Lyophilization of lipase together with ionic compounds generates highly enantioselective and solvent-sensitive lipase in organic solvents

Article abstract of DOI:10.1246/cl.2005.110

The lyophilization of lipase from aqueous enzyme solution containing ionic compounds such as disodium hydrogenphosphate resulted in a dramatic improvement of its enantioselectivity for esterification in organic solvents, compared with the ionic compound-free lipase. The enantioselectivity of the ionic compound-coated lipase was found to be fairly sensitive to the change of the solvents. Copyright

Full text of DOI:10.1246/cl.2005.110

110
Chemistry Letters Vol.34, No.1 (2005)
Lyophilization of Lipase Together with Ionic Compounds Generates Highly Enantioselective
and Solvent-sensitive Lipase in Organic Solvents
Shin-ichi Ueji,ꢀ Shu-ichi Mori,y Hiromi Yumoto, Naomi Hiroshima, and Yasuhito Ebara
Division of Natural Environment and Bioorganic Chemistry, Faculty of Human Development and Sciences,
Kobe University, Nada, Kobe 657-8501
yGraduate School of Science and Technology, Kobe University, Nada, Kobe 657-8501
(Received September 29, 2004; CL-041152)
The lyophilization of lipase from aqueous enzyme solution
CH
CH
n-BuOH
3
3
containing ionic compounds such as disodium hydrogenphos-
phate resulted in a dramatic improvement of its enantioselectiv-
ity for esterification in organic solvents, compared with the ionic
compound-free lipase. The enantioselectivity of the ionic com-
pound-coated lipase was found to be fairly sensitive to the
change of the solvents.
Lipase
Organic solvent / H2O
37 °C
X
O CH CO Bu
2
R-preference
X
O CH CO H
2
(R,S)-1-10
1:X=Et, 2:X=H, 3:X=Me, 4:X=n-Pr, 5:X=i-Pr,
6:X=n-Bu, 7:X=t-Bu, 8:X=MeO, 9:X=CF3, 10:X=Cl
Scheme 1. Lipase catalyzed esterification of 2-(4-substituted
phenoxy) propanoic acids 1–10 in organic media.
Table 1. Enantioselectivity (E value) for esterification of 1 in
isopropyl ether catalyzed by lipase lyophilized in the presence
of various ionic compounds
Although lipases are most frequently used as an important
tool in organic synthesis of optically pure compounds,1 native li-
pases are often poor enantioselective toward non-natural sub-
strates such as pharmaceuticals, agricultural, and other specialty
chemicals. This is mainly because their structures are widely dif-
ferent from those of natural substrates, triacylglycerols. To over-
come this difficulty, a variety of strategies were proposed for
controlling the enzyme enantioselectivity.2 Further develop-
ments, however, are awaited with interest as the efficient ap-
proach for exploring the enzyme enantioselectivity, because of
the increasing need of various fine chemicals and intermediates
with high stereochemical purity. Our continuing interest in seek-
ing the enzyme enantioselectivity3 led us to examine a new
method based on an easy preparation procedure.
Here, we wish to report that the enantioselectivity of
Candida rugosa lipase-catalyzed esterification of 2-(4-substitut-
ed phenoxy)propanoic acids 1–10 is markedly improved by use
of lipase lyophilized in the presence of ionic compounds such as
disodium hydrogenphosphate (Na2HPO4) or L-alanine. As an-
other remarkable feature of the lipase prepared, its E value is
found to be much more sensitive to the change of the dielectric
constant of the solvent as the reaction medium than that of native
lipase.
In a typical enzymatic reaction, the substrates 1–10
(0.36 mmol) prepared from the known method4 and a three-fold
excess of n-BuOH (1.08 mmol) were dissolved in 2 mL of iso-
propyl ether containing 0.75 vol % of water. To the solution,
Candida rugosa lipase (6.1 mg) resulting from lyophilization
in the presence of the ionic compound was added and the suspen-
sion was shaken (150–170 strokes/min) at 37 ꢁC. Lipase sample
used here was prepared by lyophilizing an aqueous enzyme solu-
tion (100 mL) containing semi-purified lipase5 (100 mg) and
Na2HPO4 (14.6 mmol/g of enzyme) or L-alanine (500 mmol/g
of enzyme). The ee was measured by HPLC on a chiral column
(Chiralcel OK, from Daicel Chemical Industries).
For the model reaction (Scheme 1), the lyophilized lipase
catalyzed preferentially the R enantiomer of all the substrates
used. Initially, various ionic compounds were tested as the addi-
tives in the lyophilization of lipase to investigate the enhance-
ment effect of the enantioselectivity (E value)7 for the model re-
Ionic compounda
Time/h Conv./% ee/% E value
Noneb
Na2HPO4 (14.6 mmol)
27
24
38.2
39.9
37.5
37.4
35.4
33.0
35.9
35.4
36.6
36.6
38.2
85.0
99.1
97.9
91.6
99.3
98.2
98.4
98.0
96.7
96.5
92.0
77.3
21
463
170
40
c
d
Na5P3O10 (5.84 mmol)
24
DNA (ca.0.04 mmol)
L-Ala (500 mmol)
D-Ala (500 mmol)
L-Leu (200 mmol)
D-Leu (200 mmol)
L-Phe (50 mmol)
D-Phe (50 mmol)
Gly (500 mmol)
117
21
520
175
211
169
104
98
21
17.5
17.5
18
18
18
43
NH2–(CH2)4–CO2He (500 mmol) 210
2.32
7.9
aThe value in the parenthesis indicates the optimal concentration (mmol/
g of enzyme) to maximize the E value, among their different concentra-
tions examined (for example, E ¼ 202 at 7.3 mmol of Na2HPO4 and E ¼
252 at 29.2 mmol of Na2HPO4). bSemi-purified native lipase. cDisodium
e
hydrogenphosphate. dSodium tripolyphosphate. 5-aminovaleric acid.
action using 1 (Table 1). As can be seen from the E value listed
in Table 1, Na2HPO4 and L-alanine in the lipase preparation
brought about ca. 20-fold jump in the E value in isopropyl ether,
as compared with native lipase. On the other hand, the enhance-
ment effect in the E value is small when the same concentrations
of these ionic compounds were only added to the reaction mix-
ture (for example, E ¼ 22 at 0.4 vol % of aq Na2HPO4, E ¼ 23
at 0.75 vol % of aq Na2HPO4, and E ¼ 13 at 1.1 vol % of aq
Na2HPO4).
To our knowledge, this is the first example of the dramatic
enhancement of the lipase enantioselectivity caused by the
incorporation of the ionic compounds during lyophilization of
native lipase,8 although there have been several interesting
reports on the effect of simple inorganic salts on the activation
of enzymes.9 Furthermore, the different behavior in the E value
between L- and D-amino acids may be mainly attributed to a dif-
ferent fashion in the chiral interaction between each enantiomer
of the amino acid with dipolar ion and the charges on the surface
of lipase (see Table 1).
Copyright ꢀ 2005 The Chemical Society of Japan

Chemistry Letters Vol.34, No.1 (2005)
111
Table 2. Enantioselectivity (E value) for esterification of 2–10
in isopropyl ether catalyzed by lipase lyophilized with Na2HPO4
(14.6 mmol/g of enzyme) or L-Ala (500 mmol/g of enzyme)
500
400
300
200
100
None
Na2HPO4
Na5P3O10
L-Ala
X
Ionic compound Time/h Conv./% ee/% E value
Nonea
2
3
4
5
6
7
8
9
H
119
38.2
59.5
5.6
b
Na2HPO4
L-Ala
—
690
50
26.4
36.7
32.9
34.9
38.2
41.4
37.3
35.6
41.2
39.9
37.9
42.4
38.2
34.9
39.9
34.9
39.8
9.5
79.9
80.6
99.0
98.0
71.2
96.8
95.9
89.1
98.3
98.1
62.6
88.6
94.8
53.2
66.3
88.4
84.4
86.2
96.8
53.1
91.2
92.1
64.1
95.0
92.0
12
15
Me
None
Na2HPO4
L-Ala
None
50
49
327
169
n-Pr
i-Pr
19.5
9.1
1.5
2.5
3.5
4.5
Na2HPO4
L-Ala
22
26
129
85
Dielectric constant /
ε
Figure 1. Plot of E value for lipase-catalyzed esterification
against dielectric constant (") of solvent. The values of dielectric
constant of the solvent examined were taken from Ref. 10: n-
hexane (1.88), n-heptane (1.92) isooctane (1.95), cyclohexane
(2.22), benzene (2.28), dibutyl ether (3.08), isopropyl ether
(3.88), and t-butyl methyl ether (4.5).
None
24
22
28
Na2HPO4
L-Ala
None
240
204
24
n-Bu
t-Bu
MeO
CF3
310
47
6.3
Na2HPO4
L-Ala
None
33
68
120
435
60
4.3
7.5
26
of the lipase active site allowing an easier access for the R enan-
tiomer, and not for the S enantiomer. Thus, the opposite direction
in their initial rates is ascribed to the marked enhancement of the
enantioselectivity.
This electrostatic association would be strongly controlled
by the bulk property, dielectric constant, of the solvent as the re-
action medium. Indeed, as is seen in Figure 1, the enantioselec-
tivity of the ionic compound-coated lipase is found to be
changed profoundly on switching the solvent from n-hexane
(E ¼ 55 for lipase with Na2HPO4) to isopropyl ether (E ¼ 463
for lipase with Na2HPO4); this large solvent effect is thought
to be significantly amplified by the electrostatic association ap-
pended to lipase surface.
Na2HPO4
L-Ala
552
50
None
21
15
Na2HPO4
L-Ala
None
138
59
35.6
41.8
34.9
35.5
37.0
26.3
35.9
104
192
60
4.7
Na2HPO4
L-Ala
35
40
310
96
10 Cl
None
Na2HPO4
L-Ala
6.6
210
310
54
40
aSemi-purified native lipase. bThe reaction rate was too late to obtain a
reliable E value.
In conclusion, the advantage of the ionic compound-coated
lipase is that its enantioselectivity can be greatly altered by the
combination effects of the ionic compound and the solvent.
The scope of this enhancement effect in lipase enantioselec-
tivity was evaluated by the model reaction using the other sub-
strates 2–10 with a wide variety of substituents (Table 2). The
data summarized in Table 2 shows that the E value enhancement
brought about by the lipase preparation in the presence of
Na2HPO4 or L-alanine was observed for all the substrates, except
for 2 and 8 using Na2HPO4, although a few substrates displayed
a lowering of the reaction rate.
We then investigated the effect of the ionic compound on in-
itial rate for each enantiomer of 1 (Table 3). In the presence of
the lyophilized lipase with Na2HPO4, the initial rate for the R
enantiomer (correctly reacting substrate) was largely accelerat-
ed, whereas the S enantiomer (incorrectly reacting one) was de-
celerated, relative to native lipase. The direction of the initial
rate observed is explained by assuming that the electrostatic as-
sociation between the ionic compound and the charges on lipase
surface may influence the conformation and/or the local polarity
References and Notes
1
For recent reviews, see: A. M. Klibanov, Nature, 409, 241 (2001);
P. Berglund, Biomol. Eng., 18, 13 (2001); F. Bordusa, Chem. Rev.,
102, 4817 (2002).
2
3
For recent reviews, see: F. Theil, Tetrahedron, 56, 2905 (2000);
U. T. Bornscheuer, Curr. Opin. Biotechnol., 13, 543 (2002).
T. Okamoto and S. Ueji, Chem. Commun., 1999, 939; K. Watanabe
and S. Ueji, J. Chem. Soc., Perkin Trans. 1, 2001, 1386; S. Ueji, M.
Nishimura, R. Kudo, R. Matsumi, K. Watanabe, and Y. Ebara, Chem.
Lett., 2001, 912.
4
5
D. T. Witiak, T. C.-L. Ho, and R. E. Hackney, J. Med. Chem., 11,
1086 (1968).
The semi-purified lipase was previously prepared by dialyzing
and lyophilizing from crude material (Sigma Chemical Co.). The
MALDI-TOF MS spectrum of the lipase showed a single parent
peak, m/z 60.2 kD, which is consistent with the molecular weigh
of Candida rugosa lipase.6
6
7
R. J. Kazlauskas, TIBTECH, 12, 464 (1994).
C.-S. Chen, Y. Fujimoto, G. Girdaukas, and C. J. Sih, J. Am. Chem.
Soc., 104, 7294 (1982).
The water contents of native lipase and Na2HPO4-coated lipase
estimated by the Karl Fischer method under the reaction conditions
were 573 and 769 mg, respectively. This difference of the water
content seems not to be the main factor for enhancing the E value
dramatically.
Table 3. Initial rate of esterification of each enantiomer of 1 in
isopropyl ether catalyzed by native lipase and lipase lyophilized
with Na2HPO4 (14.6 mmol/g of enzyme)
8
Initial rate (mmol hꢂ1mgꢂ1
)
Ionic compound
VR/VS
b
b
VR
VS
Nonea
Na2HPO4
8.0
41.0
0.45
0.14
18
293
9
For a recent review, see: M.-Y. Lee and S. Dordick, Curr. Opin.
Biotechnol., 13, 376 (2002).
aSemi-purified native lipase. bThe VR and VS denote the initial rates of
the R and S enatniomers, respectively.
10 C. Reichardt, ‘‘Solvents and Solvent Effects in Organic Chemistry,’’
VCH, Weinheim (1988).
Published on the web (Advance View) December 18, 2004; DOI 10.1246/cl.2005.110