Investigation and optimisation of the biochemical enantio- and diastereoselective reduction of Ethyl-2-cyclohexanonecarboxylate using Yeast cells. Thesis submitted to the department of chemistry, Fachhochschule beider Basel (FHBB), Muttenz, Switzerland in partial fulfillment for a degree in chemistry. February 1999. By Alpha Mohamed Salieu Jalloh J.J.Wepferstrasse 5, CH-8202, Schaffhausen Tel: +52 644 4103 (or Cellphone 078 628 9100) e-mail:sjalloh@cilch.jnj.com

Examiner: Prof. Dr. E. Hüngerbühler, FHBB, Muttenz, Switzerland.

Supervisor: Dr. C. De Virgilio, University of Basel, Basel, Switzerland.

Expert: Dr. P. Wenk, Baselland State Laboratories, Baselland, Switzerland.

Introduction:

The use of baker's Yeast in the biochemical reduction of asymmetric ketones to enantiomerically pure compounds is well known[1,2]. However, the yields of most reductions are not good enough. Nevertheless, few take the pain of going one step further and try other yeast strains (wild types) simply because baker's yeast is not only cheap and easily available but also well documented.

The "FHBB-Yeast-Project" is working to increase the palette of substrate molecules that yield (-hydroxyesters using Yeast. (-hydroxyesters serve as chiral starting materials for the synthesis of (-lactams [3], insect pheromones[4] and carotenoids[5].

The reduction of Ethyl-2-cyclohexanonecarboxylate (adduct) to enantiomerically pure products using baker's yeast has left much to be desired[6].

In this work, wild Yeast strains were carefully chosen, cultivated and studied for appropriateness to reduce Ethyl-2-cyclohexanonecarboxylate. The synthetic process was then carried out in the laboratory and a way to optimise the enantio- and diastereoselectivity sought.

Aim:

The aim is to study the growth pattern of H. polymorpha, S. cerevisiae (strain in baker's Yeast) and a third strain (Strain3) in suitable growth medium, to determine the concentration of adduct and product that is dangerous to the Yeast cells (toxicity) and use these information to optimise the reduction of Ethyl-2-cyclohexanonecarboxylate. The "specific yield" of the reaction which lied at 0.3 g. litre-1. h-1[6] should be improved.

Method:

Microbiological study of the Yeast strains:

Yeast cultures of S. saccharomyces, H. polymorpha and Strain3 in 10% sucrose growth medium with known start cell density were left to grow at 27°C (H. polymorpha 37°C) in an incubator for three days. The cell count was registered about every two hours. From these, the cell doubling time and the duration of the various growth phases were determined.

In further experiments, known amounts of adduct and product were each added into Yeast cultures of Strain3, S. saccharomyces and H. polymorpha with known start cell densities and left to grow at 27°C (H. polymorpha 37°C). 100 ?l of the cultures were then spread on sterile Agar plates and left in an incubator for three days. The number of cell colonies that grew compared to that of a blank gives information on the toxicity of the adduct or product. Sterile conditions are a prerequisite for reliable results.

Reduction of Ethyl-2-cyclohexanonecarboxylate using Yeast:

Yeast cultures of S. saccharomyces, H. polymorpha and Strain3 in 10% sucrose were cultivated and left to grow till stationary phase sets in. 24 ml of adduct was reduced by 2 liters of culture in 24 hrs. (conditions withheld!). Both chemical and optical yields were determined using Gas Chromatography (GC). The efficiency of the various strains were compared with each other and with baker's Yeast.

Further, the influence of pH on the diastereoselectivity of Strain3 was investigated. Finally, an anti-hydrogen addition over the enol moiety on the adduct by Strain3 was studied.

Results:

	Growth rate [h^-1]	td [h]	Time lag phase [h]	Time to stat. Phase [h]
S. cerevisiae in sucrose	0.17	1.8	~1.8	~20
H. polymorpha in sucrose	0.23	1.3	~2	~30
H. polymorpha in glycerine	0.17	1.8	~2	~35
Strain3 in sucrose	0.12	2.5	~3.5	~40

	LC-50 cis product (exposure period of 2h) [ml per 100 ml culture]	LC-50 adduct (exposure period of 2h) [ml per 100 ml culture]
S. cerevisiae	1.20	1.60
H. polymorpha	1.35	1.2
Strain3	1.30	2.5

	Yield [%]	cis:trans	e.e. cis product[%]	e.e. trans product[%]
Klipfel's yeast in the growth phase	-	97:3	94	100
Klipfel's yeast in the stationary phase	79	98:2	95	100
S. cerevisiae culture	35	93:7	91	100
H. polymorpha culture	43	94:6	97	-
Strain3	98	6:94	88	100

Last word:

The transformation of the adduct to (1S, 2S)-Ethyl-2-hydroxy-1-cyclohexanecarboxylate by Strain3 was very efficient. It surpasses all previous hopes that were there. Moreover, this compound costs more than twice the price of the initial target (i.e. (1R, 2S)-Ethyl-2-hydroxy-1-cyclohexanecarboxylate)[7].

Literature:

[1] Kometani T, Yoschii H, Matsuno R (1996) J. Mol. Catal. B 1:45

[2] Csuk R, Glänzer B (1991) Chem Rev. 91:49

[3] Tschaen DM et. al. (1988) Tetr. Lett. 29:2779

[4] Mori K (1989) Tetr. 45:3233

[5] Kramer A, Pfader H (1982) Helv. Chim. Acta. 65:293

[6] Hungerbühler, E. et. al, FHBB, unpublished material, Muttenz, Switzerland.