received in revised form
The lipase-catalyzed resolution of (R,S)-pyrazolides containing a 2-aryl substituent to the ␣-chiral cen-
ter has been successfully extended to (R,S)-N-2-methylalkanoyl-3-(2-pyridyl)pyrazoles (1–4) containing
different alkanoyl-chain lengths. The best reaction condition for CALB-catalyzed hydrolysis of (R,S)-N-2-
methylheptanoyl-3-(2-pyridyl)pyrazole (1) in water-saturated MTBE at 35 C is selected, leading to an
excellent enantioselectivity (VR/VS > 100) with improved initial specific activities in comparison with that
of (R,S)-N-2-phenylpropionyl-3-(2-pyridyl)pyrazole. The thermodynamic analysis for the hydrolysis of
2
2 November 2010
◦
Accepted 22 November 2010
Available online 27 November 2010
1
demonstrates great influences of water content and solvent hydrophobicity on varying the ehthalpic
Keywords:
and entropic contributions in water-saturated and anhydrous MTBE and IPE, and leads to an excellent
enthalpy–entropy compensation relationship ꢀꢀS = 3.113ꢀꢀH + 33.86 (r = 0.999). Moreover, a thor-
ough kinetic analysis for all substrates indicates that a critical valeroyl-chain length for obtaining the
enantiomer discrimination and improved lipase activity for the fast-reacting (R)-pyrazolide is needed.
Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved.
(R,S)-N-2-methylalkanoyl-3-(2-
2
pyridyl)pyrazoles
Lipases
Hydrolysis
Alcoholysis
Kinetic and thermodynamic analysis
1
. Introduction
Lipases as versatile biocatalysts have been employed for the
and susceptible to nucleophilic attack, especially when accepting
an proton from the imidazolium of catalytic histidine in the rate-
limiting acylation step [14,15].
preparation of chiral alcohols, amines, carboxylic acids, or their
derivatives for synthesizing a variety of fine chemicals includ-
ing pharmaceuticals [1,2]. In order to obtain the fine-tuning of
enzyme active-site structure to enhance the enantiomer discrim-
ination toward targeted racemates, various approaches of using
substrate engineering [3–6], medium engineering [7,8], enzyme
engineering [9–11], or their combinations have been proposed.
Recently, a kinetic resolution process using (R,S)-azolides, i.e.
If the rationale of obtaining improved enzyme performance is
valid for the (R,S)-pyrazolides containing an aryl substituent to
the ␣-chiral center, one may wonder if the resolution platform
can be extended to other substrates without containing the aryl
moiety mainly causing the enantiomer discrimination when com-
paring with the other substituent such as a methyl, ethyl, bromo,
or methoxy group in the ␣-chiral center [14]. By assigning (R,S)-
N-2-methylalkanoyl-3-(2-pyridyl)pyrazoles as the substrates, it is
aimed to investigate the alkanoyl-chain length on affecting the
lipase activity and enantioselectivity in water-saturated or anhy-
drous organic solvents. The resultant (R)- or (S)-2-methylalkanoic
acid products have been used as drugs, pesticides, flavors and fra-
grances, sweeteners, and synthetic building blocks [16]. They can
be prepared mainly via Candida rugosa lipase-catalyzed hydrolysis
of (R,S)-2-methylalkanoyl alkyl esters, esterification or transester-
ification of the corresponding acids or vinyl esters by alcohol in
alkane solvents [16–24]. Depending on the alkanoyl-chain length
and reaction conditions, low to good enantioselectivity with in gen-
eral low enzyme activity due to the substrate inhibition from acid
or alcohol was found.
(
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 [12–14]. The
results indicated that the activity and enantioselectivity of an
immobilized Candida antarctica lipase B (CALB) in water-saturated
or anhydrous methyl tert-butyl ether (MTBE) greatly increased,
if (R,S)-pyrazolides containing a leaving 3- or 3,4-substituted-
pyrazole moiety were employed for preparing optically pure
carboxylic acids or esters containing an 2-aryl group to the ␣-chiral
center. This may be attributed to the unique pyrazolide structure
that contains an unshared electron pairs on the acyl-substituted
nitrogen N(1), making the carbonyl carbon atom more electrophilic
The best reaction condition for lipase-catalyzed hydrolysis
of (R,S)-N-2-methylheptanoyl-3-(2-pyridyl)pyrazole in water-
saturated solvents is first selected and extends to the alcoholysis
by methanol in anhydrous solvents. The thermodynamic anal-
∗ Corresponding author. Tel.: +886 3 2118800x3415; fax: +886 3 2118668.
E-mail address: tsai@mail.cgu.edu.tw (S.-W. Tsai).
1
381-1177/$ – see front matter. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2010.11.012

2
46
Y.-S. Lin et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 245–249
O
R
CALB
O
O
N
R
+
R
N
H O
2
N
N
OH
N
N
or
(R)-acid
(S)-pyrazolide
(R,S)-pyrazolide
O
R
CALB
O
N
+
N
R
CH OH
3
OCH3
N
(
R)-ester
(S)-pyrazolide
R:
(1)
(2),
(3)
(4)
C H
5
C H
,
C H
C H
,
5
11
4
9
3
7
2
Scheme 1. Lipase-catalyzed enantioselective resolution of (R,S)-N-2-methylalkanoyl-3-(2-pyridyl)pyrazoles.
ysis for each enantiomer is then carried out. The hydrolytic
anhydrous MgSO for 12 h, filtered and concentrated under reduced
4
resolution is also applied to other (R,S)-N-2-methylalkanoyl-
pressure, giving the desired oily products. All the synthesized sub-
strates were confirmed from the 1H NMR spectra recorded at
400 MHz on Brucker AC-400 spectrometer in DMSO-d6 solution
3
-(2-pyridyl)pyrazoles containing
length for doing the comparison from the kinetic analysis
Scheme 1).
a
shorter alkanoyl-chain
(
with TMS as an internal standard.
1
(R,S)-N-2-methylheptanoyl-3-(2-pyridyl)pyrazole (1); H NMR
(
DMSO-d /TMS) ı: 0.83 (3H, t), 1.23–1.53 (11H, m), 3.87 (1H, q),
6
2
. Materials and methods
7
(
.15 (1H, d), 7.43–7.46 (1H, q), 7.91–7.94 (1H, m), 8.0 (1H, d), 8.47
1H, d), 8.66 (1H, d). The abbreviations d, q, m and s were the peak
multiplicities of doublet, quartet, multiplet and single, respectively.
R,S)-N-2-methylhexanoyl-3-(2-pyridyl)pyrazole (2); 1H NMR
2.1. Materials
(
Lipase MY from Candida rugosa and Novozym 435 as a Can-
(DMSO-d /TMS) ı: 0.85 (3H, t), 1.24–1.55 (9H, m), 3.88 (1H, q), 7.15
6
dida antartica lipase B (CALB) immobilized on acrylic resins
were provided by Meito Sangyo (Tokyo, Japan) and Novo
Nordisk (Bagsvaerd, Denmark), respectively. Dimethyl sulfoxide-
d6 (DMSO-d ) containing 1% (v/v) tetramethylsilane (TMS) for
NMR analysis was from Cambridge Isotope Laboratories (Andover,
MA). Other chemicals of analytical grade were commercially
available: (R,S)-2-methylbutyric acid and (R,S)-2-methylvaleric
acid from TCI (Tokyo, Japan); (R,S)-2-methylheptanoic acid from
Acros (Geel, Belgium); (R,S)-2-methylhexanoic acid and 3-(2-
pyridyl)-1H-pyraozle from Alfa Aesar (Ward Hill, MA); benzene,
cyclohexane (CYC), dipropylether (IPE), hexane (HEX), isopropanol
(
1H, d), 7.44–7.46 (1H, q), 7.92–7.95 (1H, m), 8.0 (1H, d), 8.47 (1H,
d), 8.67 (1H, d).
R,S)-N-2-methylvaleroyl-3-(2-pyridyl)pyrazole (3); 1H NMR
DMSO-d /TMS) ı: 0.88 (3H, t), 1.25–1.57 (11H, m), 3.91 (1H, q),
(
1
H
6
(
6
7
(
.17 (1H, d), 7.45–7.47 (1H, q), 7.93–7.95 (1H, m), 8.0 (1H, d), 8.49
1H, d), 8.68 (1H, d).
R,S)-N-2-methylbutanoyl-3-(2-pyridyl)pyrazole (4); 1H NMR
DMSO-d /TMS) ı: 0.91 (3H, t), 1.24 (3H, t), 1.59 (2H, s), 3.82 (1H,
(
(
6
q), 7.15 (1H, d), 7.44–7.47 (1H, q), 7.92–7.95 (1H, m), 8.0 (1H, d),
8
.49 (1H, d), 8.67 (1H, d).
(
IPA), isooctane (ISO), and methyl t-butyl ether (MTBE) from
2.3. Analysis
Tedia (Fairfield, OH); calcium hydride and methanol from
Riedel-deHaen (Seelze, Germany). Anhydrous IPE and MTBE
were prepared by adding calcium hydride to both solvents
for 24 h.
The hydrolysis or alcoholysis of (R,S)-pyrazolides was mon-
itored by HPLC using a chiral column from Daicel (OJ-H;
Tokyo, Japan) that was capable of separating the internal
standard of benzene (BEN). UV detection at 270 nm was
employed for quantification at the room temperature. The
mobile phase composition (HEX:IPA:acetic acid, v/v/v) were
2.2. General procedures for substrates synthesis
9
4
9.8:0.1:0.1 for 1, 98:2:0 for 2 and 3, and 99.4:0.1:0.5 for
. The retention time for the internal standard and enan-
To 2.5 ml benzene containing 1 mmol racemic acid, 1.2 mmol
-(2-pyridyl)pyrazole, and 4 mmol triethylamine was added drop-
3
tiomers were BEN:(S)-1:(R)-1 = 2.3:8.5:10.0 min, BEN:(S)-2:(R)-
2 = 2.2:3.5:3.8 min, BEN:(R)-3:(S)-3 = 2.2:3.5:3.8 min, and BEN:(S)-
4
dissolved in ethanol were determined at 589 nm on a Atago AP-100
polarimeter.
wise a mixture containing 0.5 ml benzene and 1 mmol thionyl
◦
chloride at 0 C with stirring for 2 h. The resultant mixture
:(R)-4 = 2.6:10.7:12.4 min. Optical rotations of the acid product
was filtered, 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 solu-
tion (3 × 10 ml). The organic phase was separated, dried over

Y.-S. Lin et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 245–249
247
Table 1
Effect of lipase source, solvent, and temperature on enantioselective resolution of (R,S)-N-2-methylheptanoyl-3-(2-pyridyl)pyrazole.
◦
Lipase
Solvent
Temp. ( C)
VR/(Et) (mmol/h g)
VS/(Et) (mmol/h g)
VR/VS or VS/VR
Time (h)
Xt (%)
ees (%)
Hydrolysis in water-saturated solvents
Lipase MY
CALB
CALB
CALB
CALB
CALB
CALB
CALB
CALB
CYC
CYC
ISO
25
25
25
25
25
35
45
35
45
3.12E−5
7.69E−2
3.92E−1
2.20E−2
3.19E−2
5.58E−2
1.17E−1
4.88E−2
1.14E−1
4.20E−4
5.02E−4
2.08E−3
7.39E−5
1.65E−4
3.90E−4
1.40E−3
3.90E−4
1.71E−3
13
153
21.6
3.3
1.0
8.5
8.5
3.4
2.3
3.4
1.5
3.1
48.1
56.6
47.1
48.6
43.7
46.1
43.3
43.8
3.6
92.8
100.0
86.8
94.5
85.9
95.2
81.8
83.2
188
298
193
143
84
IPE
MTBE
MTBE
MTBE
IPE
125
66
IPE
Alcoholysis by 100 mM methanol in anhydrous solvents
CALB
CALB
CALB
CALB
CALB
CALB
MTBE
MTBE
MTBE
IPE
IPE
IPE
25
35
45
25
35
45
3.13E−1
6.66E−1
9.21E−1
1.08E−1
2.60E−1
6.16E−1
2.36E−3
9.00E−3
1.44E−2
1.38E−3
4.26E−3
1.08E−2
132
74
64
78
61
57
5.4
2.1
1.9
4.7
2.4
1.9
53.7
54.0
51.5
50.3
49.6
51.2
96.9
100.0
98.1
92.6
89.4
91.7
Reaction conditions: 10 ml solvent containing 3 mM racemate at 400 rpm; 20 mg/ml lipase employed for hydrolysis; 4 mg/ml CALB for alcoholysis except for 10 mg/ml in IPE
◦
−1
at 25 and 35 C. Symbol E−1 as 10 . The temperature-variation of water content in water-saturated organic solvents can be found from [26–28], e.g. 467.7 mM and 110.6 mM
◦
in water-saturated MTBE and IPE, respectively, at 35 C. It is reasonable to assume no water dissolving in anhydrous solvents after adding calcium hydride into the solvent
for 24 h.
2.4. Effects of lipase, solvent, temperature, and substrate structure
(R)-acid) [25], one might determine the (R)-preference for CALB and
S)-preference for Lipase MY.
(
To 10 ml water-saturated CYC containing 3 mM of 1 was added
2
0 mg/ml of Lipase MY or CALB for performing the hydroly-
◦
3. Results and discussion
sis at 25 C. Samples were removed from the reaction medium
at different time intervals for HPLC analysis, from which the
time-course conversions XR and X , initial rates for both enan-
3.1. Effects of lipase source, solvent, and temperature
S
tiomers VR and VS based on several conversion determinations,
racemate conversion Xt, and enantiomeric excess for the sub-
strate ees were determined. Similar experiments were carried
out for CALB in water-saturated ISO, IPE or MTBE. In order to
study the temperature effect on CALB performances, the same
Table 1 demonstrates effects of lipase source, solvent, and tem-
perature on the kinetic resolution of (R,S)-N-2-methylheptanoyl-
-(2-pyridyl)pyrazole on changing the initial specific activities
VR/(Et) and V /(Et), enantioselectivity in terms of VR/V or V /VR,
3
S
S
S
Xt, and ees at a specified reaction time. In comparison with Lipase
MY-catalyzed esterification of (R,S)-2-methylheptanoic acid with
1-hexadecanol in CYC at the room temperature (E = 23, Xt = 15.1%,
ees = 16.1% at 5.4 h) [19], a better enzyme activity and enantioselec-
reaction was carried out in water-saturated IPE or MTBE at
◦
3
5 or 45 C. The alcoholytic resolution via CALB was also per-
◦
formed at 25–45 C, except that 100 mM methanol as an acyl
acceptor was added to 10 ml anhydrous IPE or MTBE containing
tivity (VR/V = 153, Xt = 48.1%, ees = 92.8% at 3.3 h) for CALB is shown
S
3
mM of 1.
◦
for the hydrolysis of 1 in water-saturated CYC at 20 C, regardless
More experiments were carried for investigating the substrate
of giving an opposite stereo-preference to Lipase MY. A change of
the reaction medium to ISO but not IPE or MTBE results in 5-fold
enhancements of VR/(Et) and slight increasing of the enzyme enan-
tioselectivity. However, by considering the low water solubility in
ISO and CYC that is disadvantageous for carrying the hydrolytic res-
olution, IPE and MTBE are selected for the temperature screening.
structure on the enzyme activity and enantioselectivity, in which
the alkanoyl-chain length was reduced from 7 to 4. In order to com-
pare the kinetic behaviors at 35 C, the hydrolysis of varying the
◦
substrate concentration of 1–4 in water-saturated MTBE was car-
ried out. From the time-course conversions XR and X , and hence
S
the specific initial rates VR/(Et) and V /(Et), the kinetic constants
S
◦
An increase of reaction temperature to 45 C has resulted in
k2R/KmR, k2S/KmS, and E value (i.e. k2RKmS/k2SKmR) were estimated
enhancements of VR/(Et) and V /(Et), but leading to decreasing
S
[
14].
of the enantioselectivity, in water-saturated IPE and MTBE. By
considering the higher water solubility and enzyme activity with
2.5. Product separation for determining lipase enantioselectivity
VR/V value higher than 100, CALB-catalyzed hydrolysis in water-
saturated MTBE at 35 C is selected as the best reaction condition.
S
◦
After removing the lipase employed in the kinetic analysis, the
In comparison the hydrolysis of (R,S)-N-2-phenylpropionyl-
◦
resultant solutions of Xt about 70% were collected, added to a
biphasic medium containing 5 ml benzene and 10 ml aqueous solu-
tion (0.1 N NaOH), and stirred for 20 min. The aqueous phase was
separated, added to 10 ml benzene containing 0.45 ml HCl solu-
tion (37%), and stirred for 20 min. The organic phase was then
3-(2-pyridyl)pyrazole in water-saturated MTBE at 45 C
−
2
−5
(VR/(Et) = 1.27 × 10 mmol/h g,
V /(Et) = 2.63 × 10 mmol/h g,
S
and VR/V = 482) with that of 1 [14], the lower CALB activity with
S
higher enantioselectivity may be attributed to the more hindered
2-phenyl substituent for decreasing the substrate affinity and
proton transfer in the active site.
separated, dried over anhydrous MgSO for 12 h, filtered and con-
4
centrated under reduced pressure to give the acid product. From
In comparison with the hydrolytic resolution, the methanolysis
the optical rotations of [␣]D = −9.1 (c 0.11, EtOH) for (R)- and (S)-
in anhydrous MTBE or IPE has yielded enhanced VR/(Et) and V /(Et),
but leading to decreasing of the enantioselectivity, especially at low
S
-methylvaleric acid mixture (Lit. [␣]D2 = −20.1 (c 5.5, Et O) for
0
2
2
the (R)-acid) [17], [␣]D = −13.0 (c 0.38, EtOH) for (R)- and (S)-2-
temperature. For example for the alcoholysis in anhydrous MTBE at
methylhexanoic acid mixture (Lit. [␣]D2 = −20.2 (c 5.3, Et O) for
0
25 C, 9.8- and 14.3-fold higher of VR/(Et) and V /(Et), respectively,
◦
2
S
the (R)-acid) [17], and [␣]D = −9.7 (c 0.31, EtOH) for (R)- and (S)-2-
methylheptanoic acid mixture (Lit. [␣]D = −15.6 (c 0.55, EtOH) for
and hence 1.4-fold lower of VR/V , were estimated from Table 1.
S
These are very similar to the kinetic behaviors by using (R,S)-N-

2
48
Y.-S. Lin et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 245–249
Table 2
Thermodynamic analysis for CALB-catalyzed hydrolysis of (R,S)-N-2-methylheptanoyl-3-(2-pyridyl) pyrazole.
Solvent
ꢀHR (kJ/mol)
ꢀHS (kJ/mol)
A + ꢀSR (J/mol K)
A + ꢀSS (J/mol K)
−ꢀꢀH (kJ/mol)
−ꢀꢀS (J/mol K)
−ꢀꢀG (kJ/mol)
Hydrolysis in water-saturated solvents
MTBE
IPE
51.1
73.0
83.9
130.4
142.6
211.6
209.0
347.1
32.8
57.3
66.0
145.4
12.5
12.5
Alcoholysis in anhydrous solvents
MTBE
IPE
42.7
68.6
71.6
81.1
134.0
211.6
191.3
217.6
28.8
12.4
56.6
5.4
11.3
8.9
◦
Reaction conditions given in Table 1; −ꢀꢀG calculated at 35 C.
2
-phenylpropionyl-3-(2-pyridyl)pyrazole as the substrate, and can
be attributed to the water adsorbed in the active site near the bulky
-(2-pyridyl)pyrazole moiety on impeding the substrate affinity in
enzyme molecules on changing the number of degrees of freedom.
Yet in Table 2, one can still obtain the enthalpy–entropy compen-
2
3
sation relationships ꢀSR = 2.900ꢀHR + 4.187 − A (r = 0.959) and
2
water-saturated MTBE [14]. Apparently, more studies are needed
for determining whether the hydrolysis or the alcoholysis is the
best resolution platform.
ꢀS = 2.704ꢀH − 6.982 − A (r = 0.988).
S
S
The excellent enthalpy–entropy compensation phenomena
ꢀꢀS = 3.113ꢀꢀH + 33.86 (r = 0.999) might be attributed to the
2
narrow range of parameters investigated, i.e. similar solvents and
acyl acceptors of water and methanol. It is interesting to find the
increase of −ꢀꢀH from 12.4 kJ/mol of anhydrous IPE to 28.8 kJ/mol
of MTBE, implying that the polar MTBE is favorable for enhancing
interactions between the fast-reacting (R)-enantiomer at the tran-
sition state and enzyme residues to lower the enthalpy. Apparently,
the addition of water in water-saturated IPE is advantageous for
giving the interactions mainly for (R)-enantiomer, yielding −ꢀꢀH
of 57.3 kJ/mol, as some water will bind to the active site. How-
ever, more water added and hence adsorbed in water-saturated
MTBE also contributes the enthalpic gain for both (R)- and (S)-
enantiomers at the transition state, leading to slight increasing of
−ꢀꢀH from 28.8 to 32.8 kJ/mol. More data on using other solvents
and substrates are needed in the future in order to confirm the eluci-
dation and obtained empirical relationships. By further comparing
3
.2. Thermodynamic analysis
The transition state theory relates the microscopic rate con-
stant of an elementary step to the Gibbs free energy difference
between reactant’s ground state and the activated transition
state [29]. By assuming Michaelis–Menten kinetics for each
enantiomer in the acylation step, the equation relating the
kinetic constants k2i and Kmi and the difference of Gibbs
free energies between ground and transition states ꢀGi is
expressed as k /K = (ꢁkBT/h)exp(−ꢀG /RT) with h, kB, R, T, ꢁ
2i
mi
i
as Plank’s constant, Boltzmann’s constant, gas constant, abso-
lute temperature, transmission coefficient, respectively, and hence
ln(E) = −ꢀꢀG/RT = −ꢀꢀH/RT + ꢀꢀS/R with −ꢀꢀG, −ꢀꢀH, and
◦
−
ꢀꢀS as the differences of Gibbs free energies (ꢀGR and ꢀG ),
−ꢀꢀH and −ꢀꢀG at 35 C for each case, the enantiomer discrimi-
S
enthalpies (ꢀHR and ꢀH ), and entropies (ꢀSR and ꢀS ) between
nation is found to be enthalpy-driven, especially for the hydrolysis
in water saturated IPE.
S
S
the transition states of both enantiomers. For the temperature
varying in a narrow range, the equation is approximated as
ln(k /K ) = −ꢀH /RT + (A + ꢀS )/R, with A = R ln(ꢁkBT/h) regarded as
2
i
mi
i
i
a constant.
3.3. Kinetic analysis
Good liner relationships between ln(VR/V ) and ln(k /Kmi) and
, i = R and S, in water-saturated or anhydrous solvents in Table 1
S
2i
−
1
T
The best reaction condition for CALB-catalyzed hydrolysis
of 1 in water-saturated MTBE is extended to other (R,S)-
N-2-methylalkanoyl-3-(2-pyridyl)pyrazoles containing a shorter
alkanoyl-chain length. Some results are tabulated in Table 3, in
which very similar enzyme performances with excellent enan-
tioselectivity are demonstrated when using 1–3 as the substrates.
However by using 4 as the substrate, more than 20-fold increases of
V /(Et)/(S ) and 6-fold decreases of VR/(Et)/(SR) in comparison with
were found, from which ꢀH , (A + ꢀS ), −ꢀꢀH, and −ꢀꢀS were
i
i
estimated and represented in Table 2. The thermodynamic param-
eter ꢀHR or ꢀH increases by replacing the anhydrous solvent by
S
water-saturated ones, implying that the stabilization effect due to
interactions of more dissolved water with substrate, solvent, and
enzyme molecules at the ground state is higher than that at the
transition state, especially for ꢀH of the slow-reacting enantiomer
S
S
S
in water-saturated IPE. On the contrary, the unfavorable enthalpic
contribution due to desolvation of polar water (or methanol) at
the transition state should be compensated from the favorable
enthalpic gain of the polar group with water (or methanol) and
those for 1 are obtainable and yield no enantioselectivity. This can
be attributed to the slight difference of ethyl and methyl moieties
to the ␣-chiral center on discriminating (R)-1 and (S)-1. However,
when the ethyl moiety is replaced by a propyl group for 3, about 1.5-
solvent, leading to a lower ꢀHR (or ꢀH ) in the more polar water-
fold enhancements of VR/(Et) with 49.6-fold decreases of V /(Et),
S
S
saturated (or anhydrous) MTBE relative to IPE. In general, it is
and hence giving VR/V as 74.
S
difficult to elucidate the variations of ꢀSR and ꢀS and hence ꢀꢀS
with the reaction condition, owing to the complicated entropic con-
tributions from interactions between water, solvent, substrate, and
In order to shed insights into effects of alkanoyl-chain length
on the hydrolytic resolution, the kinetic analysis based on
Michaelis–Menten mechanism was carried out. Fig. 1 illustrate
S
Table 3
◦
Effect of substrate structure on CALB-catalyzed hydrolysis at 35 C.
Entry
(SR) or (SS) (mM)
VR/(Et) (mmol/h g)
VS/(Et) (mmol/h g)
VR/VS
Time (h)
Xt (%)
ees (%)
1
2
3
4
4.4
5.5
2.0
4.2
1.78E−1
2.01E−1
4.11E−2
2.75E−2
1.41E−3
1.52E−3
5.54E−4
2.75E−2
126
132
74
3.0
4.0
4.1
4.2
59.7
51.4
50.7
45.1
100.0
87.8
78.0
0.0
1
Reaction conditions: 10 ml water-saturated MTBE at 400 rpm. Symbol E−1 as 10−1. From the liner relationships in Fig. 1, one can compare CALB activity for each enantiomer
via VR/(Et)/(SR) or VR/(Et)/(SR).

Y.-S. Lin et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 245–249
249
2.5
2.0
1.5
1.0
0.5
0.0
the best reaction condition of CALB-catalyzed hydrolysis in
water-saturated MTBE at 35 C is selected, in which an excellent
◦
enantioselectivity (VR/V > 100) with improved enzyme activities
S
is obtainable when comparing with V /(Et) and VR/(Et) using (R,S)-
S
N-2-phenylpropionyl-3-(2-pyridyl)pyrazole as the substrate. The
thermodynamic analysis demonstrates the great influence of water
content and solvent hydrophobicity on varying the ehthalpic and
entropic contributions in water-saturated and anhydrous MTBE
and IPE, and leads to an excellent enthalpy–entropy compensation
relationship between ꢀꢀS and ꢀꢀH. The resolution platform is
successfully extended to other substrates of 2 and 3, but not 4. A
thorough kinetic analysis for all substrates indicates that a critical
valeroyl-chain length is needed for obtaining the enantiomer
discrimination and improved lipase activity for the fast-reacting
(R)-enantiomer.
Acknowledgements
0
20
40
60
80
Financial supports from National Science Council (Grant No. NSC
99-2221-E-182-028) are appreciated.
(
S ) or (S ) (mM)
R S
Fig. 1. Variations of VR/(Et) and 40VR/(Et) with (SR) and (SS) for CALB-catalyzed
hydrolysis in water-saturated MTBE for (R)-1 (ꢀ), (S)-1 (᭹), (R)-2 (ꢁ), (S)-2 (ꢀ),
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(
—) Best-fit results.
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1