Effects of chiral additives on enantioselectivity for lipase-catalyzed esterifications in an organic solvent. A remarkable enhancement of its enantioselectivity due to cooperative effects of two kinds of additives

Article abstract of DOI:10.1246/bcsj.75.2239

The enantioselectivity for the lipase-catalyzed esterifications in an organic solvent was found to be enhanced by addition of (+)- or (-)-camphor as a chiral additive. A more remarkable enhancement of its enantioselectivity was brought about by the cooper

Full text of DOI:10.1246/bcsj.75.2239

c. Jpn.
Bull. Chem. Soc. Jpn., 75, 2239–2240 (2002) 2239
e Chemical
ciety of Ja-
n
Short Articles
e Chemical
ciety of Ja-
n
combine broad substrate specificity with high enantioselectivi-
ty.
Effects of Chiral Additives on Enan-
tioselectivity for Lipase-Catalyzed
Esterifications in an Organic Sol-
vent. A Remarkable Enhancement
of Its Enantioselectivity Due to Co-
operative Effects of Two Kinds of
Additives
Here, we report the effect of camphor as a chiral additive on
the enantioselectivity for the lipase-catalyzed esterification in
an organic solvent. Furthermore, the use of the combined ad-
ditives, camphor and a small amount of water, was found to
display a more remarkable enhancement of enantioselectivity.
As a model reaction, we chose the esterification of 2-(4-sub-
stituted phenoxy)propionic acids with 1-butanol catalyzed by
Candida rugosa lipase MY in isopropyl ether containing 0.15
vol% of water (Scheme 1). In this reaction, the lipase cata-
lyzed preferentially the R enantiomer of all the substrates used.
2-Phenoxypropionic acids are well-known herbicides and also
have other biological activities.10
Initially, we investigated the effects of (+)- or (−)-camphor
as a chiral additive on the enantioselectivity in the model reac-
tion. Table 1 summarizes the results of the variation of the
enantioselectivity (E value11) at ca. 40% conversion in the es-
terification of the substrates 1–5 by addition of 5 mol% of
camphor. Also, the enantioselectivity for 3 is strongly depen-
dent on the content (mol%) of (+)- or (−)-camphor in the re-
action medium (for (+)-camphor, E = 24 at 2.5 mol%, E = 27
at 5.0 mol%, E = 30 at 7.5 mol%, and E = 39 at 10 mol%; for
(−)-camphor, E = 36 at 2.5 mol%, E = 42 at 5.0 mol%, E =
44 at 7.5 mol%, and E = 58 at 10 mol%). The data of Table 1
shows that the addition of (−)-camphor produces a more re-
markable enhancement of the enantioselectivity than that of
(+)-camphor for all the substrates 1–5. Furthermore, the dif-
ferent behavior in the enantioselectivity between (+)- and (−)-
camphors suggests that each enantiomer of camphor may in-
teract with the lipase molecule in a different fashion.
02
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SJA8
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S. Ueji et al.
Shin-ichi Ueji,* Hideyuki Sakamoto, Keiichi
Watanabe,† Takashi Okamoto,† and Shuichi Mori
02
Division of Natural Environment and Bioorganic Chemistry,
Faculty of Human Development and Sciences,
Kobe University, Nada, Kobe 657-8501
02
0.3
†The Graduate School of Science and Technology,
Kobe University, Nada, Kobe 657-8501
(Received May 17, 2002)
The enantioselectivity for the lipase-catalyzed esterifi-
cations in an organic solvent was found to be enhanced by ad-
dition of (+)- or (−)-camphor as a chiral additive. A more
remarkable enhancement of its enantioselectivity was
brought about by the cooperative effects of two kinds of addi-
tives: (−)-camphor and water.
To ascertain whether the carbonyl group of camphor plays
an important role in the interaction with the lipase, bornane
(obtained from reduction of camphor) was tested as an additive
under the same conditions as the addition of camphor in the
model reaction, thus resulting in the disappearance of the
enantioselectivity enhancement (E = 20). Taking into account
this observation, one could speculate that the camphor effect
on the enantioselectivity may be mainly ascribed to a change
in a flexibility and/or in a local conformation around the bind-
ing site of the lipase through an electrostatic interactions in-
volving a hydrogen bonding between the carbonyl group of
camphor and the lipase molecule.
To elucidate a mechanism of the enantioselectivity enhance-
ment, we investigated the initial rate for each enantiomer of 3
in isopropyl ether by addition of 5 mol% of (−)-camphor
(Table 2). As is seen in Table 2, although (−)-camphor
brought about the decrease in the initial rates for both enanti-
omers as compared with that for no additive conditions, the
larger deceleration was observed for the incorrectly binding S-
enantiomer. Such a large difference in their initial rates is at-
tributed to the enantioselectivity enhancement.
Enhancing the enantioselectivity of enzymes remains a
challenging problem in the field of the enzyme technology.
Although various strategies have been introduced for this pur-
pose, use of additives has been worthy of remark because it is a
simple and a cheap way to alter enzyme functions.1–8 Recent-
ly, a review concerning the additive effects on enzyme-cata-
lyzed reactions has appeared.9 Indeed, the satisfactory results
of the enzyme reactions have been achieved by using certain
additives in the reaction mixture.9 In particular, the extensive
investigations of the additive effects have been directed mainly
towards lipases employed for organic synthesis, because they
Our next step was to investigate the cooperative effects of
two kinds of additives, 5 mol% of (+)- or (−)-camphor and
0.125 vol% of water, upon the enantioselectivity of 3 in the
model reaction. Simultaneous addition of these additives pro-
duced a more remarkable enhancement of the enantioselectivi-
ty than expected from the sum of each effect (Table 3). To our
Scheme 1. Lipase-catalyzed esterification of 2-(4-substituted
phenoxy)propionic acids 1–5 with 1-butanol in isopropyl
ether.

2240 Bull. Chem. Soc. Jpn., 75, No. 10 (2002)
© 2002 The Chemical Society of Japan
Table 1. Effects of (+)- or (−)-Camphor as a Chiral Additive on the Enantioselectivity at ca. 40% Conversion in the Lipase-Cata-
lyzed Esterification of 2-(4-Substituted Phenoxy)propionic Acids 1–5 in Isopropyl Ether
No additive
(+)-Camphor (5 mol%)
(−)-Camphor (5 mol%)
Substrate
X
Time/h
97
40
30
50
Convn.%
ee/%
34
82
85
81
E
Time/h
100
37
Convn.%
ee/%
37
83
E
Time/h
166
50
Convn.%
ee/%
45
86
E
3.5
24
42
28
18
1
2
3
4
5
H
CH3
CH3CH2
CH3(CH2)2
CH3(CH2)3
45
41
43
44
43
2.6
18
24
18
7.6
41
41
42
43
44
2.7
19
27
18
8.5
40
41
45
40
41
33
53
80
87
81
67
42
67
92
90
88
82
76
65
Table 2. Effects of (−)-Camphor as Additive on the Initial
Rate for Each Enantiomer of 3 in the Lipase-Catalyzed
Esterification in Isopropyl Ether
Isopropyl ether and (+)- and (−)-camphors were purchased from
Wako Pure Chemical Industries, Ltd., Japan. Racemic 2-(4-sub-
stituted phenoxy)propionic acids 1–5 were prepared by the reac-
tion of the corresponding 4-substituted phenol and ethyl 2-bro-
mopropionate, according to the known method.14
Lipase-Catalyzed Esterification. The substrates 1–5 (0.36
mmol) and 1-butanol (1.08 mmol, 3 equiv) were dissolved in iso-
propyl ether (2 mL). To the solution, a small amount of the addi-
tives ((+)- or (−)-camphor, or water) was added, followed by ul-
trasonic dispersion, and then Candida rugosa lipase (30 mg) was
added. The suspension was shaken (170 strokes/min) at 37 °C.
The E value was calculated from the enantiomeric excess (ee) for
the butyl ester produced, according to the literature.11 The ee was
measured by HPLC on a chiral column (Chiralcel OK, from
Daicel Chemical Industries Co. Ltd., Japan).
Initial rate/µM h−1
Additive
VR/VS
VR
VS
None
(−)-Camphor
0.70
0.57
0.080
0.053
8.8
11
Table 3. Cooperative Effects of Two Kinds of the Additives,
5 mol% of (+)- or (−)-Camphor and 0.125 vol% of Water
upon the Enantioselectivity at ca. 40% Conversion in the
Lipase-Catalyzed Esterification of 3 in Isopropyl Ether
Additive
Time/h
ee/%
E
None
H2O
(+)-Camphor + H2O
(−)-Camphor + H2O
27
10
11
14
85
91
93
96
24
26
46
84
Initial Rate for Lipase-Catalysed Esterification.
Accord-
ing to our method, (R)- or (S)-ethyl 2-(4-ethylphenoxy)propionate
3 was prepared.4 Each enantiomer was submitted to the model re-
action under the same additive conditions. At an appropriate time
interval, aliquots were withdrawn, and the supernatant was ana-
lyzed by HPLC on a chiral column to determine the conversion.
Five data points (less than 10% conversion) were collected to de-
termine the initial rate coefficient > 0.96.
knowledge, this is the first example of the enantioselectivity
enhancement brought about by the cooperative effects of two
kinds of additives. This observation can be explained by as-
suming that the flexible conformation of the lipase caused by
water addition is liable to associate with the carbonyl group of
camphor. As to the effect of added water on enzymes in organ-
ic solvents, it is known that water acts as a lubricant to increase
the enzyme’s flexibility, due to its ability to form multiple hy-
drogen bonds.12,13 In fact, in anhydrous isopropyl ether, the
enantioselectivity enhancement due to camphor becomes
much smaller (data not shown).
For a brief test of the validity of the combined use of these
additives, Candida rugosa lipase AY also displayed a marked
increase in the enantioselectivity in the model reaction (E = 45
for no additive, E = 54 for (+)-camphor additive (5 mol%), E
= 57 for (−)-camphor additive (5 mol%), E = 57 for water
additive (0.125 vol%), E = 66 for (+)-camphor (5 mol%) and
water additive (0.125 vol%), and E = 73 for (−)-camphor (5
mol%) and water additive (0.125 vol%)).
References
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In conclusion, our approach based on the combined use of
the additives demonstrates the feasibility of a more effective
way to improve the outcome of enzyme reactions, as compared
with the use of a single additive. In this approach, we recom-
mend the combination of a hydrophobic additive and a hydro-
philic one.
9
F. Theil, Tetrahedron, 56, 2905 (2000).
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Experimental
Materials. Lipase MY and lipase AY were supplied from
Meito Sangyo Co., Ltd., and Amano Pharmaceutical Co., Ltd.,
Japan, respectively, and were used without further purification.