Asymmetric reduction of aliphatic and cyclic ketones with secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus: effects of substrate

catalysis today Catalysis Today 22 ( 1994) 607-620

Asymmetric reduction of aliphatic and cyclic ketones with secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus: effects of substrate structure and temperature Changsheng Zheng, Van T. Pham, Robert S. Phillips* Departments of Chemwtry and Biochemistry and Centerfor Metalloenzyme Studies, University of Georgia, Athens, GA 30602-2.556,USA

Abstract The reduction of aliphatic ketones catalyzed by a secondary alcohol dehydrogenase (SADH) from Thermoanaerobucter ethanolicus affords (S)-alcohols in high enantiomeric purities, when the chain has six or more carbons. With 2-pentanone, the reduction gives (S)-2-pentanol, and (R)-stereoselectivity is observed in the case of 2-butanone at 37°C. The rate of the reduction of aliphatic methyl ketones decreases about three-fold for each additional methylene increment. A temperature dependent reversal of enantiospeficity is observed in the oxidation of enantiomers of 2-butanol. A linear dependence of -R7lnE with temperature is observed for 2-butanol, 2-pentanol, and 2-hexanol. The cofactor analogues, thionicotinamide adenine dinucleotide phosphate (SNADP) and acetylpyridine adenine dinucleotide phosphate (APADP) gave higher enantioselectivity in the reduction of 2-butanone to (R)-2-butanol. For the reduction of cyclic ketones, SADH is enantiospecific for (S) isomers of cyclic alkyl ketones, and the transfer of hydrogen is stereoselective for the Re face to give ( 1s) cyclic alcohols. The facial stereospecificity of SADH for hydride transfer to NADP was determined by NMR, and it was found to be re-specific ( ‘A face’). In order to explain the stereoselectivity of SADH catalyzed reductions, a model is proposed that emphasizes the importance of the stability of substrate conformation and the steric interaction between substrate, enzyme and coenzyme.

1. Introduction

Alcohol dehydrogenase has been one of the most intensively investigated from horse liver enzymes in biotransformation [ 1 ] . Alcohol dehydrogenases (HLADH) [ 21, yeast [ 31, and Thermoanaerobium brockii (TBADH) [ 41, have * Correspondmg

author

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been studied and extensively

utilized in organic syntheses. Studies of alcohol dehydrogenases from different biological sources or new applications of known alcohol dehydrogenases are often reported. For examples, alcohol dehydrogenases from. Geotrichurn candidum [5], Pseudomonas sp. [6], and Lactobacillus kefr [71, and using Baker’s yeast in aqueous media [ 81 have been reported recently. However, the study or application of alcohol dehydrogenases from thermophilic bacteria has attracted increasing biotechnological interest, particularly due to their high the~ostability. Of these, TBADH has been used frequently in recent chemoenzymatic syntheses [4e]. Most applications of alcohol dehydrogenases are applied to the reduction of prochiral carbonyl compounds, in order to prepare optically active chiral secondary alcohols. In a preceding paper, we described a synthetically useful secondary alcohol dehydrogenase (SADH) from Thermoanaerobacter ethanolicus, a thermophilic anaerobic bacterium grown on D-glucose, which catalyzes the reduction of ketoesters to afford (S) -hydroxyesters with high enantioselectivity [ 93. In other studies, aimed at expanding the range of useful enzyme stereoselectivity, we found temperature and coenzyme dependencies of the stereosele~tivity of SADH in the reduction of 2-butanone or 2-~ntanone, and oxidation of enantiomers of 2-butanol or 2pentanol [ 10,111. In this paper, we review our earlier studies and present the results of the asymmetric reduction of aliphatic ketones and cyclic ketones with SADH from T. ethunolicus either as a homogeneous, cell free extract or following immobilization on a solid support.

2. Results 2.1. A~iphatic ketones The general procedure employed in SADH-catalyzed reduction reactions utilizes a catalytic amount of NADP” and an excess of 2-propanol, which conveniently serves both as a cosolvent for the solubilization of ketones and also as a reducing agent to recycle the expensive coenzyme, NADPH. The enzyme was used either as the crude cell-free extract or in partially purified form. The immobilized enzyme was prepared through direct coupling of the enzyme to polyacrylamide-oxirane beads. Table 1 shows the results of reduction of a series of acyclic ketones with SADH. The absolute configuration of products was determined by optical rotation (entry I and entry 7) or by comparing the retention time of trifluoroacetyl (TFA) Lproline esters of alcohol products in gas chromatographic (GC) analysis as previously described [ lob]. It is worth noting that in GC on the Chirasil-Val capillary column the (R, L) diastereomeric TFA L-proline esters had shorter retention times than the (S, L) diastereomeric esters. Table 1 exhibits a number of interesting points. First, 2-butanone was reduced to the (R)-alcohol, while 2-pentanone and the rest of the ketones were reduced to the (S)-alcohols (entry 2-8)) as expected

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Table 1 Asymmetric reduction of acyclic ketones with SADH from T. ethanolicus Ketone

Alcohol

Methods

Rel. rateb

Abs. Config.

ee (%)

2-Butanone

2-Butanol

100

R R

2 3

3-Butyn-2-one 2-Pentanone

3-Butyn-2-01 2-Pentanol

4

3-Hexanone 2-Hexanone 2-Heptanone 6-Methyl-5-heptene-2-one 2-Octanone ‘LA-Pentanedione Acetophenone I-Phenyl-1,3-butanedione

3-Hexanol 2-Hexanol 2-Heptanol Sulcatol 2-Octanol No reaction No reaction No reaction

A B B A B A A A A A A A A

28 13 62 44 50 96 96 98 92 99

5 6 I 8 9 10

11

s 33 3.3 11 4 1

S S S S S S S

*Method A: reduction of ketones using immobili~d SADH; Method B: reduction of ketones with SADH as a homogeneous, ceil-free extract. bathe reactions were stopped when the conversion reached 50% and the relative rates were determined by GC analysis. where the reduction rate of 2.butanone was arbitrarily assigned as 100%.

from our previous results [ lOa,b] . Second, the reduction of all short chain ketones, 2-butanone, 3-butyn-2-one and 2-pentanone, gave alcohols with low enantiomeric purities, while the other aliphatic ketones afforded alcohol products with much higher enantiomeric purities (ee’s from 92 to 99%). Finally, in the series from 2butanone to 2-heptanone the rate of the reduction decreases about three-fold for each methylene added on the methyl ketone. However, when a methyl ketone is changed to a ethyl ketone, the relative rate is decreased lo-fold (entry 3 and 4). Similar results were reported by Keinan et al. in the reduction of acyclic ketones with TBADH [4a]. The reduction of aromatic ketones and 1,3-diketones is not catalyzed by SADH, as no reaction products were observed with acetophenone, pentane-2,4-dione, or l-phenylbutane-1,3-dione. The inactivity of the 1,3-diketones is puzzling, as /3ketoesters are readily reduced by SADH [ 91. This lack of activity may be due to the higher enol content of pentane-2,4-dione in solution. 2.2. Efects ofte~per~t~r~ on Stere~s~Le~t~vi~ In order to understand the mechanistic basis for the unusual inversion of stereoselectivity observed in Table 1, we examined the effect of temperature on the kinetic parameters of oxidation of the enantiomers of secondary alcohols by SADH. Surprisingly, we found that (S) -2-butanol is the favored substrate at temperatures below 26”C, while (R) -2-butanol is preferred at higher temperatures [ lO,a,b] . Furthermore, the stereospecificity for (R) -2-butanol increases as the temperature is increased above 26°C. In contrast, for 2-pentanol and 2-hexanol, the (S)-enan-

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610

4000

-2000

/

280

’

290

300

i 310

Temperature,

/ 320

330

/’ 340

I<

Rg. I. Effect of temperature on enantiospecificity of oxidation of secondary alcohols by SADH. Filfed circles: Zbutanol. Open squares: 2-pentanol. Filled squares: 2-hexanol. Filled hexagon: (R)-2-butanol from 2-butanone reduction. Filled triangle: (S)-2-pentanol from 2-pentanone reduction. Filled diamond: (S)-2-hexanol from 2hexanone reduction.

tiomer is preferred at all temperatures studied, and the E value slowly decreases with increasing temperature. The E values obtained in prep~tive reductions of ketones at 37°C are in good agreement with those obtained in kinetic experiments of alcohol oxidation at 37°C (Fig. 1) , as expected. A linear dependence of - RTlnE with absolute temperature is observed (Fig. 1). These results are consistent with the theoretical basis for the effects of temperature on stereochemistry, which is published in detail elsewhere [ lOb,c] . We also found that replacement of NADP by the acetylpy~dine analogue (APADP) or the thionicotinamide analogue (SNADP), or by NAD, resulted in an improvement of the stereoselectivity of 2-butanone reduction by SADH [ 11 I. At 37”, the E value of the (R) -2-butanol increased from 1.3 to 3.7, 2.9 or 4.6, when NADP was replaced by APADP, SNADP, or NAD, respectively. At 47”, the E values went from 1.9 to 9.4,6.6, or 7.0. Thus, the effects of temperature and cofactor are additive, and the (R)-2-but~ol obtained at 47’ with APADP has an enantiomerit purity greater than 80% ee. We also performed kinetic measurements with the cofactor analogues, and the data showed linear dependencies of - R7lnE with T, similar to Fig. 1. In contrast, reduction of 2-pentanone in the presence of the cofactor analogues resulted in reduced enantiomeric purity of the (S) -2-pentanol product. 2.3. Cyclic ketones The reductions of cyclic alkanones catalyzed by SADH were carried out in an immobilized column reactor. Unconverted ketones and product alcohols were sep-

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arated by HPLC, and the assignment of cis or truns configurations of products was determined with GC analysis by comparison with standard samples. The absolute configuration and the stereochemical purity of cyclic alcohol products were determined on the basis of GC analysis and by comparison of optical rotation with literature. The results of SADH catalyzed reductions are compared with HLADH catalyzed reductions in Table 2. The rate of reduction of racemic 3-methylcyclohex~one by both enzymes is much faster than that of (R) -3-methyl~y~lohex~~ne. This result implies that (S)-3-methylcyclohexanone is much more reactive than the (R) -enantiomer. Thus, in the reduction of ( -f ) -3-methylcyclohexanone to 60% conversion using SADH, the isolated unreacted ketone was identified as (R)-3methylcyclohexanone ( 86% ee) . When the conversion was controlled at 30%, ( IS, 3s) -( + ) -3-methylcyclohexanol (truns) was obtained in high purity ( > 99% ee) . Reduction of (R) -3-methylcyclohexanone by SADH yielded ( lS, 3R) -c&alcohol (86% de) and ( lR, 3R)-trans-alcohol (7%). The reduction of 4-methylcycloTable 2 Comparison of enzymatic reductions of cycloalkanones Substrate

Reactton

Time

Yield (%)

using

Product

SADH and HLADH

alcohol

(W

0

IO

29’

0

(IWS)

>99

38

31

0

i iS.35)

-49

60

flS,3Rl

93

(iR.;R)

7

(IS,3R)

91

(IR.3R)

Q

SADH

cl% 6

HLADH

0

SADH

110

HLADH

“0,

0

SADH

48

99

HLADH

0

49

30

70

0

(IS,ZS)i99

0

(I S.1S) ‘99

0

(iS,3SI

i

0

SADH

0

HLADH

0

SADH

4 0

HLADH

SARH

?I

HLADH

120

20

IX

168 168

I68 168

22

799

5

66

(lS,3R>

,99

0

0

‘s

I

aA 200-ml reaction mixture was used. The reaction was allowed to proceed to approximately 30% conversion. bathe reaction was performed according to literature procedures [ 71.

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hexanone catalyzed by HLADH gave a mixture of 70% rruns- and 30% c&4methylcyclohexanols [ 16a], whereas reduction catalyzed by SADH afforded > 99% truns-4-methylcyclohexanol. 2-Methylcyclohexanone is a poor substrate for HLADH, as the relative rate is 50 times slower than 3-methylcyclohexanone [ 16a]. However, using SADH as the catalyst, truns-( lS, 2s) -2-methylcyclohexanol (73.7% ee) was obtained in 20% yield. The unreacted ketone was isolated and identified as ( + ) -(R) -2-methylcyclohexanone. HLADH exhibits regiospecificity for the cyclohexanone function in compounds which also contain a cyclopentanone function [ 181. In the present work, we found that SADH reduces cyclopentanones readily, albeit at much lower rates than cyclohexanones. However, we found previously that cyclopentanol is oxidized by SADH faster than cyclohexanol [ lob]. The reduction of ( rf: )-3-methylcyclopentanone yields ( lS, 3S)-3methylcyclopentanol (rruns) . From our results, the relative rate of reduction of ( + )-(R) -3-methylcyclopentanone by SADH is much slower than ( + )-3-methylcyclopentanone, demonstrating that SADH is enantiospecific for the (S) -ketone in both cyclopentanones and cyclohexanones. Overall, for the reduction of simple cycloalkanones, SADH catalyzed the transfer of the hydrogen of NADPH to the Re face of the carbonyl group of cyclic ketones to give ( 1s) -alcohol products. This stereoselectivity is similar to that of HLADH, but with a broader substrate range.

2.4. Cofactor stereospecificity

The stereochemistry of hydride transfer to the coenzyme, NADP, was determined by the enzyme-catalyzed transfer of deuteride from 2-propanol-ds to NADP followed by NMR analysis. The diastereotopic protons at C4 of NADPH are distinguishable by NMR. Thepro-R hydrogen shows a higher chemical shift (2.77 ppm), than the pro-S hydrogen (2.67 ppm) [ 121. Thus, by observing only the presence of a single peak at 2.60 ppm after transfer of the deuteride to the cofactor (NADPH shows an AB system at 2.74-2.54 ppm), we conclude that the deuteride (and analogously, the hydride) is transferred to and from the Re face of the cofactor. This is the same facial stereospecificity exhibited by LADH and TBADH [ 121.

PADPi

Fig. 2. SADH from T efhar2olicus-catalyzed ribose.

PADPi

reduction of NADP. PADPR, 2-phosphate

adenosine diphosphate

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lS,2R

I ADP

Scheme 1.

Scheme 2.

613

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3. Discussion Based on the results presented in Table 1, there appear to be large and small alkyl-binding sites in the enzyme active site. In space, the small site is in proximity to the carboxamide chain of NADPH, due to the A face stereospecificity. The Zn2+ in the enzyme active site is likely involved in coordinating the carbonyl oxygen of substrates. The small binding site can readily accommodate methyl and ethyl groups. tolerates n-propyl, but will not accept more bulky substituents [ lob], whereas the large site can accept groups as large as n-pentyl. The inverted stereoselectivity for reduction of 2-butanone at elevated temperatures is thus due to the ethyl group projecting into the small alkyl site and the methyl group occupying the large alkyl site. However, at temperatures below 26°C the ‘normal’ binding is favored, with the ethyl group sitting in the large site. In contrast, we were surprised that the reduction of 3-butyn-2-one afforded (S)-3-butyn-l-01, even though it is smaller than 2-butanone in volume. In fact, the effective length of the alkynyl group in 3-butyn-2-one is longer than the ethyl substituent in 2-butanone, due to the linear geometry of the triple bond and the folded conformation of the ethyl group. Similar ‘pocket’ models have been proposed in our previous work on oxidation of secondary alcohols with SADH [ lob] and by Keinan et al. in their studies of TBADH [4a]. In order to explain the stereochemistry of SADH catalyzed reduction of the cyclic ketones, we propose a model that emphasizes the importance of the steric interaction between substrate, enzyme and coenzyme (Scheme l), which is an extension of Prelog’s diamond lattice model [ 191, but is different from Jones’ cubic model [ 201 for the reduction of cyclic ketones by HLADH. The diamond lattice model focused on the analysis of the product conformations, whereas the cubic site model mainly analyzed the steric interaction of the substrate in the cubic space of the enzyme binding site. There are several important points in our proposed model. The transferredpro-R hydride in coenzyme NADPH is close to the carbonyl, and the transfer of hydride to carbonyl must occur from the exo side, in accordance with ‘steric approach control’ [21], since NADPH is a bulky reagent. Zn*+ stabilizes the transition state by ligating with the substrate oxygen and amino acids in the enzyme active site. The steric interaction between the (R) -alkyl substituent of cyclic ketones with the small alkyl binding pocket and the carboxamide of NADPH determines that alcohols with ( 1s) -configuration are formed preferentially. This steric restraint was first recognized by Prelog in his pioneering studies of the stereoselectivity of HLADH [ 191. For reduction of 3-methylcyclohexanone, the higher reaction rate of (3s) -3-methylcyclohexanone compared with its (3R) -isomer is due to the equatorial conformation of the (3S)-methyl group in the reaction transition state, whereas the (3-R) -methyl group will be axial, resulting in severely unfavorable 1,3-diaxial interactions in the transition state (Scheme 1) . (3s) -3-Methylcyclohexanone is one of the most active substrates for reduction by SADH. In contrast, the higher activity of (2s) -2-methylcyclohexanone requires that the 2-methyl substituent be preferentially axial. Although it would be expected that the equatorial

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2-methyl would be confo~ationally more stable, there will be gauche interactions with the oxygen which may raise the transition-state energy (Scheme 1) . The high diastereoselectivity for reduction of 4-methylcyclohexanoue to ~~a~~-4-methylcyclohexanol implies that the 4-methyl substituent must be axial during reduction (Scheme 1). Since there is no significant 1,4 interaction with the methyl and the alcohal oxygen in either axial or equatorial orientations, this preference must reflect a steric requirement of the enzyme binding site. The acyclic alcohols can be analyzed by this model as well. Either (R) or (S)-Zbutanol can be aligned on the cyclohexane skeleton of ( IS, 3S)-3-methyleyclohexanol in an extended conformation (Scheme 2). However, ~~)-2-pe~t~ol can be aligned in an extended conformation with the methyls superimposed, while (R) -2-pentanol must adopt a folded conformation (Scheme 2). Thus, the very low rate of formation of (R)alcohols from IL-hexanone and longer chain ketones may be a reflection of both unfavorable steric and ~onfo~ational interactions. In conclusion, SADH from T. e~~~oZ~c~s shows high stereoselectivity in the reduction of aliphatic methyl ketones, while substrate size-induced reversal of stereosele~tivity was observed. Other cyclic ketones such as 3-methylcy~lohexanone are reduced with high stereospecificity and diastereoselectivity. Our results demonstrate that SADH from T. ethanoEicus is a versatile catalyst for production of a wide variety of optically active alcohols.

4. Experimental 4.1. General Ketones were purchased from Aldrich Chemical Co., and purities were checked by gas chromatography before use. NADP was purchased from United States Biochemical Co., and dithiotheitol was purchased from Research Organ&, Inc. T&r, Red A Agarose, 2-prop~ol-~* and oxirane acrylic beads (approx. 150 pm macroporous particles) were obtained from Sigma Chemical Co. ‘H NMR spectra were recorded at 250 MHz on aBruker AC-250 spectrometer with CDCl, containing CHCl, ( 6 7.26) as an internal reference, except that the ‘H NMR spectra of NADPH and NADPD were obtained at 400 MHz on a Bruker AMX-400 s~~trometer. Optical rotations were measured at the sodium D-line on a Perkin-Elmer 141 polarimeter. GLC analyses were performed on a Varian 3300 (FID detector) equipped with a 25 m X 0.32 mm ID Chirasil-Val capilla~ column. Separations by flash chromatography were achieved with Merck :ilica gel, and separations by HPLC were carried out with a Rainin Dynamax 60 A Si column (21.4 X 250 mm) I Enzyme assays were performed on a Gilford Response II UV/Vis spectrophotometer with a the~oel~t~c cell block for tem~~ture control.

616

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4.2. Enzyme preparation 7: ethanolicus JW200wt (ATCC 31550) was grown at 55°C according to our previous method [ lOb,22]. Generally, from a 20 1carboy, 25-33 g of cells, which contained between 95%-70% of SADH activity (1200-1500 units) and between 5-30% primary alcohol dehydrogenase activity (PADH) were obtained. With our improved method, T. ethanolicus was grown at 50°C in a 400-l New Brunswick fe~entor for 20 to 22 h from 10% innoculum, and 600 g of cells with more than 99% of SADH activity (48 000 total units) were obtained [9]. The two alcohol dehydrogenase activities were assayed spectrophotometrically at 40°C with 2-propan01 (for SADH) and 1-butanol (for PADH) as the substrates as described [ lob]. The content of PADH and SADH in cell extracts was determined by comparison of the initial velocities of production of NADPH by enzymatic oxidation of lbutanol and 2-propanol. Crude cell extract which contained a small amount of primary alcohol dehydrogenase did not affect the reduction of ketones using immobilized enzyme or free enzyme form. However, when there is a significant amount of contamination with the primary alcohol dehydrogenase, they can be separated by chromatography on a Red A Agarose column [ lob]. For kinetic studies, SADH was purified as previously described [ lob].

4.3. Determination

of the stereospeci$city

of hydride transfer

The following were combined and incubated at 37°C: 100 ~1 of 2-propanol-ds, 7 mg of NADP, 1 ml of 50 mM potassium phosphate water-d, buffer, pH 8, and purified SADH alcohol dehydrogenase (2 units). After 2 days, the solution was filtered to remove protein with microp~ition system MPS-1 using a YM-10 membrane (Amicon). ‘H NMR (DzO), 2.60 (s). For standard NADPH, 7 mg NADPH was dissolved in 1 ml 50 mM potassium phosphate water-d, buffer, pH 8, ‘H NMR, 2.54-2.74 (AB system). 4.4. Preparation of immobilized SADH Oxirane acrylic beads ( 10 g) were shaken gently for 4 h with SADH (2500 units) in phosphate buffer (50 ml) under an N2 atmosphere. The mixture was left to stand at room temperature overnight, then was loaded onto a double-wailed glass column (20 cm X 2.5 cm). The column was washed with aqueous NaCl ( 1 M), followed by aqueous Tris-WC1 buffer (pH=7,5, 50 mM) containing 10 mM 2mercaptoethanol. The column reactor was then ready for use. When not in use, the column should be kept closed at 4°C in 50 mM Tris-HCI (pH = 8) buffer containing 10 mM 2-mercaptoethanol and 0.5 M (NH,) $O+ 4.5. General procedure A: reduction

of

ketones using immobilized SADH

A reaction mixture (100 ml) containing 1% (v/v) substrate, 10% to 30% (v/ v) Z-propanol (depending on the so~ubility of the ketones), 50 mM Tris-HCI

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617

buffer (pH = 8 >,0.05 mM NADP and 3 mM 2-mercaptoethanol was passed through the immobilized SADH column reactor (which was kept at 37°C by circulating water) by using a peristaltic pump at a flow rate between 16 ml/h and 60 ml/h. For acyclic ketones, when the reaction reached 50% conversion, the column effluent was saturated with ammonium sulfate and extracted with Et,O. For cyclic ketones, conversion yields depend on the different substrate. The organic layer was dried over MgS04 and evaporated at reduced pressure. The resulting mixture of aliphatic alcohol and ketone can be separated by flash chromatography (Merck Si gel, 230400 mesh, hexane/ethyl acetate, 9 : I ) . For cyclic ketone reaction mixtures, separations by HPLC were carried out with a Dynamax 60 A Si column (21.4 mm X 250 mm) using hexane/ethylacetate (70 : 130,5 ml/min). The collection of fractions in separation was controlled by GC analysis. The structure of products was determined by ‘H NMR spectra or by comparison with authentic samples, whenever possible. 4.6. General procedure B: reduction of ketones with SADH as a homogeneous, cell free extract Flasks loaded with 100 ml solution containing the following: ketone substrate ( 1 ml), 2-propanol (20 ml), cell extract containing SADH ( 100 units), NADP (0.05 mM), 2-mercaptoethanol (4 mM), and Tris-HCl buffer (pH 8, 50 mM), were placed in a water bath at 37°C. The reduction was followed by GC and worked up as described in method A when no more product formed. 4.7. Preparation of ( - )-N-(trijluoroacetyl)

L-prolyl chloride

N-trifluoroacetyl (N-TFA) L-prolyl chloride was prepared in the following manner, which is a variation of the methods by Wells [ 231 and Souter [ 241. To a solution of L-proline (5 g, 0.043 mol) in 100 ml of dry methanol at 0°C were added tetramethylguanidine (10.56 g, 0.092 mol, 2.1 equiv.) and ethyl trifluoroacetate ( 11.94 g, 0.084 mol, 1.9 equiv.) , and the reaction was stirred at room temperature for 24 h. The reaction solution was then concentrated, acidified with 50 ml 2 N HCl, extracted with 3 X 50 ml of ethyl acetate, dried over MgS04, and concentrated to afford N-TFA-r_-proline ( 10.90 g, 99%). To N-TFA proline in a ice bath, 50 ml of freshly distilled thionyl chloride was added. After standing for 30 min, the excess thionyl chloride was evaporated in vacua and the residue was dissolved in 150 ml of dry methylene chloride (0.028 mol/ml) . The prepared reagent was tested with R- ( + ) -2-butanol and ( f ) -2-butanol, and the error was within +_1.5%. 4.8. Determination

of optical purity of the alcohol products

Alcohol (20-30 mg) mixed with equivalent ( - ) -N- (trifluoroacetyl)-L-prolyl chloride solution in 5 ml dry CH$& was stirred for 2 h at room temperature and

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subsequently analyzed by GC on a Chirasil-Val capillary column [ 70°C (2 min) / 3°C per min/200”C]. The optical purity was determined by comparison with authentic samples, whenever possible. 4.9. Determination

of cis-trans ratio in reduction of cyclic ketones

( f )-3-Methyl-, ( k )-2-methyl-, 4-methylcyclohexanone and ( + )-3-methylcyclopentanone were each reduced with NaBH, in methanol, and the resulting mixture of alcohols was used as standard for gas chromatographic analysis of products of enzymatic reduction. The assignment of retention times to the cis- or trans-alcohol followed from the greater percentage of equatorial isomer in the reduction mixture and consequently greater peak area in GC. The percentage of each isomer in the chemical reduction was in accord with that reported [ 251. The stereochemistry of enzymatic reaction products was determined by GC analysis and comparison with standard samples [ GC program: 70°C (2 min) / 1°C per min/ 12O”C, or 50°C (2 min) / 1°C per min/ 100°C trans-alcohols have longer retention times than cis-alcohols] . 4.10. Physical properties (R) -2-butanol: 28% ee (method A), 13% ee (method B) ; (S) -2-pentanol: 44% ee (method A), 50% ee (method B) ; (S) -3-hexanol: 96% ee; (S) -2-hexanol: 96% ee; (S)-2-heptanol: 98% ee; (S) -2-octanol: 99% ee as determined by conversion to WA L-proline esters and comparison of the ratio of area integration of diastereoisomers in GC analysis. The absolute configurations were determined by comparing the retention time of TFA L-proline esters with standard samples. (S)-3-Butyn-l-01: 60% ee as determined by conversion to TFA L-proline ester followed by GC analysis. [a] “D - 24.08” (c = 0.55, dioxane). The absolute configuration was assigned on the basis of the literature values of optical rotation for (S) isomer{[a]17D-35.670(c=2.2,dioxane),99%ee} [13]. (S)-6-Methylhept-5-en-2-01: 92% ee as determined by conversion to a TFA Lproline ester followed by GC analysis. [a)] I’D+ 11.56” (c=4.3, CHCI,). The absolute configuration was determined by comparison of literature values of optical rotation for the (S) enantiomer { [a] 23D + 17.4“ (neat) } [ 141. ( lS, 3S)-3-Methylcyclohexanol: > 99% trans, 98% ee as determined by comparison of optical rotation, [ a] 17D+ 3.64” (c = 2.8, CH,OH) . The absolute stereochemistry was assigned on the basis of analysis of the reduction of ketones and by comparison of literature value of optical rotation for ( + )-trans-3-methylcyclohexanol { [ cr] 14D+ 3.70” (neat) ) [ 261. (R) -3-Methylcyclohexanone: 86% ee as determined by comparison of the optical rotation ( [a] 25D + 14.35” (c = 9.67, CHC13, 99% ee for the (R) isomer), [ 141 [c#‘D+ 12.35” (c=O.95, CHC13).

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( lS, 3R) -3-Methylcyclohexanol: 93% cis, 86% de as determined by GC analysis. [aI 17D- 6.50” (c = 4.85, CH,OH). The absolute stereochemistry was assigned by GC analysis and by comparison of literature value of optical rotation for ( lS, 3R)3-methylcyclohexanol { [ a] $$ - 4.22”) [ 141. 4-Methylcyclohexanol: > 99% trans as determined by GC analysis. ( lS, 2s) -2-Methylcyclohexanol: > 99% trans, 73.7% ee as determined by comparison of optical rotation, [a] I’D + 29.13” (c = 1.05, CH,OH). The absolute configuration was assigned on the basis of analysis of the reduction of ketones and by comparison of literature value of optical rotation, [a] 17D- 39.5” (c = 0.43, CH,OH) ,99% ee for ( lR, 2R)-truns-form [ 141. (R)-2-Methylcyclohexanone: 20.2% ee as determined by comparison of the optical rotation ( [Q] 25D + 12.3” (c = 2.67, CH,OH, 99% ee for the (S) isomer) [ 141, [ a] “D - 2.46” (c = 0.46, CH,OH). ( lS, 3s) -3-Methylcyclopentanol: > 99% truns, as determined by GC analysis [ a] 17D+ 6.02” (c = 0.24, CH,OH). The absolute configuration was assigned on the basis of analysis of the reduction of ketones.

Acknowledgement We thank Nancy Bond and William Reid for supplying a culture of T. ethanolicus and for assistance in growth of the organism. This work was partially supported by a Biotechnology Grant from the University of Georgia Research Foundation.

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