Asymmetric hydrolysis of enol esters with two esterases from Marchantia polymorpha

Article abstract of DOI:10.1016/S0957-4166(00)00046-X

Two esterases participating in the asymmetric hydrolysis of α-alkylated enol acetates to α-chiral ketones were isolated from the cultured cells of Marchantia polymorpha. These two esterases had opposite enantioselectivities and both of them reversed the stereoselectivity of protonation into the enol intermediate in the hydrolysis when the chain length and the bulkiness of α-substituents were increased. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0957-4166(00)00046-X

Tetrahedron: Asymmetry 11 (2000) 1063±1066
Asymmetric hydrolysis of enol esters with two esterases from
Marchantia polymorpha
Toshifumi Hirata,* Kei Shimoda and Tsuyoshi Kawano
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama,
Higashi-Hiroshima 739-8526, Japan
Received 25 December 1999; accepted 24 January 2000
Abstract
Two esterases participating in the asymmetric hydrolysis of a-alkylated enol acetates to a-chiral ketones
were isolated from the cultured cells of Marchantia polymorpha. These two esterases had opposite enantio-
selectivities and both of them reversed the stereoselectivity of protonation into the enol intermediate in the
hydrolysis when the chain length and the bulkiness of a-substituents were increased. # 2000 Elsevier
Science Ltd. All rights reserved.
Optically active a-substituted ketone derivatives are widely employed as chiral synthons in
asymmetric reactions and there is a continuing interest in the development of ecient procedures
1
2
3
to prepare them in enantiomerically pure form. Recently, it has been found that yeast and the
4
cultured cells of M. polymorpha were capable of performing the asymmetric hydrolysis of a-
alkylated cyclohexanone enol esters to give a-substituted chiral ketones and that hydrolytic
enzymes derived from yeast need an enantioselectivity-promoting factor to di€erentiate the
5
enantiotopic face in the protonation of the enol intermediate. We have now investigated the
enzymes which are able to catalyze the asymmetric hydrolysis of enol acetates in the plant cell
cultures of M. polymorpha.
Two hydrolytic enzymes named esterases I and II participating in the hydrolysis of enol esters
6
were isolated from the cultured cells of M. polymorpha. Several cyclohexanone enol acetates
,8
(
1±7)7 were subjected to enzymatic hydrolysis with these esterases to clarify the e€ect of various
substituents at the b-position to the acetoxyl group on the enantiomeric ratio and the catalytic
activity of enzymes. The absolute con®guration and enantiomeric excess of the resulting ketone
9
were determined by the circular dichroism (CD) spectra of the products1
0±14
and the peak area of
15
the corresponding enantiomers in GLC analyses on CP cyclodextrin b 236M-19. Hydrolysis of
enol acetates, 1±3, by esterase I gave the corresponding optically active ketones (8±10) whose CD
*
Corresponding author.
0957-4166/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(00)00046-X

1
064
T. Hirata et al. / Tetrahedron: Asymmetry 11 (2000) 1063±1066
curves were positive (Table 1). The acetate 1 was the best substrate for esterase I, allowing us to
achieve the highest enantiomeric excess (>99% e.e.) and yield (99%). In the case of 2, the ethyl
group at the a-position to the acetoxyl group markedly reduced the enantiomeric excess of the
product (14% e.e.). Substrate 3 with isopropyl group as the a-substituent was hydrolyzed in 10%
yield, and protonation at the a-position gave a poor enantioselectivity (17% e.e.). The chiral
preference of esterase I was retained among these substrates: the protonation of the enol inter-
mediates from 1±3 occurred preferentially from the same enantiotopic face of the C±C double
bond. However, when t-butyl, n-propyl, n-pentyl and benzyl groups were introduced into the
b-position to the acetoxyl group of the substrates (4±7), the CD curves of the corresponding
products, 18±21, were negative. This result indicates that the stereoselectivity of esterase I in the
protonation of these enol intermediates is reversed by long chain (Cꢀ3) and bulky substituents at
the b-position to the acetoxyl group. It is noteworthy that esterase I converted 7 into optically
active 21 in high enantiomeric excess (99%), although the hydrolysis of 7 with hydrolytic enzymes
from yeast gave racemic ketone.
Table 1
Enantioselectivity in the hydrolysis of enol acetates by esterase I
On the other hand, the conversion yield and enantiomeric purity in the hydrolysis with esterase
II are very low in comparison to the case of esterase I (Table 2). In the hydrolysis of 1±3, the
protonation of the enol intermediate following the hydrolysis of these substrates occurred
stereoselectively from the same enantiotopic face of the C±C double bond. In the cases of the enol
acetates 4±7, the protonation of the enol intermediate occurred preferentially from the reverse
side of the C±C double bond with respect to the hydrolysis of 1±3. Interestingly, the stereo-
selectivity of esterase II was opposite to that of esterase I.

T. Hirata et al. / Tetrahedron: Asymmetry 11 (2000) 1063±1066
1065
Table 2
Enantioselectivity in the hydrolysis of enol acetates by esterase II
Thus, two hydrolytic enzymes were isolated from the cultured cells of M. polymorpha and were
con®rmed to be capable of discriminating the enantiotopic face of the C±C double bond of the
enol intermediate in the hydrolysis. The enantioselectivities in the protonation of the enol inter-
mediate were opposite between these enzymes. The enantioselectivity of both enzymes reversed in
the hydrolysis of the substrates with long side chain, bulky t-butyl or benzyl group at the
b-position to the acetoxyl group, compared with the substrates having short side chains. Such
inversion of the enantioselectivity may be explained by the occurrence of a turnover of the sub-
strate in the active site of the enzymes due to the steric hindrance o€ered by the a-substituents.
Recently, Matsumoto et al. reported that the hydrolytic enzymes from yeast and commercially
available lipases from microorganisms could exhibit enantioselectivity in the hydrolysis of enol
5
acetate only in the presence of an enantioselectivity-promoting factor. Hydrolytic enzymes from
plant cell cultures of M. polymorpha appear to be di€erent from those from microorganisms.
Acknowledgements
The authors thank the Instrument Center for Chemical Analysis of Hiroshima University for
1
the measurement of H NMR, GC±MS and CD spectra.
References
1
. (a) Tomioka, K.; Koga, K. In Asymmetric Synthesis; Morrison, J. D., Ed.; Academic Press: New York, 1983; Vol.
, p. 201. (b) Ender, D. In Asymmetric Synthesis; Morrison, J. D., Ed.; Academic Press: New York, 1984; Vol. 3,
p. 275.
2
2
. (a) Duhamel, L.; Duhamel, P.; Launay, J. C.; Plaquevent, J. C. Bull. Soc. Chim. Fr. 1984, II-421. (b) Fehr, C.
Chimia 1991, 45, 253. (c) Waldmann, H. Nachr. Chem. Tech. Lab. 1991, 39, 413. (d) Duhamel, L.; Fouquay, S.;
Plaquevent, J. C. Tetrahedron Lett. 1986, 27, 631. (e) Gerlach U.; Hunig, S. Angew. Chem., Int. Ed. Engl. 1987, 26,
1
283. (f) Potin, D.; Williams, K.; Rebek Jr., J. Angew. Chem., Int. Ed. Engl. 1980, 29, 1420. (g) Kumar, A.;
Salunkhe, R. V.; Rane, R. A.; Dike, S. Y. J. Chem. Soc., Chem. Commun. 1991, 485. (h) Henin, F.; Muzart, J.;
Pete, J.-P.; Piva, O. New J. Chem. 1991, 15, 611. (i) Matsumoto, K.; Ohta, H. Tetrahedron Lett. 1991, 32, 4729. (j)
Henin, F.; Muzart, J. Tetrahedron: Asymmetry 1992, 3, 1161. (k) Takeuchi, S.; Miyoshi, N.; Hirata, K.; Hayashida,
H.; Ohgo, Y. Bull. Chem. Soc. Jpn. 1992, 65, 2001. (l) Yasukata, T.; Koga, K. Tetrahedron: Asymmetry 1993, 4,

1
066
T. Hirata et al. / Tetrahedron: Asymmetry 11 (2000) 1063±1066
35. (m) Fuji, K.; Tanaka, K.; Miyamoto, H. Tetrahedron: Asymmetry 1993, 4, 247. (n) Haubenreich, T.; Hunig, S.;
Schultz, H.-J. Angew. Chem., Int. Ed. Engl. 1993, 32, 398. (o) Fuji, I.; Lerner, R. A.; Janda, K. D. J. Am. Chem.
Soc. 1991, 113, 8528. (p) Reymond, J.-L.; Lever, J.-L.; Lerner, R. A. Angew. Chem., Int. Ed. Engl. 1994, 33, 475.
. (a) Matsumoto, K.; Tsutsumi, S.; Ihori, T.; Ohta, H. J. Am. Chem. Soc. 1990, 112, 9614. (b) Kume, Y.; Ohta, H.
3
4
5
6
Tetrahedron Lett. 1992, 33, 6367.
. (a) Hirata, T.; Shimoda, K.; Ohba, D.; Furuya, N.; Izumi, S. Tetrahedron: Asymmetry 1997, 8, 2671. (b) Hirata,
T.; Shimoda, K.; Ohba, D.; Furuya, N.; Izumi, S. J. Mol. Cat. B: Enzymatic 1998, 5, 143.
. (a) Matsumoto, K.; Oishi, T.; Nakata, T.; Shibata, T.; Ohta, K. Biocatalysis 1994, 9, 97. (b) Matsumoto, K.;
Kitajima, H.; Nakat, T. J. Mol. Cat. B: Enzymatic 1994, 9, 97.
. Homogenates of the cultured cells of M. polymorpha in 100 mM phosphate bu€er (pH 7.0) were centrifuged at
1
00 000 g to give a cell-free extract, which was treated with ammonium sulfate (60±80% satd) to give a crude
enzyme preparation. Butyl-Toyopearl column chromatography of the crude enzyme preparation gave a good
separation of the two di€erent esterases. Further puri®cation by chromatography on a diethylaminoethyl-Toyo-
pearl column and then a Sephadex G-75 column gave homogeneous esterases as judged by SDS±PAGE: esterase I,
molecular mass ca. 54 000, dimeric form composed of two identical 27 000 subunits; esterase II, molecular mass ca.
45 000, dimeric form composed of two identical 22 500 subunits.
7
. Cyclohexanone enol acetates, 1±7, were prepared by treatment of their corresponding ketones with perchloric acid
and acetic anhydride.8
. Gall, M.; House, H. O. Org. Synth. Vol. I 1988, 121.
8
9
. In a typical experiment 2-methylcyclohexanone enol acetate 1 (5 mg) and Triton X-100 (5 mg) were dissolved in 2
ꢁ
ml of the sodium phosphate bu€er containing enzyme (pH 7.0). The mixture was shaken at 300 rpm and 35 C.
After 0.5 h the reaction mixture was extracted with n-pentane and the product was identi®ed by direct comparison
with the authentic sample by GLC and GC±MS analyses. The other substrates (2±7) were subjected to the
enzymatic hydrolysis by the same procedure. It was con®rmed that neither non-enzymatic hydrolysis nor racemization
of the product occurred under the incubation conditions.
1
0. The CD data of the products obtained in the hydrolysis with esterase I are as follows; 8: [ꢀ]288 +990 (c 0.25,
1
1
12
MeOH) {lit. [ꢀ]288 � 987 for R enantiomer (15)}; 9: [ꢀ]288 +351 (c 0.25, MeOH) {lit. [ꢀ]288 +2200}; 10: [ꢀ]288
1
3
14
+
(
2
361 (c 0.12, MeOH) {lit. [ꢀ] +2126}; 18: [ꢀ] � 86 (c 0.15, MeOH) {lit. [ꢀ]288 +1690 for R enantiomer
2
88
288
12
11)}; 19: [ꢀ]288 � 2485 (c 0.25, MeOH) {lit. [ꢀ]288 +2480 for S enantiomer (12)}; 20: [ꢀ]288 � 669 (c 0.14, MeOH);
1
2
1: [ꢀ]288 � 1990 (c 0.15, MeOH) {lit. [ꢀ]288 +1750 for R enantiomer (14)}. The CD data of the products obtained
in the hydrolysis with esterase II are as follows; 12: [ꢀ]288 +99 (c 0.09, MeOH); 13: [ꢀ]288 +173 (c 0.17, MeOH);
4: [ꢀ]288 +278 (c 0.15, MeOH). The CD data of the products (11, 15±17) could not be obtained due to the low
transformation rate and the lack of the products.
1
1
1
1
1
1
1. Cheer, C. J.; Djerassi, C. Tetrahedron Lett. 1976, 43, 3877.
2. Meyers, A. I.; Williams, D. R.; Erickson, G. W.; White, S.; Druelinger, M. J. Am. Chem. Soc. 1981, 193, 3081.
3. Djerassi, C.; Hart, P. A.; Beard, C. J. Am. Chem. Soc. 1964, 86, 85.
4. Djerassi, C.; Hart, P. A.; Warawa, E. J. J. Am. Chem. Soc. 1964, 86, 78.
ꢁ
5. Conditions for capillary GLC analysis: column, CP cyclodextrin b 236M-19 (0.25 mmÂ25 m); injection, 180 C;
ꢁ
ꢁ
� 1
detector, 180 C; oven, 100 C; carrier gas, N (50 ml min ). Retention times for the products in the GLC were as
2
follows: 8 and 15, 11.8 and 12.8 min; 9 and 16, 12.7 and 12.9 min; 10 and 17, 23.8 and 24.0 min; 11 and 18, 18.1
and 18.4 min; 12 and 19, 27.7 and 27.9 min; 13 and 20, 72.1 and 72.8 min; 14 and 21, 60.1 and 61.2 min.