Bioreduction of α -haloacetophenones by Rhodotorula glutinis and Geotrichum candidum

Enantioselective reductions with enantiocomplementarity of α -haloacetophenones by Rhodotorula glutinis CCT 2182 and Geotrichum candidum CCT 1205 afforded the corresponding ( R )- and ( S )-halohydrins (halo = Cl, Br and I), respectively, in high chemical yields (89-99%) and enantiomeric excesses (92-99%). These halohydrins are potential chiral building blocks for the stereoselective syntheses of valuable compounds


Introduction
The reduction of α-haloketones mediated by whole cell of microorganisms has been frequently used to obtain chiral halohydrins, 1 that are potential chiral building blocks for use in stereoselective synthesis of valuable compounds such as enantiomerically pure 1,2aminoalcohols via epoxides (Scheme 1). 1 The microorganism frequently used for reduction of α-haloacetophenones is baker´s yeast (Saccharomyces cerevisiae).However, the results are only fair with fluoro-and chloroacetophenone and inferior with α-bromoacetophenone giving poor yields of the corresponding bromohydrin.Also, a dehalogenation product, i.e. acetophenone, was obtained when α-iodoacetophenone was used as substrate.1a,3 Recently, other microorganisms have been used with success for reduction of a large number of 4-substituted acetophenones.1f,4 A previous report on the microbial reduction of α-haloacetophenones gave α-bromoand α-iodo-1phenylethanols in poor yields and low enantioselectivites.1e In this work, we present results of the asymmetric bioreduction of α-haloacetophenones mediated by Rhodotorula glutinis CCT 2182 and Geotrichum candidum CCT 1205 which gave halohydrins with opposite enantioselectivity and without any dehalogenated by-products.

Results and Discussion
The bioreductions of α-haloacetophenones 1a-c mediated by the yeast Rhodotorula glutinis CCT 2182 afforded the corresponding halohydrins 2a-c with the R configuration, while the halohydrins 2a-c with the S configuration were obtained when Geotrichum candidum CCT 1205 mediated the bioreductions (Scheme 2).Excellent yields and ee were obtained in both cases as shown in Table 1.These results are superior to those reported elsewhere 1a,1e and therefore, the microorganisms used in this work should be chosen to mediate these biotransformations to obtain halohydrins 2a-c with desired configuration R or S in excellent yields and ee.To our knowledge, this is the first time that (S)-(+)-and (R)-(-)-2-bromo-1-phenylethanol 2b and (S)-(+)-and (R)-(-)-2-iodo-1-phenylethanol 2c are obtained in high yields and enantioselectivity.Both Rhodotorula glutinis CCT 2182 and Saccharomyces cerevisiae give products following the Prelog rule, 5 which predicts that in general, hydrogen transfer to the prochiral ketone occurs from the pseudo re-face (Scheme 3) were L represent a large and S a small substituent group. 6n the contrary, the Geotrichum candidum CCT 1205 gives anti-Prelog products.This ability of Geotrichum candidum to furnish anti-Prelog reduction products has been observed elsewhere.4a,7

Scheme 3
The α-haloacetophenones have been used as mechanistic probe in the reduction reactions of NADH-dependent horse liver alcohol dehydrogenase, 8 for identification of reductants in sediments 9 and even in the whole cells. 3This probe enables differentiation between reduction processes which proceed via hydride transfer (H -) or by a multistep electron transfer (e -, H • or e -, H + , e -as has been suggested). 10Acetophenone is the reduction product obtained by electron transfer while optically active halohydrin is obtained when an enzyme mediates a hydride transfer process.
In this work, the yields of optically active halohydrins were high and no dehalogenation product was obtained when iodoacetophenone was used as substrate; therefore, the hydride transfer is the unique mechanism observed in the reduction of α-haloacetophenones mediated by both Rhodotorula glutinis CCT 2182 and Geotrichum candidum CCT 1205.

Conclusions
In conclusion, Rhodotorula glutinis CCT 2182 and Geotrichum candidum CCT 1205 should be used to mediate reduction of α-haloacetophenone to obtain halohydrins 2a-c with the desired configuration R or S in excellent yields and ee's.Achieving these asymmetric bioreductions with enantiocomplementarity is remarkable and highlights the potential of such an approach.

Experimental Section
General Procedures.IR spectra ware recorded on a Bomem MB Series spectrometer. 1 H and 13 C NMR spectra were recorded on a Varian Gemini 300 spectrometer in CDCl 3 .The gas chromatographic analyses were performed using a Shimadzu GC/MS Class 5000 and with helium as carrier gas, with a chiral GC-column CHIRASIL-DEX (30 m x 0.25 mm x 0.25 µm).Optical rotation was measured with a J-720, VRDM306 JASCO, 589.3 nm spectropolarimeter.Ketones 1a-b were acquired from Aldrich Co. Ketone 1c was obtained by reacting 1a with NaI in acetone at rt.The racemic 2a-c were obtained by reacting the corresponding 1a-c with NaBH 4 in water/methanol at rt.All other reagents and solvents were reagent grade.

Growth conditions for yeast culture
The yeasts Rhodotorula glutinis CCT 2182 and Geotrichum candidum CCT 1205 were stored at Fundação André Tosello Pesquisa e Tecnologia. 11Rhodotorula glutinis was cultivated in YM (yeast-malt extract) nutrient broth (400 mL) at 30°C and Geotricum candidum was cultivated in ME (malt extract) nutrient broth (400 mL) at 28°C.Both yeasts were incubation for 2 days on an orbital shaker (200 rpm) before use.Sterile material was used to perform the experiments and the yeasts were manipulated in a laminar flow cabinet.