Rate enhancement of lipase-catalyzed reaction in supercritical carbon dioxide

Article abstract of DOI:10.1246/cl.2005.1102

Lipase-catalyzed kinetic resolution of various 1-arylethanols was performed in supercritical carbon dioxide (scCO₂), in hexane and without solvent. The use of scCO₂ enhances reactivity especially when a cross-linked enzyme aggregate (CLEA) was used. Copyright

Full text of DOI:10.1246/cl.2005.1102

1102
Chemistry Letters Vol.34, No.8 (2005)
Rate Enhancement of Lipase-catalyzed Reaction in Supercritical Carbon Dioxide
Tomoko Matsuda,ꢀ Kazuhiko Tsuji,y Takashi Kamitanaka,y Tadao Harada,y Kaoru Nakamura,yy and Takao Ikariyayyy
Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501
yDepartment of Materials Chemistry, Ryukoku University, Otsu, Shiga 520-2194
yyInstitute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011
yyyDepartment of Applied Chemistry and Frontier Collaborative Research Center, Tokyo Institute of Technology,
Meguro-ku, Tokyo 152-8552
(Received April 18, 2005; CL-050534)
Lipase-catalyzed kinetic resolution of various 1-aryletha-
ing or withdrawing ability of the substituents.
nols was performed in supercritical carbon dioxide (scCO2), in
hexane and without solvent. The use of scCO2 enhances reactiv-
ity especially when a cross-linked enzyme aggregate (CLEA)
was used.
The enantioselectivity evaluated by E-value8 was extremely
high (E > 1000) for the reaction in scCO2 or hexane except for
the case of 1c. However, when the solvent was not used, enantio-
selectivity decreased for cases 1d and 1f.
OH
OH
O
Supercritical carbon dioxide (scCO2) has been used as a sol-
vent for organic synthesis due to its environmental friendliness,
high diffusivity that increases the rate of diffusion-limited reac-
tions, ease of product separation from solvents, etc.1 As a cata-
lyst for reactions, chemical catalysts have been widely used.1 Al-
though not as extensively as chemical catalysts, biocatalysts
have also been used due to high chemo-, regio-, and enantiose-
lectivities and high reactivity.2,3
Among biocatalytic reactions in scCO2, lipase-catalyzed ki-
netic resolution is one of the most important reactions because
asymmetric synthesis is possible. Therefore, in this study, de-
tailed examinations of substrate specificity for kinetic resolution
by Candida antarctica lipase B, especially using 1-arylethanols,
were conducted using a batch scCO2 system because many
1-arylethanols have been used as intermediates for the synthesis
of pharmaceuticals, agrochemicals, and natural products.4 The
reaction proceeded with very high enantioselectivity, and more-
over, it was found that reactivity was improved by using scCO2
over the cases using hexane5 and without solvent. Interestingly,
when two different methods of enzyme preparation (Novozym
and CLEA), derived from the same enzyme (Candida antarctica
lipase B), were compared, rate enhancement was more obvious
when CLEA was used.
+
+
O
X
X
(R)-1a-f
2
(S)-1a-f
O
O
OH
Candida antarctica
lipase
+
scCO2,
hexane or
non-solvent
X
X
1a: X = H
1b: X = p-Br
1c: X = p-CH3
1d: X = p-CF3
1e: X = m-CF3
1f: X = m,m-(CF3)2
(S)-1a-f
(R)-3a-f
Figure 1. Kinetic resolution of 1-arylethanols by lipase.
Next, different preparations of the same lipase from Candi-
da antarctica, CLEA, were used for the kinetic resolution of 1a
in scCO2 or in hexane as well as without solvent. CLEA 103 is
the lipase preparation suitable for transformations in aqueous
solvents (hydrolysis), and CLEA 301 is developed for transfor-
mation in organic media (transesterification).9 For the reaction
in scCO2, both preparations almost equally catalyzed the reac-
tion with high efficiency. The conversion was higher than those
using Novozym when the same enzyme weight was used. The
intriguing point is that the rate acceleration of the CLEA cata-
lyzed reaction using scCO2 is more prominent than cases using
Novozym. When Novozym was used, conversion improved
from 25% (hexane or non-solvent) to 31% (scCO2) (Table 1);
but when CLEA was used, it improved from 13–35% (hexane
or non-solvent) to over 46% (scCO2) (Table 2).
Although the reasons for the rate enhancement using scCO2
for Novozym and CLEA catalyzed reactions remain unclear at
present, plausible causes may be related to three factors: 1) des-
olvation of enzyme and/or substrate as reported to cause rate ac-
celeration for homogeneous reactions using lipid-coated enzy-
mes,2a,2b 2) conformational changes of the enzyme by adsorp-
tion, absorption, and/or binding of CO2 to the enzyme partially
through formation of carbamates from CO2 and the free amine
groups on the surface of the enzyme as reported previous-
ly,2d,3c,3d,10 or 3) the high diffusivity of scCO2.11 Because rate ac-
celeration using CLEA (enzyme aggregate) was larger than that
using Novozym, high diffusivity of scCO2 may be more impor-
Using a lipase from Candida antarctica as a catalyst and
vinyl acetate 2 as an acetyl donor, kinetic resolution of 1-phenyl-
6
ethanol derivatives 1 was conducted in scCO2 or in hexane as
well as without solvent. As shown in Figure 1, (R)-1 selectively
reacted, resulting in the formation of (R)-3 and unreacted (S)-1.
Absolute configurations were determined by comparing the GC
retention times with the authentic samples prepared by the meth-
ods reported in the literature.4a,7
The results using an immobilized lipase preparation, Novo-
zym, are listed in Table 1. The conversions of scCO2 reactions
under pressure of 9 MPa were higher than in hexane or under
non-solvent conditions. The rates of the reactions at 13 MPa
were similar to those at 9 MPa (data not shown). The effect of
the position of the substituents on the phenyl ring was obvious.
The conversion was in the order of: 1b (p-Br), 1c (p-CH3), 1d
(p-CF3) > 1a (H) > 1e (m-CF3) > 1f (m,m-(CF3)2). When
there is a substituent at the para position, conversion was better
than unsubstituted substrate 1a, regardless of the electron donat-
Copyright Ó 2005 The Chemical Society of Japan

Chemistry Letters Vol.34, No.8 (2005)
1103
Table 1. Novozym catalyzed kinetic resolution of 1 in various
solvents
The authors greatly appreciate the discussions and the gift of
CLEA given from Prof. Roger Sheldon and Dr. Rob Schoevaart
of CLEA Technologies.
(S)-1a (R)-3 Conv.b
Substrate/Conditions
E
ee/% ee/%
/%
References and Notes
1a
1a
1a
scCO2 9 MPa
Hexane
Non-solvent
45.8 99.9
32.9 >99.9
32.9 >99.9
31
25
25
>1000
>1000
>1000
1
P. G. Jessop, T. Ikariya, and R. Noyori, Nature, 368, 231
(1994); P. G. Jessop, T. Ikariya, and R. Noyori, Science,
269, 1065 (1995); P. G. Jessop, T. Ikariya, and R. Noyori,
Chem. Rev., 99, 475 (1999); M. F. Sellin, I. Bach, J. M.
1b
1b
1b
scCO2 9 MPa
Hexane
Non-solvent
90.2
75.3 >99.9
56.0 >99.9
99.8
48
43
36
>1000
>1000
>1000
´
Webster, F. Montilla, V. Rosa, T. Aviles, M. Poliakoff,
and D. J. Cole-Hamilton, J. Chem. Soc., Dalton Trans.,
2002, 4569; J. M. DeSimone, Science, 297, 799 (2002); B.
M. Bhanage, Y. Ikushima, M. Shirai, and M. Arai, Chem.
Commun., 1999, 1277.
1c
1c
1c
scCO2 9 MPa
Hexane
Non-solvent
80.4
57.0
57.1
96.1
98.5
97.9
46
37
37
125
230
167
2
a) T. Mori and Y. Okahata, Chem. Commun., 1998, 2215.
b) T. Mori, A. Kobayashi, and Y. Okahata, Chem. Lett.,
1998, 921. c) A. J. Mesiano, E. J. Beckman, and A. J.
Russell, Chem. Rev., 99, 623 (1999). d) Y. Ikushima, N.
Saito, M. Arai, and H. W. Blanch, J. Phys. Chem., 99,
8941 (1995). e) N. Mase, T. Sako, Y. Horikawa, and K.
Takabe, Tetrahedron Lett., 44, 5175 (2003). f) J. Ottosson,
L. Fransson, J. W. King, and K. Hult, Biochim. Biophys.
Acta, 1594, 325 (2002). g) M. D. Romeroa, L. Calvoa, C.
Albaa, M. Habulin, M. Primozic, and Z. Knez, J. Supercrit.
Fluids, 33, 77 (2005). h) E. Celia, E. Cernia, C. Palocci, S.
Soro, and T. Turchet, J. Supercrit. Fluids, 33, 193 (2005).
i) E. Catoni, E. Cernia, and C. Palocci, J. Mol. Catal. A:
Chem., 105, 79 (1996).
1d
1d
1d
scCO2 9 MPa
Hexane
Non-solvent
65.3
52.6 >99.9
18.9 97.4
99.6
40
34
16
>1000
>1000
92
1e
1e
1e
scCO2 9 MPa
Hexane
Non-solvent
30.6 >99.9
7.4 >99.9
3.8 >99.9
24
7
4
>1000
>1000
>1000
1f
1f
1f
scCO2 9 MPa
Hexane
Non-solvent
23.2 >99.9
10.5 >99.9
19
10
10
>1000
>1000
154
10.8
98.8
3
a) T. Matsuda, K. Watanabe, T. Harada, K. Nakamura, Y.
Arita, Y. Misumi, S. Ichikawa, and T. Ikariya, Chem.
Commun., 2004, 2286. b) T. Matsuda, R. Kanamaru, K.
Watanabe, T. Harada, and K. Nakamura, Tetrahedron Lett.,
42, 8319 (2001). c) T. Matsuda, R. Kanamaru, K. Watanabe,
T. Kamitanaka, T. Harada, and K. Nakamura, Tetrahedron:
Asymmetry, 14, 2087 (2003). d) T. Matsuda, T. Harada,
K. Nakamura, and T. Ikariya, Tetrahedron: Asymmetry, 16,
909 (2005).
Conditions: 40 ꢃC, 2 h, Enzyme: 5.0 mg, substrate: 100 mg,
vinyl acetate: 0.50 mL, solvents (scCO2 or hexane): 10 mL.
b
aRecovered alcohols. Conversion to 3 based on the starting
amount of 1. No detectable side reaction occurred. Higher
conversions were obtained with longer reaction times.
Table 2. CLEA (cross-linked enzyme aggregate) catalyzed ki-
netic resolution of 1a in various solvents
4
a) K. B. Hansen, J. R. Chilenski, R. Desmond, P. N. Devine,
E. J. J. Grabowski, R. Heid, M. Kubryk, D. J. Mathre, and
R. Varsolona, Tetrahedron: Asymmetry, 14, 3581 (2003).
b) D. Badone and U. Guzzi, Bioorg. Med. Chem. Lett., 4,
1921 (1994).
(S)-1aa (R)-3a Conv.b
Enzyme
Conditions
E
ee/% ee/%
CLEA103 scCO2 9 MPa 85.5 99.6
15.4 >99.9
34.2 99.5
/%
46
13
26
>1000
>1000
540
CLEA103 Hexane
CLEA103 Non-solvent
5
6
Lipase-catalyzed reaction in hexane is usually faster than
that in other hydrophilic solvents.
Visual inspection of the reaction mixture in an autoclave
equipped with sapphire windows showed that the reactants
and products were all soluble in scCO2 under the reaction
conditions at 9 and 13 MPa.
CLEA301 scCO2 9 MPa 93.8 >99.9
27.3 >99.9
52.8 99.5
48
21
35
>1000
>1000
624
CLEA301 Hexane
CLEA301 Non-solvent
Conditions are shown in Table 1. aRecovered alcohol.
bConversion to 3a based on the starting amount of 1a. No
detectable side reaction occurred.
7
8
K. Nakamura and T. Matsuda, J. Org. Chem., 63, 8957
(1998).
C.-S. Chen, Y. Fujimoto, G. Girdaukas, and C. J. Sih, J. Am.
Chem. Soc., 104, 7294 (1982). E value was used to evaluate
enantioselectivity and determined by using ee values of the
substrate (ees) and product (eep); Conv. ¼ ð1 ꢁ ðeep=
ðeep þ eesÞÞÞ ꢂ 100 E ¼ lnf1 ꢁ conv.=100 ꢂ ð1 þ eep=100Þg=
lnf1 ꢁ conv.=100 ꢂ ð1 ꢁ eep=100Þg.
tant for reactions by CLEA than that by Novozym; for CLEA re-
actions, the enzyme inside the aggregate probably catalyzed the
reaction more efficiently in scCO2 than in hexane or under non-
solvent conditions due to the high diffusivity of scCO2. The de-
gree of rate enhancement is also different between the substrates,
so the reasons are probably rather complex. The development
of methods to understand the mechanism are in progress in our
laboratory.
9
Personal communication with Dr. Rob Schoevaart.
10 K. Nakamura, T. Hoshino, and H. Ariyama, Agric. Biol.
Chem., 55, 2341 (1991).
11 O. Kajimoto, Chem. Rev., 99, 355 (1999).
Published on the web (Advance View) July 2, 2005; DOI 10.1246/cl.2005.1102