K. Watanabe et al.
Bull. Chem. Soc. Jpn., 77, No. 3 (2004)
547
The concentration of each enantiomer was varied from 0.006–0.05
mmol and the concentration of 1-butanol was kept constant (0.15
mmol). Each enantiomer was submitted to the model reaction un-
der the same additive conditions. At an appropriate time interval,
aliquots were withdrawn and the supernatant was analyzed by
HPLC on a chiral column to determine the initial rate at each sub-
strate concentration. The values of Km and Vmax were obtained
from the Michaelis–Menten equation. All the experimental data
points obtained from the Lineweaver–Burk plot gave a straight line
with the correlation coefficient >0:97. The values were reproduci-
ble to about Æ5% on repeated runs.
the enantioselectivity. The improvement of the enantioselectiv-
ity is mainly attributed to the lower reactivity of the S enan-
tiomer for the alcohol bearing the longer alkyl chain, while
the reactivity for the R enantiomer is increased by increase of
the lipase flexibility brought about by addition of water. Thus,
this result obtained also supports the model that for the S enan-
tiomer, the serious steric difficulty in attacking the acyl-enzyme
would cause the small sensitivity of the Vmax value to the lipase
flexibility. For lipase-catalyzed reactions, the choice of the al-
cohol as a nucleophile is found to be a useful method to im-
prove the enantioselectivity.
ESR Measurement. The active site (serine) of the semi-puri-
fied lipase was spin-labeled with 4-(ethoxyfluorophosphoryloxy)-
2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO-4-EPF) purchased
from SIGMA, according to the procedure reported by Morrisett
and Broomfield.12 It can be assumed that the spin label has attached
to the active site, because the spin-labeled lipase showed a clear de-
crease in the enzymatic activity for the esterification of 1. Typical-
ly, about 35% of the active sites are considered to be labeled, as
calculated from the residual enzymatic activity. All the ESR meas-
Conclusions
For lipase-catalyzed reactions in organic solvents, the in-
crease of the lipase flexibility brought about by addition of wa-
ter is found to be favorable for the induced fit motion of the li-
pase for the correctly binding R enantiomer of the substrate 1,
thus resulting in the improvement of the lipase enantioselectiv-
ity. In the induced fit motion, the CH ꢀ association between
ÁÁÁ
ꢀ
urements were carried out at room temperature (ca. 25 C) on a
Bruker EMX081 spectrometer at X-band frequency in organic sol-
vents containing 0.3 vol % of water.
amino acids side chains around the lipase’s active site and
the aromatic ring of the R enantiomer is assumed to stabilize
the complex between the enzyme and the substrate. Further-
more, on the basis of the model concerning the acyl-enzyme
structure for the incorrectly binding enantiomer, the long alkyl
chain alcohols that act as nucleophiles are found to improve li-
pase enantioselectivity markedly.
The success of the model should be of interest to organic
chemists and useful for understanding the mechanism for the
optimization of the lipase-catalyzed reactions in organic
solvents.
Molecular Arrangement to Accommodate Each Enantiomer
into the Lipase’s Active Site. The molecular arrangement to ac-
commodate each enantiomer into the lipase’s active site was car-
ried out on the basis of the molecular modeling (Mac Spartan
Pro) using the X-ray structure of Candida rugosa lipase obtained
from the Brookhaven Data Bank.6 In the modeling for the acyl-en-
zyme structure of the correctly binding R enantiomer (overlapping
van der Waals radius was avoided), the aromatic ring of the sub-
strates 1–2 was accommodated well in the active site model and
two leucines are placed around the aromatic ring with almost
van der Waals contact.
Experimental
Materials. Lipase MY was supplied from Meito Sangyo Co.,
Ltd., Japan, and was semi-purified by dialyzing and lyophilizing
from crude materials. Organic solvents were purchased from Wako
Pure Chemical Industries, Ltd., Japan, and were dried over molec-
ular sieves for more than 24 h before use. Racemic 2-(4-substituted
phenoxy)propionic acids 1–2 were prepared by the reaction of the
corresponding 4-substitiued phenol and ethyl 2-bromopropionate
(Tokyo Kasei Kogyo Co., Ltd., Japan), followed by hydrolysis of
the corresponding esters, according to a known method.9
References
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´
ꢀ
´
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`
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3
4
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yl ether (2 mL). To the solution, 1.0 vol % of water was added, fol-
lowed by ultrasonic dispersion, and then the crude lipase (30 mg)
was added.
Michaelis–Menten Kinetic Parameters. The kinetic study
was carried out by measuring the initial rates of the transesterifica-
tion of each enantiomer of 1–2. Each enantiomer was prepared by
the lipase MY-catalyzed esterification, according to our method.11
and Y. Ebara, Chem. Lett., 2001, 912.
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5
7
M. Nishio, M. Hirota, and Y. Umezawa, ‘‘The CH/ꢀ Inter-
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