The effect of temperature on the lipase-catalyzed asymmetric protonation of 1-acetoxy-2-methylcyclohexene giving (R)-2-methylcyclohexanone

Article abstract of DOI:10.1016/j.tetasy.2004.05.008

Lipase-catalyzed enantioface-selective protonation of the enol acetate of 2-methylcyclohexanone was performed by using Burkholderia cepacia lipase immobilized on porous ceramic particles, 'Lipase PS-C II' (Amano Enzyme Inc.), in i-Pr₂O and etha

Full text of DOI:10.1016/j.tetasy.2004.05.008

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 15 (2004) 1929–1932
The effect of temperature on the lipase-catalyzed asymmetric
protonation of 1-acetoxy-2-methylcyclohexene giving
(R)-2-methylcyclohexanone
Takashi Sakai,* Akiko Matsuda, Yuko Tanaka, Toshinobu Korenaga
and Tadashi Ema
Department of Applied Chemistry, Faculty of Engineering, Okayama University, 3-1-1 Tsushima-naka,
Okayama 700-8530, Japan
Received 8 April 2004; accepted 6 May 2004
Abstract—Lipase-catalyzed enantioface-selective protonation of the enol acetate of 2-methylcyclohexanone was performed by using
Burkholderia cepacia lipase immobilized on porous ceramic particles, ‘Lipase PS-C II’ (Amano Enzyme Inc.), in i-Pr2O and ethanol,
giving (R)-2-methylcyclohexanone with 77% ee at best with 82% conversion at 0 °C. The enantioselectivity (E-value) was found to be
temperature dependent and significant in controlling the efficiency of the asymmetric protonation.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
theoretically reliable for enhancing the enantioselectivity
in the kinetic resolution of alcohols.4a–e Moreover, the
low-temperature method used in combination with a
porous ceramic (Toyonite)-immobilized lipase is effective
for enhancing both the enantioselectivity and the reac-
tion rate.4c;d;e The immobilization could prevent the
lipase from aggregation, while the dispersed lipase
molecules on the ceramic support seem to exert the
intrinsic activity. The immobilized lipase used herein is
commercially available as ‘lipase PS-C II’ (Burkholderia
cepacia) from Amano Enzyme Inc.5;6 Herein, we exam-
ine the applicability of the low-temperature method
using lipase PS-C II to the asymmetric protonation of
enol acetate 1, and have found a correlation between
E-value7 and 1/T. For example, 34% ee (E ¼ 2.0) of
product (R)-2 at 60 °C was raised up to 77% ee (E ¼ 7.7)
at 0 °C, where the use of lipase PS-C II was found to
have a crucial role in the rate enhancement. Here,
commercially available lipase PS-C II was used exclu-
sively instead of the biocatalysts reported by Ohta and
co-workers3a;b;d;e and Hirata et al.3h because the purpose
herein is to reveal the temperature effect on the enantio-
selectivity in the asymmetric protonation.
Optically active a-substituted ketones are known to be
important chiral intermediates prepared by a variety of
synthetic methods.1 Among them, chemical2a–j;l and
biochemical3a–i enantioface-selective protonations of
prochiral enol esters of ketones have been examined,
although a method with both high enantioselectivity and
operational simplicity is still being sought. Ohta et al.3b
have reported a simple method for the enantioface-
selective protonation via a biocatalytic hydrolysis of
enol esters, where Yamadazyma farinosa (formally clas-
sified as Pichia miso, then Pichia farinosa IFO 19806)
was reported to be the best biocatalyst. For example, the
reaction of 1-acetoxy-2-methylcyclohexene 1 in buffer
gave (S)-2-methylcyclohexanone (S)-2 in 90% ee. On the
other hand, Hirata et al.3h reported that an esterase
(isolated from Marchantia polymorpha)-catalyzed asym-
metric protonation of 1 in buffer also gave (S)-2 in >99%
ee. As seen in these examples, the screening of biocata-
lysts is essential in obtaining high enantiomeric excesses.
We recently proposed a ‘low-temperature method’ in the
lipase-catalyzed resolution, which is highly effective and
2. Results and discussion
We first attempted the asymmetric protonation of 1 with
lipase PS (Celite-immobilized) in the presence of added
* Corresponding author. Tel.: +81-86-251-8090; fax: +81-86-251-8092;
e-mail: tsakai@cc.okayama-u.ac.jp
0957-4166/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.05.008

1930
T. Sakai et al. / Tetrahedron: Asymmetry 15 (2004) 1929–1932
H2O (10 equiv) as a proton source in i-Pr2O at various
temperatures. As shown in Table 1 and Figure 1, the
enantiomeric excesses slightly increased from 22% to
28% ee by lowering the temperature from 60 to 0 °C;
however, the reaction rate (TTN/h:4d;e total turnover
number per hour) was dramatically decreased from 1100
to 60. The enantiomeric excess was less than 30% ee in
all cases, with the temperature effect not being obvious.
Interestingly, the configuration is (R), which is the
antipodal enantiomer of that obtained by using Yam-
adazyma farinosa3a;b or Marchantia polymorpha.3h Here,
we replaced lipase PS with lipase PS-C II (Porous
ceramic-immobilized), and found that the reaction was
accelerated by more than 6 times, while the enantio-
selectivity also improved to 33% ee as shown in entry 4.
This result encouraged us to use a bulkier proton source
such as methanol and ethanol, which was expected to
improve the enantioselectivity.
ln E
60
30
0
oC
0.8
% ee
29
0.6
0.4
0.2
0.0
20
10
2.5
3
3.5
)
4
1/ (×10—3 K—1
T
Figure 1. Correlation between ln E versus 1/T (Lipase PS on Celite,
H2O).
Further improvement of the enantioselectivity was ob-
served by replacing methanol with ethanol, which at-
tained 77% ee with 82% conversion at 0 °C (entry 14).8
As shown in Figure 2, each plot of ln E versus 1/T9 for
methanol and ethanol showed that lowering the
temperature increased the enantioselectivity, although,
without obeying an exact linear correlation. Curi-
ously, an increase of the E-value was not observed
between 30 and 10 °C in both cases. This suggests
a change of mechanism, although the details are
presently unclear. The protons can be supplied from
H2O, methanol or ethanol, whose bulkiness may be
important.10
The reaction with lipase PS-C II in the presence of
10 equiv of methanol at 60 °C gave 91% conversion with
30% ee as shown in entry 5. Lowering the reaction
temperature gradually increased the enantioselectivity,
which improved up to 70% ee with >99% conversion at
)10 °C. In these reactions, the rate acceleration by
lipase PS-C II was apparently cancelled by using
methanol, and it took as long as 65.7 h at )10 °C for
completion of the reaction, although the amount of
the lipase increased in the low temperature reactions.
Thus, lowering the temperature further seemed to be
impractical.
Table 1. Asymmetric protonation with lipase PS or PS-C II
O
O
lipase PS, or PS-C II
O
ROH(10 equiv.), i-Pr2O
R = H, Me, Et
(R)-2
1
Conditionsa
PSe/H2O
Entry
T (°C)
Lipase (mg)
Time (h)
Conv. (%)b
% Eec
E
TTN/hd
1
2
60
30
0
200
200
200
300
300
200
200
300
350
300
200
200
200
300
350
7.0
16.2
46.7
18.0
6.8
73
98
22
27
28
33
30
57
54
63
70
34
47
66
67
77
73
1.6
1.7
1.8
2.0
1.9
3.7
3.4
4.4
5.7
2.0
2.8
4.9
5.1
7.7
6.4
1100
650
60
3
26
PS-C II/H2O
PS-C IIf /MeOH
4
0
>99
91
390
960
640
350
150
90
5
60
30
10
0
6
16.4
26.5
46.8
65.7
8.7
98
88
7
8
95
9
)10
60
45
30
10
0
>99
98
PS-C IIf /EtOH
10
11
12
13
14
15
810
490
540
320
120
70
21.5
19.0
28.0
46.3
69.3
>99
97
84
82
)10
80
a Enol ester 1 (100 mg).
b Determined by GC.
c Determined by HPLC.
d Total turnover number per hour.
e Lipase PS immobilized on Celite containing ca 1.0 wt % of lipase.
f Lipase PS-C II immobilized on porous ceramic support (Toyonite).

T. Sakai et al. / Tetrahedron: Asymmetry 15 (2004) 1929–1932
1931
injection temp. 200 °C), and the enantiomeric excess
determined by HPLC [Chiralcel OB-H, hexane/
i-PrOH ¼ 100:1, flow rate ¼ 0.5 mL/min, detection
254 nm, 17.5 min for (S)-2, 19.0 min for (R)-2]. The
absolute configuration was determined to be (R) by
comparison of the sign of the specific rotation
ln E
2.5
60
30
0 —10 oC
% ee
76
2.0
1.5
1.0
0.5
0.0
64
46
24
{½aꢀ ¼ )6.0 (c 0.5, CH3OH), 54% ee} with that
D
{½aꢀ ¼ +12.2 (c 4, CH3OH), 87% ee, (S)} already re-
D
ported.11 The retention times for (S)- and (R)-2 were
confirmed by using commercially available racemic 2. In
the reaction at 60 °C, the amount of lipase was also in-
creased to shorten the reaction time, owing to the vol-
atility of the solvent.
EtOH
MeOH
2.5
3
3.5
)
4
1/T (×10—3 K—1
Figure 2. Correlation between ln E versus 1/T (Lipase PS on Toyonite,
MeOH or EtOH).
Acknowledgements
3. Conclusion
This work was supported by a Grant-in-Aid for Scien-
tific Research from the Ministry of Education, Culture,
Sports, Science and Technology of Japan.
We have reported the temperature effect on the enantio-
face-selective protonation in the lipase-catalyzed
hydrolysis of prochiral enol ester of ketone, in which
(R)-2-methylcyclohexanone was obtained at best in 77%
ee with 82% conversion at 0 °C. The existence of the
temperature effect is significant for the discussion of the
mechanism and for fine-tuning the enantioselectivity. A
combination of the low-temperature method and opti-
mization of the enzyme is the next subject for improving
further the asymmetric protonation.
References and notes
1. (a) Tomioka, K.; Koga, K. In Asymmetric Synthesis;
Morrison, J. D., Ed.; Academic: New York, 1983; Vol. 2,
pp 201–224; (b) Enders, D. In Asymmetric Synthesis;
Morrison, J. D., Ed.; Academic: New York, 1984; Vol. 3,
pp 275–339.
2. Recent examples: (a) Review: Fehr, C. Angew. Chem., Int.
Ed. Engl. 1996, 35, 2566–2587; (b) Eames, J.; Wee-
rasooriya, N. Tetrahedron Lett. 2000, 41, 521–523; (c)
Yamashita, Y.; Emura, Y.; Odashima, K.; Koga, K.
Tetrahedron Lett. 2000, 41, 209–213; Temperature effect in
4. Experimental
4.1. General
ꢀ
a chemical method: (d) Henin, F.; Letinois, S.; Muzart, J.
Tetrahedron: Asymmetry 2000, 11, 2037–2044; (e) Asensio,
ꢀ
Silica gel column chromatography was performed using
Fuji Silysia BW-127 ZH (100–270 mesh). Thin-layer
chromatography (TLC) was performed on Merck silica
gel 60 F254. Lipase PS-C II and lipase PS were purchased
from Amano Enzyme Inc. Diisopropyl ether was dis-
ꢀ
ꢀ
G.; Gil, J.; Aleman, P.; Medio-Simon, M. Tetrahedron:
Asymmetry 2001, 12, 1359–1362; Review: (f) Eames, J.;
Weerasooriya, N. Tetrahedron: Asymmetry 2001, 12, 1–24;
Review: (g) Ishibashi, H.; Ishihara, K.; Yamamoto, H.
Chem. Rec. 2002, 2, 177–188; (h) Ohtsuka, Y.; Ikeno, T.;
Yamada, T. Tetrahedron: Asymmetry 2003, 14, 967–970;
1
tilled from sodium before use. H NMR spectra were
ꢁ
ꢁ
(i) Munoz-Muniz, O.; Juaristi, E. Tetrahedron: Lett. 2003,
44, 2023–2026; (j) Futatsugi, K.; Yanagisawa, A.;
Yamamoto, H. J. Chem. Soc., Chem. Commun. 2003,
566–567; (k) Ishihara, K.; Nakashima, D.; Hiraiwa, Y.;
Yamamoto, H. J. Am. Chem. Soc. 2003, 125, 24–25; (l)
measured at 200 MHz. Enantiomeric excesses were
determined by HPLC analyses detected at 254 nm with a
chiral column (Chiralcel OB-H, Daicel Chemical
Industries). 1-Acetoxy-2-methylcyclohexene 1 was pre-
pared by a reported procedure.3b
ꢀ
Baur, M. A.; Riahi, A.; Henin, F.; Muzart, J. Tetrahedron:
Asymmetry 2003, 14, 2755–2761.
3. (a) Ohta, H.; Matsumoto, K.; Tsutsumi, S.; Ihori, T. J.
Chem. Soc., Chem. Commun. 1989, 485–486; (b) Matsu-
moto, K.; Tsutsumi, S.; Ihori, T.; Ohta, H. J. Am. Chem.
Soc. 1990, 112, 9614–9619; (c) Fujii, I.; Lerner, R. A.;
Janda, K. D. J. Am. Chem. Soc. 1991, 113, 8528–8529; (d)
Matsumoto, K.; Ohta, H. Tetrahedron Lett. 1991, 32,
4729–4732; (e) Matsumoto, K.; Oishi, T.; Nakata, T.;
Shibata, T.; Ohta, K. Biocatalysis 1994, 9, 97–104; (f)
Matsumoto, K.; Kitajima, H.; Nakata, T. J. Mol. Catal.
B: Enzym. 1995, 1, 17–21; Accounts: (g) Ohta, H. Bull.
Chem. Soc. Jpn. 1997, 70, 2895–2911; (h) Hirata, T.;
Shimoda, K.; Kawano, T. Tetrahedron: Asymmetry 2000,
11, 1063–1066; (i) Fantin, G.; Fogagnolo, M.; Guerrini,
A.; Medici, A.; Pedrini, P.; Fontana, S. Tetrahedron:
Asymmetry 2001, 12, 2709–2713.
4.2. Typical procedure for lipase PS-C II-catalyzed
asymmetric protonation in the presence of ethanol
A
mixture of 1-acetoxy-2-methylcyclohexene 13b
(100 mg, 0.65 mmol), lipase PS-C II (amount indicated
in Table 1) and 5 mL of diisopropyl ether was placed in
a test tube in a thermo-controlled bath. Ethanol (380 lL,
6.5 mmol) was quickly injected into the mixture through
a syringe. The reaction progress was monitored by TLC
(hexane/ethyl acetate ¼ 4:1). The mixture was quickly
filtered with suction to remove the lipase and the filtrate
concentrated under reduced pressure. The conversion
was analyzed by GC (PEG-3m, oven temp. 120 °C,

1932
T. Sakai et al. / Tetrahedron: Asymmetry 15 (2004) 1929–1932
4. (a) Sakai, T.; Kawabata, I.; Kishimoto, T.; Ema, T.;
7. Chen, C.-S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J.
J. Am. Chem. Soc. 1982, 104, 7294–7299, Here, we defined
the E-value (k1=k2) as follows:
Utaka, M. J. Org. Chem. 1997, 62, 4906–4907; (b) Sakai,
T.; Kishimoto, T.; Ema, T.; Utaka, M. Tetrahedron Lett.
1998, 39, 7881–7884; (c) Sakai, T.; Miki, Y.; Tsuboi, M.;
Takeuchi, H.; Ema, T.; Uneyama, K.; Utaka, M. J. Org.
Chem. 2000, 65, 2740–2747; (d) Sakai, T.; Hayashi, K.;
Yano, F.; Takami, M.; Ino, M.; Korenaga, T.; Ema, T.
Bull. Chem. Soc. Jpn. 2003, 76, 1441–1446; (e) Sakai, T.;
Matsuda, A.; Korenaga, T.; Ema, T. Bull. Chem. Soc. Jpn.
2003, 76, 1819–1821.
100 þ x
ln E ¼ lnðk1=k2Þ ¼ ln
100 ꢂ x
where k1 is the reaction rate of the faster-reacting
enantiomer, k2 is the reaction rate of the slower-reacting
enantiomer, and x is the enantiomeric purity (% ee) of the
product.
5. Lipase PS-C II is commercially available from Amano
Enzyme Inc. or Wako Pure Chemical Industries, Ltd. It is
lipase PS immobilized on Toyonite 200M, a new type of
spherical porous ceramics support (155 ꢁ 5 lm average
diameter and 60 nm average pore) having 3-(2-methyl-
propenoyloxy)propylsilanetrioxyl groups as organic
bridges on the surface [Yamashita, Y., Kamori, M.,
Takenaka, H., Takahashi, J. (Toyo Denka Kogyo Co.
Ltd, Kochi, Japan), Jpn. Kokai Tokkyo Koho, JP 09313179
(1997); U.S. Patent 6004786].
6. We recently reported that lipase PS-C II is usable at high
temperatures up to 120 °C and that it can be effectively
applied to a bulky substrate: Ema, T.; Kageyama, M.;
Korenaga, T.; Sakai, T. Tetrahedron: Asymmetry 2003, 14,
3943–3947.
8. Reaction of 1-chloroacetoxy-2-methylcyclohexene and
ethanol at 0 °C was also attempted to give only 7% ee of
(R)-2.
9. Phillips, R. S. Trends Biotechnol. 1996, 14, 13–16.
10. Certain proton-delivery systems seem to operate by the
facts that (1) the proton source (H2O or alcohol) is
requisite, and influences the reaction rate, and (2) d1-
methanol can be used as an additive to give partially
deuterated compound 1 (1H NMR). The bulkiness of the
alcohol may concern the course of proton-delivery system
to lipase molecules and/or directly to compound 1.
11. Meyers, A. I.; Williams, D. R.; Erickson, G. W.; White, S.;
Druelinger, M. J. Am. Chem. Soc. 1981, 103, 3081–
3087.