Diastereoselective reduction of ethyl α-methyl-β-oxobutanoate by immobilized Geotrichum candidum in an organic solvent

Tetrahedron Leuas.Vol.34.No.38.PP.6087-6090.1993 Printed inGnxtBritab~

Diastereoselective Immobilized

oo4o-lO39193 $6.00+ .oo Pergamen PrcBs Ltd

Reduction of Ethyl a-Methyl-P-Oxobutanoate Geotrichum candidum in an Organic Solvent

by

Kaoru Nakamura *+, Satoshi Takano ++, and Atsuyoshi Ohno + t Institutefor ChemicalResearch,KyotoUniversity,Uji, Kyoto611, Japanand t t ShofuInc., Fukuine-kamitakamatsttcho, Sakyo, Kyota605, Japan

Abstract: Diastcrcosclectivityin microbial reduction of ethyl c+methyl-P-oxobutanoatc is controlled by using immobilized microbe in an organic solvent in the presence of cyclopentanol as an additive to enhance the synselectivity. Asymmetric reduction of ketones with microbes alcohols.‘,* specific

However, serious disadvantages

configuration

is obtained

methods have been developed immobilization

and usually

including the modification

Cyclopentanol

of a substrate, 3V4the use of selective

the diastereoselectivity

of microbial

reduction

inhibitor,’

the to

If the added material Indeed, we report

by means of an added of microbial reduction of

ester. (1) with Geotrichum candidam IF0 4597* in water (substrate hydroxy ester (2) in the synlanti ratio of 35165.

= 33 mM) for 3 h gave the corresponding

of the reduction increased with the increase of reaction time: the synlanti

11/89; ix., the anti-2 was produced predominantly.

Similar phenomenon

the microbe was immobilized

with a water-adsorbent

ratio after

The 70 h was

was already observed by Buisson et

d and was claimed to be one of the methods for controlling diastereoselectivity

toward the anti-product.

polymer to and the reaction was conducted

microbial reduction gave the product with synlanti ratio of 46/54; the selectivity side.

is

several

Namely, the use of an inhibitor is effective

acts as an effective additive to increase the syn-selectivity

Reduction of ethyl 2-methyl-3-oxobutanoate anti-selectivity

of chiral

of the reduction

instead of an inhibitor, the situation will be more desirable.

here a novel method for controlling

concentration

the enantioselectivity

of microbial reduction where plural enzymes am responsible.

plays a role of an accelerator,

an a-alkyl$keto

for the preparation

of microbial reduction and to enhance enantioselectivity,

of a microbe6 and the use of organic solvent.’

control the stereochemistry

accelerator.

by a microbe

To control stereochemistry

unsatisfactory.

has widely been employed

have still existed in microbial reduction; an alcohol of only one

When

in hexane, the

shifts slightly toward the syn

To our surprise, the synlanti ratio did not change drastically after prolonged reaction in hexane contrary to

that observed in an aqueous system; the value was 35/65 even after 70 h. Thus, it seems that the reduction under an organic atmosphere

prefers to produce the syn-product

more than that in an aqueous solution.

syn-selectivity

observed herein is still unsatisfactory.

cyclopentanol

to the reduction system increases the syn-selectivity.

95/5 in the presence of 0.1 M of cyclopentanol. the syn-hydroxy

ester.

Cyclopentanol

However, the

On the other hand, it has been found that the addition of Cyclopentanol

Thus, the synlanti ratio is enhanced up to is able to shift the diastereoselectivity

toward

The results are summarized in Table 1.

exerts similar effect in an aqueous system, although the efficiency is small compared to that in

the organic system and the anti-preference

becomes dominant after prolonged reaction time.

6087

Naked microbe is

also effective formation

to the reaction in hexane, but the immobilization

enhances

the reduction rate’” as well as the

of the syn-product.

gH

CO,Et

G. candidum in water

Immobilized G. candidum in hexane

Q

-

COzEt -

OH

,+02Et

+

^i

1

anti-2 Table 1. Diasteroselectivities

syn-2

under Various Reaction Conditions SynlAnti Ratio After 3 h After 70 h

State of the Microbe

Solvent

Naked

Water

0

35/6S

Naked

Water

0.1

39161

7f93

Immobilized Immobilized

Hexane Hexane

0 0.1

46i54 9515

35165 9515

0

Cyclopentanol (M)

20

40

60

80

100

1 l/89

120

Cyclopentanol,mM

Fig. 1. Effect of cyclopentanol -o_

syn %

Although the syn-diastereoselectivity

on diastereoselectivity

and relative rate.

- 4 - - rel. rate

increases with the increase in concentration

described above, the reduction rate is not a monotonous function of the concentration concentration of cyclopentanol

of cyclopentanol of cyclopentanol:

as was when the

increases, the reduction rate increases up to 13 mM of the alcohol and then the rate

6089

decreases gradually.

Thus, diastereoselectivity

and reaction rate are controlled independently

that Geotrichum candidutn oxidizes cyclopentanol

We confirmed

suggest that cyclopentanol

Dehydrogenases

coenzyme than the others. The effects of the concentration

that afford the syn-isomer

of cyclopentanol

with the idea that certain enzymes are inhibited by cyclopentanol trapped by dehydrogenases cyclopentanol

we

responsible

apparent

are explainable

and, at the same time, accelerated by NAD(P)H

That is, the additive, cyclopentanol, Consequently,

to the reduction.

activity

selectively may have larger Km for the

on the reaction rate and diastereoselectivity

produced through ;he oxidation of cyclopentanol. reduces

Therefore,

in the present reaction system plays to keep the coenzyme in the reduced form ready to

participate in the reduction.

cyclopentanol

by cyclopentanol.

into cyclopentanone.

of the enzymes.

competes

with 1 to be

the increase in the concentration

of

On the other hand, a high concentration

of

contributes to promote the formation of NAD(P)H form NAD(P)+ through a coupled oxidation of If a group of dehydrogenases

cyclopentanol.

increase in the concentration

that afford the syn-product

selectively

of NAD(P)H than the group of dehydrogenases

of their difference in Km, the increase in the concentration

is more activated by the

that afford the anti-product because

of cyclopentnnol

will result in the increase in the syn-

selectivity. We reported that NADH-dependent

glycerol dehydrogenase

from Georrichn

cyclopentanol

and an cr-keto ester is reduced

smoothly

cyclopentanol.

lo

to keep the coenzyme

GGDH is thus

a candidate

under the catalysis

(synlanri = 4565).

in the presence

in the reduced form.

obvious that GGDH is not the enzyme that reduces 1 with a high syn-selectivity, GGDH is very slow and anti-preferential

candidum (GGDH) oxidizes of GGDH

However,

of it is

because the reduction of 1 with

The enzymes that reduce the coenzyme and those

that reduce the substrate are not necessarily the same. The preferential formation of the anti-product after prolonged reaction time as originally reported by Buisson et aZ9 may be accounted aerobic conditions,

for by the reverse oxidation of ~~2-2 initially produced.

The reaction is run under

which may inevitably tend to keep the coenzyme in the oxidized form.

Consequently,

there

are good chances for the microbe 10 oxidize the product through the reversed process of the reaction. dehydrogenases

that interact with the syn-product are more active than the others under certain concentrations

NAD(P)+, the final product after prolonged isomer.

The phenomenon

trans isomerization

of this olefin.

Cyclopentanol

form preventin g the equilibrium

that participate

in the present

reduction

as an effective additive to control the stereochemistry

In a typical experiment, added to the immobilized resulting suspension

from the oxidation

are not yet isolated,

cis-

process and,

The biocatalyst

(3.2 min).”

(1 mmol) in 6 ml of hexane was

to 3 ml of microbial suspension)

and the

Tridecane (10 mg, internal standard for was washed with ether (5 ml) and the

The anri-hydroxy

ester eluted faster (2.9 min)

The product was subjected to a column chromatography

= 2:1)12 and the absolute configuration

determined on a chiral capillary GC-column Acknowledgment.

The

The chemical yield (56 %) and sp-and ratio in the product (Y5/5 ) was measured using

(PEG, 0.25 mm x 20 m. 100 “C, He, 2 ml/min).

than the syn-isomer

role of

of other microbial reductions.

a solution of 1 (0.1 mmol ) and cyclopentanol

was shaken ( 100 stroke/min) at 30 “C for 17 h.

filtrates were combined.

the detailed

of microbial reduction is ambiguous.

microbe ( 0.5 g of the polymer (BL-100)”

GC) was added and the reaction mixture was filtered.

hexane:CH$l2

over the syn-

after prolonged photochemical

increases the amount of the reduced form of the coenzyme at

present method is worth to be tested for controlling the diastereoselectivity

a GC-column

by the anti-isomer

of

from the production of the anti-product.

Since the enzymes cyclopentanol

reaction period may be exceeded

is similar to preferential formation of cis-stilbene

the sacrifice of the amount of its oxidized consequently,

If the

on silica gel (eluent;

and e.e. of 2 (syn: 9X % (3S), anti: 95 8 (3s)) were

(Chirdldex G-TA, 0.25 mm x 30 m, 70 “C, He, 2 ml/min).

We thank Mr. Y. Hirose of Amano Pharmaceutical

Co. for his helpful suggestion

and

Osaka Yuki Co. for providing us a water-adsorbent References

and

polymer, BL-100.

notes 1990, 1.

1.

S. Servi, Synfhesis,

2.

C. J. Sih and C.-S. Chen, Angew. Chem. Int. Ed. En@., 1984,23,

3.

B.-N. Zhou, A. S. Gopalan,

4.

K. Nakamura,

K. Ushio, S. Oka, and A. Ohno, Tetrahedron Lett., 19114,25, 3979. Y. Kawai, N. Nakajima, and A. Ohno, J. Org. Chem., lY91,56,

1983,105,

F. VanMiddesworth,

570.

W.-R. Shieh, and C. J. Sih, J. Am. Chem. Sot.,

5925.

5.

K. Nakamura,

6.

K. Nakamura, M. Higaki, K. Ushio, S. Oka, and A. Ohno, Tetrahedron Lett., 1985.26,

4778.

7.

K. Nakamura,

8.

Cultivation medium of Geotrichum candidum; glycerol (3 %), yeast extract (I %), and polypeptone

4213.

K. Inoue, K. Ushio, S. Oka, and A. Ohno, J. Org. Chem., l!J91,53, 2589.

in 0.1 M potassium

phosphate

buffer (pH 6.2): N. Itoh and K. Umeda, Agric. Biol. Chem.,

(0.5 %)

1982, 46,

2159. Biocatalyst, 1990,3,

85

9.

D. Buisson, R. Azerad, C. Sanner, and M. Larcheveque,

10.

K. Nakamura,

S. Takano, and A. Ohno, Chem. Letr., 1992, 951.

11.

K. Nakamura,

T. Miyai, A. Nagar, B. R. Babu, T. Ando, and A. Ohno, Bull. Chem. Sot. Jpn.,

1990,

63, 298. 12.

The unchanged

starting material (1) should be removed

from the product prior to subject to the chiral

capillary GC, otherwise, the presence of 1 interferes the complete separation of four isomers of 2.

(Received in Japan 27 May 1993; accepted 16 July 1993)