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Screening on the use of Kluyveromyces marxianus CBS 6556 growing cellsas enantioselective biocatalysts for ketone reductions

Paola Vitale ⇑, Filippo Maria Perna, Maria Grazia Perrone, Antonio ScilimatiDipartimento Farmaco-Chimico, Università degli Studi di Bari ‘A. Moro’, Via E. Orabona 4, 70125 Bari, Italy

a r t i c l e i n f o

Article history:Received 4 October 2011Accepted 21 November 2011Available online 4 January 2012

a b s t r a c t

The versatility of Kluyveromyces marxianus CBS 6556 growing cells in the enantioselective reduction ofketone functionalities to the corresponding alcohols was exploited. In particular, methyl ketones werereduced to (S)-alcohols with ees of up to 96%. Longer chain alkyl ketones afforded, under the same exper-imental condition, (R)-alcohols with an ee of up to 84%. Interestingly, carbon–carbon double and the tri-ple bonds can also be reduced in the presence of Kluyveromyces marxianus CBS 6556 yeast. A cyclicketone, such as 2-tetralone, was also quantitatively reduced to its corresponding (S)-alcohol withee = 76%.

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1. Introduction

Optically active molecules are important building blocks for thesynthesis of many chemicals and bio-active compounds. Biocata-lyst assisted processes have some advantages over the most con-ventional asymmetric synthesis, such as chemo-, regio-, andstereoselectivities, together with very mild reaction conditions.1

Research over recent decades has led to a better understandingof biological systems and consequently increased their applicationinto chemical conversion processes, especially in the field of organ-ic synthesis.2 The use of whole cells for biocatalysed reactions hasbeen explored since the 1950s but their use can be difficult in largescale synthesis, due to complicated work-up procedures, highmedium volumes for cells cultures, the low space–time yieldsand long reaction times. Despite these disadvantages, whole-cellsare often chosen as biocatalysts because they provide a continuoussource of enzymes and cofactors,3–6 although their depletion canbe a problem. This disadvantage has recently been overcome by ge-netic engineering, in which new yeast strains have been con-structed by introducing heterologous genes to simplify the use ofcofactor-requiring enzymes (e.g., enzyme overexpression, and soon),7 and to improve the overall efficiency (mostly stereoselectivi-ty), these new techniques were recently implemented for syntheticapplications and already show great promise.

The asymmetric reduction of carbonyl compounds with yeast,in particular Saccharomyces cerevisiae, is probably one of the mostinvestigated classes of whole-cell biotransformations. Many re-search groups have focused their attention on looking for ‘non-con-ventional’ yeasts, compared to the highly investigated S.cerevisiae.5 Amongst these yeasts, Kluyveromyces marxianus strains

have not been extensively investigated as catalysts,8,9 althoughthey have several interesting features that make this species prom-ising for the industrial production of several compounds.

For the past several years, our interests have focused on usingnon-conventional yeasts to synthesize novel enantiopure com-pounds. The thermo-tolerant Kluyveromyces marxianus CBS 6556was successfully used by us for the stereoselective bioreductionof prostereogenic keto-esters to prepare clofibric acid analoguesas peroxisome proliferator-activated receptor (PPAR-alpha) li-gands.10–12 These studies allowed us to also isolate an unknownNADPH dependent ADH from Kluyveromyces marxianus CBS 6556yeast strain.13 This novel ADH efficiently mediated the reductionof 3-oxo esters with a high degree of stereoselectivity, providingchiral alcohols with an (S) absolute configuration at the newlyformed stereogenic center.

Herein, we report the continuation of such studies, in whichKluyveromyces marxianus CBS 6556 whole cells were used to ex-plore the bioreduction of various prochiral ketones, with the aimto screen the biocatalyst substrate ‘specificity’, as well its regio-chemo-, and enantioselectivities related to the molecular structureof the prochiral substrate to be reduced.

2. Results and discussion

The growing cells of Kluyveromyces marxianus CBS 6556 werechosen to study the substrate features on which it could be usedas a biocatalyst in a stereoselective reduction. Structurally differentmethyl(alkyl)ketones were chosen to check the dependence of ste-reoselectivity shown by Kluyveromyces marxianus CBS 6556 yeaston the molecular characteristics of the ketones. Two sets of ketoneswere used herein. The first were highly flexible, semi-rigid, and ri-gid methyl ketones, while the latter were ethyl, n-propyl, or i-butylketones. A cyclic ketone was also used. Fair to good conversions of

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⇑ Corresponding author.E-mail address: [email protected] (P. Vitale).

Tetrahedron: Asymmetry 22 (2011) 1985–1993

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the ketones into the corresponding alcohols were observed,although the optimization of the reaction time and other experi-mental conditions is currently in progress.

A summary of results obtained by Kluyveromyces marxianus CBS6556 growing cells-mediated methyl ketone bioreduction is re-ported in Scheme 1 and Table 1.

Phenylacetone 1a was first reduced in the presence of Kluyver-omyces marxianus CBS 6556 growing cells to the correspondingoptically active 1-phenylpropan-2-ol 2a. After 24 h incubation at30 �C, a 50% conversion of phenylacetone was obtained; theproduct had 92% ee and an (S)-absolute configuration.14,15 1-Phe-nylpropan-2-ol 2a is an important synthon in the preparation ofL-deprenyl,16 a monoamine oxidase (MAO)-B selective and irre-versible inhibitor, used in the treatment of neurodegenerative dis-orders such as Alzheimer’s and Parkinson’s disease.17,18 However,the complete phenylacetone conversion required a longer incuba-tion time (96 h). In this case, the 1-phenylpropan-2-ol isolated had90% ee. In order to compare homogeneous data, most of the reac-tions were incubated for 96 h at 30 �C, by using the same growingcell concentration.19 Under these conditions, 4-phenyl-2-butanone1b (entry 3) afforded the corresponding 4-phenylbutan-2-ol 2b ina 74% yield as a racemic mixture (enantiomeric ratio = 51:49). Thebigger distance between the carbonyl moiety and the phenylgroup, comparing the phenylacetone and 4-phenyl-2-butanone, re-sulted in a less constrained molecule. In fact, it is well known thatmost of the ADHs show high stereoselectivity for bulky–bulky sub-strates and low stereoselectivity for small-bulky substrates.20,21

As a result, no enantioselectivity was observed in the 4-phenyl-2-butanone reduction mediated by Kluyveromyces marxianus CBS6556 growing cells. These results can probably be explained byconsidering the presence of more than one ADH, operating in theyeast whole cells, with different stereo-preferences, that areresponsible for the formation of a racemic mixture.

Next, phenoxyacetone 1c (Table 1, entry 4) was reduced underthe same conditions to afford (S)-1-phenoxy-2-propanol 2c in 24 h,a 87% yield, with 80% ee.22

By comparing 4-phenyl-2-butanone with phenoxyacetone, thereplacement of a methylene with an oxygen atom allows recoveryof the enantioselectivity. This is probably due to the electronic

effects also being crucial in the enantioselection of carbonylreduction.

The 4-phenyl-3-butyn-2-one 1d reduction to (S)-4-phenyl-3-butyn-2-ol 2d occurs with lower enantioselectivity (56% ee) andyield (24%) in 4 days of incubation time. It should be noted that5% of (E)-4-phenyl-3-buten-2-one was present in the reactioncrude, derived from the triple bond reduction.23

In contrast, the (E)-4-phenyl-3-buten-2-one 1e bioreduction byKluyveromyces marxianus CBS 6556 growing cells proceeded with acomplete substrate conversion into the following products(Scheme 2): (S)-4-phenyl-2-butanol24–27 2b (90% yield and 68%ee), (S)-4-phenyl-3-buten-2-ol 2e (3% yield and 90% ee) and 4-phe-nyl-2-butanone 1b (7% yield).

Kluyveromyces marxianus CBS 6556 cells behaved almost in thesame way with 4-phenyl-3-butyn-2-one 1d and (E)-4-phenyl-3-buten-2-one 1e, because both substrates underwent two subse-quent reactions: carbonyl and carbon–carbon multiple bondreduction. A hypothesis for the bioreduction, which takes into ac-count the observed enantioselectivity, is depicted in Scheme 3.

The 4-phenyl-2-butanol 2b can be formed through two possi-ble different pathways. Carbon–carbon double bond reductionfollowed by reduction of the carbonyl double bond (a + b), orreduction of the carbonyl followed by the reduction of the car-bon–carbon double bond (c + d). The reduction of (E)-4-phenyl-3-buten-2-ol 2e to 4-phenyl-2-butanol under the same experi-mental conditions, showed that both pathways contributed,although at a different reaction rates. Hence, the reduction of(E)-4-phenyl-3-buten-2-one 1e leads to optically active (E)-4-phe-nyl-3-buten-2-ol 2b (90% ee) (via c) and to 4-phenylbutan-2-one(via a). The ee (68%) of the final product (S)-4-phenyl-2-buta-nol24–27 is the result of the sum of the allylic double bondreductions (via d) and of the carbonyl group reduction by onenon-stereoselective way (via b). These results allowed us to con-clude that from the two steps b and d, the first (via b) proceededwith almost no stereoselectivity as happened in the direct reduc-tion of 4-phenyl-2-butanone (entry 3, Table 1). However, althoughthe reaction mechanism elucidation requires further studies, thesefindings provide evidence for the probable involvement of a yeastenoate reductase.28,29 This would be, to the best of our knowledge,the first precedent of the bioreduction of a non-activated doublebond by an enoate reductase.

These results also highlighted the dependence of the enantiose-lectivity on the spacer between the aromatic ring and the carbonylfunctionality. In particular, the enantioselectivity in the productdecreased when increasing the distance between the two func-tional groups. Moreover, with a C2 spacer, the enantioselectivityobserved depended on the C2 hybridization, conjugation possibil-ity, and conformational degrees of freedom. The enantiomeric

Kluyveromyces marxianus CBS 6556

R1

O

R2R1

OH

R2 *n ngrowing cells

1a-g 2a-g

Scheme 1.

Table 1Bioreduction of prochiral ketones mediated by Kluyveromyces marxianus CBS 6556 whole cells (isolated yields)

Entry R1 R2 n Conversiona (%) Yield (%) ee (%) Time (h) Abs. Config.

1 CH3 Ph– 1 50 50 2a 92 24 (S)2 CH3 Ph– 1 100 >98b 2a 90 96 (S)3 CH3 Ph– 2 80 74 2b — 96 —4 CH3 Ph–O– 1 100 87 2c 80 24 (S)5 CH3 Ph–C„C– 0 30 24c 2d 56 96 (S)6 CH3 PhCH@CH– 0 100 3d 2e 68 d 96 (S)7 CF3 Ph– 1 80 75 2f 60 24 (S)8 CF3 Ph– 1 100 > 98 2f 58 96 (S)9 CH3 Ph– 0 70 67 2g 48 96 (S)

a Calculated by 1H NMR diagnostic signals of the unreacted ketone in the reaction crude.b 2% of 1-phenylpropan-2-yl acetate was also detected in the reaction crude (See Section 4).c 5% of (E)-4-phenyl-3-buten-2-one 1e was also isolated.d The main isolated product was the (S)-4-phenyl-2-butanol 2b (90%, ee = 68%), 3% of 4-phenyl-3-buten-2-ol 2e (90% ee) and 7% of 4-phenyl-2-butanone 1b were also

isolated by column chromatography (Scheme 2).

1986 P. Vitale et al. / Tetrahedron: Asymmetry 22 (2011) 1985–1993

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excess was higher when an a,b-unsaturated ketone was reduced(90%), although it decreased to 56% for the alkyne and becamepractically null for the saturated ketone reduction (Fig. 1 andTable 1).

The size of the substituents around the carbonyl is importantfor the reduction reaction rate and enantioselectivity shown byKluyveromyces marxianus CBS 6556 yeast. In particular, we notedthat the distance between the phenyl and carbonyl groups affectedthe product ee: the higher ee was obtained in the phenylacetonereduction with respect to acetophenone and 4-phenyl-2-butanone(Table 1).

To broaden the Kluyveromyces marxianus CBS 6556 substrateapplicability study, 1,1,1-trifluoro-3-phenyl-2-propanone 1f wasincubated, and after 24 h it was converted into the corresponding(S)-alcohol 2f (Table 1, entry 7) with 58% ee and 75% yield.

The enantiomeric excess did not change after 96 h incubationtime (Table 1, entry 8). The fluorinated phenylacetone analogues1h–j provided, after 96 h incubation with Kluyveromyces marxianusCBS 6556 yeast, the corresponding alcohols 1-(2-fluorophenyl)-2-propanol 2h, (S)-1-(4-fluorophenyl)-2-propanol 2i, and (S)-1-(3-trifluoromethyl)-phenyl)-2-propanol 2j with an ee of up to 94%(Scheme 4 and Table 2).

Although further studies are required to better define the effectof the substituent on the aromatic ring and substrates accepted byKluyveromyces marxianus CBS 6556, the stereoselectivity of most

carbonyl compound reductions seems to be predictable on thebasis of the steric hindrance of the substituent of the methylketones. These data confirmed that the Kluyveromyces marxianusCBS 6556-mediated ketone reduction followed Prelog’s rule, asfor many other ketone bioreductions performed in the presenceof yeasts: the higher steric difference of the substituents linkedto the carbonyl, meant that a higher enantioselectivity wasobserved.

In order to explore the steric hindrance tolerated for the otherside of the carbonyl functionality, the bioreduction of 1-phenyl-2-butanone 1k was accomplished. Its complete conversion intothe corresponding alcohol was observed after 72 h of incubation.It is noteworthy that (R)-1-phenyl-2-butanol 2k (ee = 84%) wasisolated.30,31 An opposite absolute configuration was obtained withrespect to the methyl ketone reduction.

1-Phenyl-2-pentanone 1l was incubated for the reduction, andwe observed both a lower conversion and enantioselectivity. Alsoin this case the product had an (R)-absolute configuration,32

revealing a different stereo-preference of Kluyveromyces marxianusCBS 6556 whole cell catalytic system in the bioreduction of longerchain alkyl ketones, under the same experimental conditions.

In order to investigate the substrates on which Kluyveromycesmarxianus CBS 6556 yeast cells were capable of inducing ketonereduction, a cyclic ketone such as 2-tetralone 1m, was reacted. Ahigh yield of (S)-2-tetralol33–35 2m was obtained with 76% ee(Table 3).

Finally, the effect of steric hindrance on the stereochemistry ofthe bioreduction was explored by incubating 4-methyl-1-phenyl-2-pentanone 1n and rac-3-phenyl-2-butanone 1o (Table 3, entry5).

The reduction of racemic 3-phenyl-2-butanone 1o afforded adiasteromeric mixture of (2S, 3R)- and (2S, 3S)-3-phenyl-2-butanol2o in 61% yield, with a diastereomeric ratio (2S,3R)/(2S,3S) = 83:17.(2S,3R)-3-Phenyl-2-butanol was the main stereoisomer (ee =96%),36,37 whereas the unreacted 3-phenyl-2-butanone was foundto be enriched in the (R)-enantiomer (50% ee), because of a resolu-tion due to a different reduction reaction rate of the two enantio-mers of 3-phenyl-2-butanone.38

The reduction of 4-methyl-1-phenylpentan-2-one gave4-methyl-1-phenyl-2-pentanol 2n with good enantioselectivity(84% ee), although its absolute configuration was not assigned.

O

(S)

OH

(S)

OH O

+ +

Kluyveromyces marxianusCBS 6556

growing cells

2b, 90% yield, ee = 68% 2e, 3% yield, ee = 90%1b, 7% yield1e

Scheme 2. Bioreduction of (E)-4-phenyl-3-buten-2-one by Kluyveromyces marxianus CBS 6556.

O

OH OH

Oa

c

d

b lowstereoselectivity

goodstereoselectivity

Scheme 3. Mechanistic hypothesis of the formation of optically active (S)-4-phenyl-2-butanol from (E) 4-phenyl-3-buten-2-one by Kluyveromyces marxianusCBS 6556.

O OO

increase enantioselectivity

E

Figure 1. Dependence of the enantioselectivity on the spacer hybridization.

Table 2Bioreduction of phenylacetone analogues by Kluyveromyces marxianus CBS 6556a

Entry Ketone R2 Yield (%) ee Abs. Config.

1 1a Ph– >98 2a 90 (S)2 1h 2-FC6H4– >98 2h 94 (S)b

3 1i 4-FC6H4– 93 2ic 94 (S)4 1j 3-CF3C6H4– >98 2j 92 (S)

a Incubation conditions: 30 �C, 2000 rpm, 96 h; in all the experiments 100% ofketone conversion was observed.

b The absolute configuration was attributed by conversion into the correspondingMosher’s esters 4h, 5h, Scheme 5; see Sections 2.1 and 4.

c 7% of 1-(4-fluorophenyl)propan-2-yl acetate 3i, was recovered from the reac-tion mixture (ee = 90%); see Section 4.

P. Vitale et al. / Tetrahedron: Asymmetry 22 (2011) 1985–1993 1987

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2.1. Mosher’s esters analysis

Although we found a stereo-preference of Kluyveromyces marxi-anus CBS 6556 for the reduction of phenylacetone analogues intothe corresponding (S)-alcohols, Mosher’s ester analysis was usedto deduce the absolute configuration of 1-(2-fluorophenyl)-2-pro-panol 2h at the new stereogenic center.

Since the (S)-MTPA ester group affects the chemical shifts of theR1 and R2 groups of the secondary alcohol in a predictable way, the(R)-MTPA ester group had an opposite effect on d; the absoluteconfiguration of the stereogenic centre can be reliably deducedby analyzing the sign of DdSR values.39,40

a) The two MTPA esters have to be described in the conforma-tional model which successfully correlates the knownresults: it shows the carbonyl group syn-coplanar with theO–CO bond, with the trifluoromethyl (CF3) substituent ofthe MTPA moiety and with the methine proton of the sec-ondary alcohol moiety.

b) After calculation of Dd = dS�dR for the diagnostic protons, thenucleus that shows Dd >0, must be shifted downfield fromthe OCH3; conversely, the proton with Dd <0, must beshifted upfield because it faces the phenyl ring.

1H NMR spectroscopy was used to determine the configurationon the basis of different chemical shift of a-methoxy-a-trifluoro-methylphenylacetic (MTPA) diastereomeric ester protons.

Both diastereomeric (S)- and (R)-MTPA esters of the opticallyactive 1-(2-fluorophenyl)propan-2-ol (Scheme 5) 5h and 4h,respectively, were prepared and studied with regards to their dif-ferences in the chemical shifts (DSR values) of the diagnostic 1HNMR data of two esters.

DdSR(CH2) = 2.97�2.92 = +0.05; DdSR(CH3) = 1.35�1.38 = �0.03On the basis of Dd analysis, performed with reference to CH3

and CH2, the absolute configuration of (S)-1-(2-fluorophenyl)-2-propanol 2h was deduced.

3. Conclusions

In conclusion, we have found that with Kluyveromycesmarxianus CBS 6556 yeast (Fig. 2), phenylacetone analogues canbe reduced with high enantioselectivity to the corresponding(S)-alcohols; a reversal of stereoselectivity occurs by increasingthe alkyl chain of the ketone, affording (R)-alcohol enrichedmixtures. Interestingly, whole cells of Kluyveromyces marxianusproved to be effective for the reduction of multiple bonds, espe-cially double bonds. Although some aspects need further study, anew enoate reductase seems to be involved in the bioreductionof a non-activated CAC double bond.

This study also provides interesting insight on the enzymaticpool present in Kluyveromyces marxianus CBS 6556 yeast, whichcan be further exploited for the stereoselective synthesis of opti-cally active building-blocks. In particular, this biocatalyst repre-sents an alternative method to ‘classic’ stereoselective reductionreaction approaches that often require both high temperaturesand pressures and special equipment, or milder reducing agents(e.g., silanes)41 with expensive transition metal (Rh, Ru, Ir, Ti, Zn,Cu, Fe) containing catalysts, complexed with optically active bio-mimetic ligands such as BINAP, SEGHPHOS, etc.42,43

Although a wider range of substrates needs to be investigated tofully evaluate the steric and electronic effects that affect the yield

Table 3Bioreduction of phenylacetone analogues by Kluyveromyces marxianus CBS 6556 (isolated yields)

Entry Substrate Conversion a (%) Yield 2k–p (%) ee Abs. Config.

1O

1k90 87 2k 84 (R)

2O

1l60 58 2l 50 (R)

3

O1m 100 >98 2m 76 (S)

4O 1n 15 10 2n 84 n.d.

5

O1o

70 61 2o (dr = 83:17)9690

(2S,3R)(2S,3S)

a Calculated by 1H NMR diagnostic signals of the unreacted ketone in the reaction crude.

CH3

O

R2CH3

OH

R2

growing cells

1h-j 2h-j

Kluyveromyces marxianusCBS 6556

Scheme 4.

F

OH

CH3

?

(S)-MPTA-Cl (R)-MPTA-Cl

4h 5h

(S) CF3O

H3CO Ph

OCH3F

(R) CF3O

Ph OCH3

OCH3F

* *

2h

Scheme 5.


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and stereoselectivity of this yeast-mediated reduction, this methodcould already be used for the preparation of intermediates of morecomplex chemical architectures both in organic and pharmaceuti-cal syntheses.16,44

Since the reduction of the carbon–carbon triple bond occurs at alower rate compared to the reduction of a carbonyl, Kluyveromycesmarxianus CBS 6556 could be a useful catalyst for the synthesis ofoptically active propargyl alcohols,45,46 which are valuable for thepreparation of various natural products such as alkaloids, phero-mones, prostaglandins, steroids, antibiotics, vitamins, andsesquiterpenes.47

Multiple bond reduction by this yeast could be an interestingnew field of investigation, since its high potential in creatingtwo stereogenic centers in one reaction, is often achieved inasymmetric synthesis by homogeneous catalysis and/or byorganocatalysis.48

Further studies are currently in progress by using the restingcells of the biocatalyst, in order to define the process parameters,and for a further characterization of the enzymes responsible forthe stereoselective biotransformations, through enzyme isolationand purification, or immobilization on suitable supports. More-over, new frontiers in molecular biology could also be helpful inoptimizing the biocatalyst not only for carbonyl bioreduction pro-cesses, but also for processes involving the multiple bond reduc-tion and the formation of new carbon–carbon bonds.

4. Experimental

4.1. General methods

Melting points were taken on an electrothermal apparatus. 1HNMR and 13C NMR spectra were recorded on a Varian Inova400 MHz spectrometer and chemical shifts are reported in partsper million (d). 19F NMR spectra were recorded by using CFCl3 asan internal standard. The absolute values of the coupling constantsare reported. FT-IR spectra were recorded on a Perkin–Elmer 681spectrometer. GC analyses were performed on a HP 6890 model,Series II by using a HP1 column (methyl siloxane; 30 m �0.32 mm � 0.25 lm film thickness). Analytical thin-layer chroma-tography (TLC) was carried out on pre-coated 0.25 mm thick platesof Kieselgel 60 F254; visualization was accomplished by UV light(254 nm) or by spraying a solution of 5% (w/v) ammonium molyb-date and 0.2% (w/v) cerium (III) sulfate in 100 mL 17.6% (w/v) aqsulfuric acid and heating to 473 K until blue spots appeared. Chro-matography was conducted by using silica gel 60 with a particlesize distribution 40–63 lm and 230–400 ASTM. Petroleum etherrefers to the 40–60 �C boiling fraction. GC–MS analyses wereperformed on HP 5995C model and elemental analyses on anElemental Analyzer 1106-Carlo Erba-instrument. MS-ESI analyseswere performed on Agilent 1100 LC/MSD trap system VL.

Optical rotation values were measured at 20 �C using a Perkin–Elmer 341 polarimeter with a cell of 1 dm path length; the concen-

tration (c) is expressed in g/100 mL. The enantiomeric ratios weredetermined by HPLC analysis using a Daicel Chiralcel OD-H column(250 � 4.6 mm), or by GC-analyses performed on a Hewlett–Pack-ard 6890 Series II chromatograph equipped with a Chirasil-DEX CB(250 � 0.25 lm) capillary column, column head pressure = 18 psi,He flow 2 mL/min, split ratio 100/1, T (oven) from 90 to 100 �C.

All the chemicals and solvents were commercial grade and puri-fied further by distillation or crystallization prior to use. The race-mic alcohols 2a–o were prepared by NaBH4 reduction in EtOH in91–95% yields.49 All the optically active alcohols 2a–o obtainedfrom Kluyveromyces marxianus CBS 6556 bioreductions had analyt-ical and spectroscopic data identical to the racemic compoundsprepared by synthesis, or to those commercially available. The pro-gress of the reactions was monitored by TLC and/or GC.

4.2. Microorganism and cultures

Kluyveromyces marxianus CBS 6556 was obtained from publictype culture collections (Centraalbureau voor Schimmelcultures,Delft, The Netherlands), and was cultivated under aerobicconditions in a medium containing 0.3% yeast extract, 0.3% maltextract, 0.5% peptone, 1% glucose. Agar–agar (2%) was added tothe same medium for cell preservation on agar slants.

4.3. Blank experiments

A 1 L flask containing 400 mL of the culture medium was stirredat 30 �C on an orbital shaker at 200 rpm. Ketones 1a–o (100 mg)dissolved in 1 mL of ethanol were added. The reaction was moni-tored by GC analysis and stopped after 4 days. The content of theflask was extracted with ethyl acetate and analyzed.

4.4. Bioreduction of 1a–o by using Kluyveromyces marxianusgrowing cells: general procedure

Kluyveromyces marxianus CBS 6556 was stored on agar slants at4 �C. For inoculum preparation, a 250 mL conical Erlen–Meyer flaskcontaining 100 mL of yeast maintenance medium (YMM) (previ-ously autoclaved at 121 �C, 1 atm, for 15 min), was inoculated witha single loopful of the microorganisms from the agar slants. Theflask was then incubated aerobically at 30 �C in a rotary shakerat 200 rpm for 24 h. Next, the growing cultures were inoculated(5% v/v) into a 250 mL conical Erlen–Meyer flask containing100 mL of YMM and incubated for additional 24 h under the sameconditions. These cultures were then used as a final inoculum (1%v/v) into a 1000 mL flask containing 400 mL of YMM. After 24 h ofincubation at 30 �C in the shaker (200 rpm), a mixture of 100 mg ofthe ketone 1a–o dissolved in 1 mL of absolute ethanol was added.The reactions were monitored by GC, by collecting suspension ali-quots of 1 mL after 1, 3, 4 and 5 days of reaction from each flask:after extraction with ethyl acetate (2 mL), the organic phase wasanalyzed by GC. After the appropriate conversion, the suspension

stereoselectivity- +

O

CH3n m

R= EWG

0 > n > 1m < 2

EWG on carbonyl

n > 2m > 2

R

size differences betweenthe two carbonyl substituents

conformational flexibilityof alkyl chains

Figure 2. Structural determinants for the stereoselectivity in the bioreductions mediated by Kluyveromyces marxianus CBS 6556.


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was centrifuged (3000 rpm, 6 min, 4 �C), and the aqueous phaseextracted with ethyl acetate (4 � 150 ml). The yellow organicphase was dried over Na2SO4, filtered and evaporated under re-duced pressure. The residue was purified by silica gel column chro-matography using petroleum ether and ethyl acetate (90:10 or80:20) as eluent to yield the desired alcohols.50,51

In order to measure the enantiomeric ratios,52 the correspond-ing acetates of the alcohols 2h–i, 2o were quantitatively obtainedby acetylation with acetic anhydride/Et3N. The absolute configura-tions of alcohols 2a–g, 2k–m, 2o obtained from the bioprocesswere determined by the comparison of their specific rotations withthose previously reported in the literature or by comparison of theretention times with previously published data.

4.4.1. (S)-1-Phenyl-2-propanol 2a14,15,25

50–98% Yield; ½a�20D ¼ þ20:1 (c 0.6, CHCl3), ee = 92% (HPLC OD-

H, Hexane/IPA = 90:10, 0.6 mL/min, k = 254 nm) (S)-isomer:8.4 min, (R)-isomer: 10.8 min; mmax (neat) 3368, 3027, 2968,2929, 1604, 1496, 1453, 1373, 1118, 1080, 940, 742, 699 cm�1;dH (400 MHz, CDCl3) 7.34–7.21 (5H, m, ArH), 4.06–3.99 (1H, m,CH), 2.80 (1H, dd, J 13.6, 4.8, CH2), 2.69 (1H, dd, J 13.6, 8.1, CH2),1.63–1.56 (1H, br s, OH, exchanges with D2O), 1.25 (3H, d, J 6.2,CH3); dC (100 MHz, CDCl3) 138.1, 129.4, 128.5, 126.5, 68.9, 45.7,22.8; m/z 136 (M+, 1%), 92 (100), 91 (70), 65 (13), 45 (23).

4.4.2. (S)-4-Phenyl-2-butanol 2b24–27

74–90% Yield; ee = 68% (HPLC OD-H, Hexane/IPA = 90:10,0.6 mL/min, k = 254 nm) (R)-somer: 8.2 min, (S)-isomer:10.6 min);27 mmax (neat) 3350, 3085, 3062, 3026, 2965, 2926,2860, 1603, 1495, 1454, 1374, 1178, 1128, 1083, 1055, 1031,954, 745, 698 cm�1; dH (400 MHz, CDCl3) 7.31–7.17 (5H, m, ArH),3.83 (1H, sextet, J 6.2, CH), 2.80–2.64 (2H, m, CH2), 1.83–1.73(2H, m, CH2), 1.40–1.38 (1H, br s, OH, exchanges with D2O), 1.23(3H, d, J 6.2, CH3); dC (100 MHz, CDCl3) 142.0, 128.4 (2Cortho +2Cmeta), 125.8, 67.5, 40.8, 32.1, 23.6; m/z 150 (M+, 8%), 132 (48),117 (100), 105 (13), 92 (34), 91 (86), 79 (10), 78 (24), 77 (17), 65(13), 45 (16).

4.4.3. (S)-1-Phenoxy-2-propanol 2c22

87% Yield; ½a�20D ¼ �1:8 (c 1, EtOH), ½a�20

D ¼ þ24:3 (c 1, CHCl3),ee = 80% (HPLC OD-H, Hexane/IPA = 90:10, 0.8 mL/min,k = 254 nm) (R)-somer: 9.8 min, (S)-isomer: 18.6 min); mmax (neat)3391, 3060, 3040, 2966, 2921, 2873, 1599, 1586, 1495, 1456,1383, 1330, 1290, 1245, 1170, 1150, 1076, 1042, 953, 935, 863,810, 752, 691 cm�1; dH (400 MHz, CDCl3) 7.32–6.91 (5H, m, ArH),4.25–4.17 (1H, m, CH), 3.95 (1H, dd, J 9.2, 7.7, CH2), 3.81 (1H, dd,J 9.2, 3.3, CH2), 2.43 (1H, br s, OH, exchanges with D2O), 1.30 (3H,d, J 6.2, CH3); dC (100 MHz, CDCl3) 158.5, 129.5, 121.1, 114.5,73.2, 66.3, 18.7; m/z 152 (M+, 0.2%), 108 (1), 94 (10), 77 (1).

4.4.4. (S)-4-Phenyl-3-butyn-2-ol 2d45,53,54

24% Yield; ½a�20D ¼ �13:5 (c 1, CHCl3), ee = 56% (HPLC OD-H, Hex-

ane/IPA = 90:10, 0.8 mL/min, k = 254 nm) (R)-somer: 8.7 min, (S)-isomer: 18.6 min;54 mmax (neat) 3350, 3053, 2981, 2930, 2866,2226, 1598, 1489, 1440, 1370, 1330, 1286, 1105, 1073, 1037,932, 852, 755, 691 cm�1; dH (400 MHz, CDCl3) 7.45–7.27 (5H, m,ArH), 4.77 (1H, q, J 6.6, CH), 2.02 (1H, br s, OH, exchanges withD2O), 1.57 (1H, d, J 6.6, CH3); dC (100 MHz, CDCl3) 131.6, 128.3,128.2, 122.6, 90.9, 84.0, 58.8, 24.4; m/z 145 (M+, 6%), 146 (3), 131(10), 103 (6), 77 (3).

4.4.5. (S)-(E)-4-Phenyl-3-buten-2-ol 2e25,55

3% Yield; er = 95:5 (HPLC OD-H, Hexane/IPA = 90:10, 0.8 mL/min, k = 254 nm) (R)-isomer: 10.0 min, (S)-isomer: 13.8 min;27,54

mmax (neat) 3339, 3059, 3026, 2971, 2925, 2873, 1598, 1493,1449, 1368, 1140, 1069, 967, 748, 693 cm�1; dH (400 MHz, CDCl3)

7.40–7.20 (5H, m, ArH), 6.56 (1H, d, J 15.9, CH), 6.28 (1H, dd, J15.9, 6.3, CH), 4.52–4.45 (1H, m, CH), 2.40–2.20 (1H, br s, OH, ex-changes with D2O), 1.38 (1H, d, J 6.4, CH3); dC (100 MHz, CDCl3)136.7, 133.6, 129.3, 128.6, 127.6, 126.4, 68.8, 23.4; m/z 148 (M+,61%), 133 (34), 131 (18), 130 (82), 129 (100), 128 (65), 127 (27),116 (12), 115 (85), 105 (99), 104 (15), 103 (27), 102 (16), 91(56), 79 (14), 78 (23), 77 (42), 65 (12), 63 (14), 55 (17), 51 (24),50 (10), 43 (31).

4.4.6. (S)-1,1,1-Trifluoro-3-phenyl-2-propanol 2f49

75–98% Yield; er = 80:20, (GC-Chirasil-DEX CB, T = 100 �C) (R)-isomer: 3.89 min, (S)-isomer: 4.06 min;49 mmax (neat) 3400, 3067,3034, 2932, 1606, 1497, 1391, 1274, 1166, 1129, 1099, 1031,919, 882, 803, 749, 702 cm�1; dH (400 MHz, CDCl3) 7.38–7.26(5H, m, ArH), 4.19–4.10 (1H, m, CH), 3.07 (1H, dd, J 14.01, 2.9,CH2), 2.85 (1H, dd, J 14.01, 10.3, CH2), 2.19–2.18 (1H, br s, OH, ex-changes with D2O); dC (100 MHz, CDCl3) 135.6, 129.4, 128.8, 127.2,124.8 (q, 1JC-F 281.8); 71.5 (q, 2JC-F 30.8), 36.0 (q, 3JC-F 1.7); d19F

(CDCl3, 376 MHz) -83.9 (d, J 6.2); m/z 190 (M+, 33%), 92 (9), 91(100), 65 (10).

4.4.7. (S)-1-Phenylethanol 2g14,27,56

67% Yield; ½a�20D ¼ �22:2 (c 1, CHCl3),14 er = 74:26 (HPLC OD-H,

Hexane/IPA = 90:10, 0.8 mL/min, k = 254 nm) (R)-isomer:7.53 min, (S)-isomer: 8.36 min;54 mmax (neat) 3346, 3080, 3063,3030, 2973, 2927, 2875, 1602, 1493, 1451, 1410, 1369, 1304,1262, 1204, 1077, 1029, 1011, 997, 898, 760, 699 cm�1; dH

(400 MHz, CDCl3) 7.39–7.26 (5H, m, ArH), 4.9 (1H, q, J 6.5, CH),1.93–1.88 (1H, br s, OH, exchanges with D2O), 1.66 (3H, d, J 6.5,CH3); dC (100 MHz, CDCl3) 145.8, 128.5, 127.4, 125.4, 70.4, 25.1;m/z 122 (M+, 4%), 107 (10), 105 (1), 79 (10), 78 (2), 77 (6), 51 (2),43 (1).

4.4.8. (S)-1-(2-Fluorophenyl)-2-propanol 2h57

98% Yield; ½a�20D ¼ þ25:2 (c 1, CHCl3) er = 97:3; mmax (neat) 3340,

3066, 3040, 2971, 2926, 1616, 1583, 1490, 1453, 1373, 1230, 1180,1113, 1080, 1050, 936, 856, 826, 753 cm�1; dH (400 MHz, CDCl3)7.26–7.02 (4H, m, ArH), 4.12–4.04 (1H, m, CH), 2.85 (1H, dd, J13.6, 5.2, CH2), 2.75 (1H, dd, J 13.6, 7.6, CH2), 1.6 (1H, br s, OH, ex-changes with D2O), 1.25 (3H, d, J 6.2, CH3); dC (100 MHz, CDCl3)161.3 (d, 1JC-F 244.9), 131.7 (d, 3JC-F 5.3), 128.2 (d, 3JC-F 8.4), 125.4(d, 2JC-F 16.0), 124.0 (d, 4JC-F 3.1), 115.3 (d, 2JC-F 22.9), 67.9, 38.8,22.9; d19F (CDCl3, 376 MHz) �122.1(m); m/z 154 (M+, 1%), 110(100), 109 (74), 83 (13), 45 (15). The enantiomeric ratio was mea-sured after conversion of alcohol 2h into the corresponding (S)-1-(2-fluorophenyl)propan-2-yl acetate 3h: 90% yield; (GC-Chirasil-DEX CB, T = 100 �C) (S)-isomer: 26.23 min, (R)-isomer: 28.03 min;mmax (neat) 3040, 2981, 2934, 2873, 1736, 1617, 1586, 1493,1456, 1373, 1243, 1181, 1133, 1110, 1089, 1057, 1029, 1013,956, 840, 814, 756, 720 cm�1; dH (400 MHz, CDCl3) 7.24–7.17(2H, m, ArH), 7.10–6.98 (2H, m, ArH), 5.14 (1H, sestet, J 6.3, CH),2.94–2.84 (2H, m, CH2), 1.98 (3H, s, COCH3), 1.23 (3H, d, J 6.3,CH3); dC (100 MHz, CDCl3) 170.5, 161.3 (d, 1JC-F 245.7), 131.7 (d,3JC-F 4.6), 128.3 (d, 3JC-F 7.6), 124.4 (d, 2JC-F 16.0), 123.8 (d, 4JC-F

3.8), 115.2 (d, 2JC-F 22.1), 70.4, 34.9, 21.2, 19.4; d19F (CDCl3,376 MHz) �122.1 (m); m/z (ESI+): 219 [M+Na]+.

4.4.9. (S)-1-(4-Fluorophenyl)-2-propanol 2i52

93% Yield; ½a�20D ¼ þ8:6 (c 0.7; CHCl3); mmax (neat) 3346, 3040,

2966, 2920, 1600, 1509, 1416, 1373, 1263, 1220, 1156, 1113,1096, 1080, 943, 930, 850, 810, 763 cm�1; dH (400 MHz, CDCl3)7.19–6.97 (4H, m, ArH), 4.03–3.95 (1H, m, CH), 2.76 (1H, dd, J13.6, 4.8, CH2), 2.67 (1H, dd, J 13.6, 8.1, CH2), 1.6 (1H, br s, OH, ex-changes with D2O), 1.23 (3H, d, J 6.2, CH3); dC (100 MHz, CDCl3)161.6 (d, 1JC-F 244.1), 134.1 (d, 4JC-F 3.8), 130.7 (d, 3JC-F 7.6), 115.2(d, 2JC-F 20.6), 68.8, 44.8, 22.8; d19F (CDCl3, 376 MHz) -121.1 (m);


1190


m/z 154 (M+, 4%), 110 (100), 109 (81), 83 (15), 45 (18). The enan-tiomeric ratio was measured after conversion of alcohol 2i intothe corresponding (S)-1-(4-fluorophenyl)propan-2-yl acetate 3i:52

er = 97:3 (HPLC OD-H, Hexane/IPA = 99:1, 0.8 mL/min,k = 254 nm) (S)-isomer: 19.93 min, (R)-isomer: 21.34 min; mmax

(neat) 3040, 2980, 2933, 2866, 1737, 1602, 1511, 1449, 1373,1246, 1159, 1134, 1100, 1090, 1057, 1016, 956, 819, 764,706 cm�1; dH (400 MHz, CDCl3) 7.18–7.13 (2H, m, ArH), 7.00–6.95 (2H, m, ArH), 5.07 (1H, sestet, J 6.6, CH), 2.88 (1H, dd, J 13.7,6.8, CH2), 2.73 (1H, dd, J 13.7, 6.4, CH2), 2.0 (3H, s, COCH3), 1.21(3H, d, J 6.6, CH3); dC (100 MHz, CDCl3) 170.4, 161.6 (d, 1JC-F

244.1), 133.2 (d, JC-F 3.1), 130.7 (d, JC-F 7.6), 128.6 (d, JC-F 38.1),115.1 (d, JC-F 21.4), 71.3, 41.3, 21.2, 19.3; d19F (CDCl3, 376 MHz)�121.1 (m); m/z (ESI+): 219 [M+Na]+.

4.4.10. (S)-1-[3-Trifluoromethyl)-phenyl]-2-propanol 2j58

98% Yield; ½a�20D ¼ þ19:4 (c 1, CHCl3), er = 96:4 (HPLC OD-H,

Hexane/IPA = 99.8:0.2, 0.8 mL/min, k = 254 nm) (S)-isomer:32.01 min, (R)-isomer: 35.40 min; mmax (neat) 3351, 3073, 3046,2973, 2926, 1613, 1596, 1490, 1450, 1376, 1333, 1200, 1160,1123, 1073, 940, 906, 866, 796, 750, 703 cm�1; dH (400 MHz,CDCl3) 7.51–7.40 (4H, m, ArH), 4.10–4.02 (m, 1H, CH), 2.84 (1H,dd, J 13.6, 5.1, CH2), 2.77 (1H, dd, J 13.6, 7.9, CH2), 1.6 (1H, br s,OH, exchanges with D2O), 1.26 (3H, d, J 6.2, CH3); dC (100 MHz,CDCl3) 139.5, 132.8 (q, JC�F 1.3), 130.7 (q, JC�F 32.6), 128.8, 126.0(q, JC�F 3.8), 124.1 (q, JC�F 272.4), 123.3 (q, J 3.8), 68.6, 45.3, 23.0;d19F (CDCl3, 376 MHz) �66.9 (s); m/z 204 (M+, 1%), 185 (12), 160(100), 159 (27), 140 (45), 109 (15), 91 (28), 45 (25).

4.4.11. (R)-1-Phenyl-2-butanol 2k30,31

87% Yield; ½a�20D ¼ �30:0 (c 1, CHCl3), er = 92:8, (HPLC OD-H,

Hexane/IPA = 90:10, 0.5 mL/min, k = 254 nm) (S)-isomer:10.42 min, (R)-isomer: 12.31 min; mmax (neat) 3390, 3084, 3068,3028, 2963, 2934, 2876, 1604, 1495, 1454, 1337, 1183, 1122,1079, 1016, 975, 911, 851, 742, 699 cm�1; dH (400 MHz, CDCl3)7.35–7.22 (5H, m, ArH), 3.80–3.70 (1H, m, CH), 2.85 (1H, dd, J13.6, 4.4, CH2), 2.66 (1H, dd, J 13.6, 8.4, CH2), 1.65–1.47 (2H: 1H,m, CH and 1H: br s, OH, exchanges with D2O), 1.01 (3H, d, J 7.5,CH3); dC (100 MHz, CDCl3) 138.6, 129.4, 128.5, 126.4, 74.0, 43.6,29.5, 10.0; m/z 150 (M+, 1%), 121 (7), 103 (8), 93 (8), 92 (100), 91(58), 77 (6), 65 (9), 59 (11).

4.4.12. (R)-1-Phenyl-2-pentanol 2l32

58% Yield; ½a�20D ¼ �1:85 (c 0.5, EtOH), ½a�20

D ¼ �8:1 (c 0.4, CHCl3),er = 25:75, (HPLC OD-H, Hexane/IPA = 90:10, 0.8 mL/min,k = 254 nm) (S)-isomer: 5.78 min, (R)-isomer: 6.69 min; mmax (neat)3369, 3080, 3060, 3028, 2958, 2927, 2872, 1602, 1550, 1495, 1454,1123, 1080, 1030, 1015, 843, 737, 699 cm�1; dH (400 MHz, CDCl3)7.38–7.30 (2H, m, ArH), 7.27–7.21 (3H, m, ArH), 3.90–3.80 (1H,m, CH), 2.83 (1H, dd, J 13.6, 4.0, CH2), 2.65 (1H, dd, J 13.6, 8.4,CH2), 1.60–1.37 (5H, m, 2CH2 + br s, OH, exchanges with D2O),0.94 (3H, d, J 6.6, CH3); dC (100 MHz, CDCl3) 138.6, 129.4, 128.5,126.4, 72.4, 44.0, 38.9, 18.9, 14.1; m/z 164 (M+, 1%), 121 (4), 103(5), 92 (100), 91 (44), 65 (7), 55 (15), 43 (7).

4.4.13. (S)-2-Tetralol 2m33–35

98% Yield; ½a�20D ¼ �36:5 (c 0.5, CHCl3), er = 88:12; mmax (neat)

3339, 3060, 3018, 2926, 2840, 1688, 1603, 1580, 1495, 1453,1437, 1361, 1340, 1291, 1232, 1112, 1049, 963, 842, 813,745 cm�1; dH (400 MHz, CDCl3) 7.15–7.08 (4H, m, ArH), 4.21–4.12(1H, m, CH), 3.13–3.07 (1H, m, CH), 3.00–2.93 (1H, m, CH), 2.90–2.75 (2H, m, CH2), 2.11–2.04 (1H, m, CH), 1.89–1.80 (1H, m, CH),1.79–1.72 (1H, br s, OH, exchanges with D2O); dC (100 MHz, CDCl3)135.6, 134.2, 129.5, 128.6, 125.9, 125.8, 67.2, 38.4, 31.5, 27.0; m/z148 (M+, 12%), 131 (12), 130 (100), 129 (42), 128 (12), 115 (27),105 (14), 104 (59), 103 (18), 91 (16), 78 (14), 77 (10), 65 (5), 51

(5); the enantiomeric ratio was measured after conversion of alco-hol 2m into the corresponding (S)-1,2,3,4-tetrahydronaphthalen-2-yl acetate 3m:59 87% Yield; (GC-Chirasil-DEX CB, T = 120 �C) (S)-iso-mer: 53.20 min, (R)-isomer: 55.28 min; mmax (neat) 3063, 3021,2938, 2848, 1732, 1496, 1454, 1436, 1366, 1242, 1111, 1034, 910,747 cm�1; dH (400 MHz, CDCl3) 7.15–7.06 (4H, m, ArH), 5.24–5.17(1H, m, CH), 3.12 (1H, dd, J 16.5, 4.9, CH), 2.99–2.80 (3H, m,1CH + 2CH2), 2.06 (3H, s, CH3), 2.05–1.89 (2H, m, CH2).

4.4.14. 4-Methyl-1-phenyl-2-pentanol 2n60

10% Yield; er = 92:8; (HPLC OD-H, Hexane/IPA = 99:1, 0.8 mL/min, k = 254 nm) major-isomer: 5.57 min, minor-isomer:6.54 min; mmax (neat) 3557, 3391, 3086, 3063, 3028, 2955, 2929,2870, 1601, 1496, 1468, 1454, 1385, 1367, 1136, 1081, 1030,747, 699 cm�1; dH (400 MHz, CDCl3) 7.35–7.30 (2H, m, ArH),7.27–7.21 (3H, m, ArH), 3.91–3.88 (1H, m, CH), 2.82 (1H, dd, J13.6, 4.0, CH), 2.63 (1H, dd, J 8.4, 13.6, CH), 1.89–1.79 (1H, m,CH), 1.52–1.44 (2H, m, CH + 1H, br s, OH, exchanges with D2O),1.35–1.27 (1H, m, CH), 0.95 (3H, d, J 6.6, CH3), 0.92 (3H, d, J 6.6,CH3); dC (100 MHz, CDCl3) 138.6, 129.4, 128.5, 126.4, 70.6, 46.0,44.5, 24.6, 23.4, 22.0; m/z 178 (M+, 0.3%), 145 (1), 121 (5), 117(2), 103 (6), 93 (8), 92 (100), 91 (42), 77 (4), 69 (11), 65 (7), 43 (7).

4.4.15. (2S,3R)-3-Phenyl-2-butanol 2o36

51% Yield; ½a�20D ¼ þ19:55 (c 1.4, EtOH),61,62 ee = 96%; mmax (neat)

3366, 3028, 2966, 1601, 1494, 1452, 1377, 1260, 1101, 913, 799,700 cm�1; dH (400 MHz, CDCl3) 7.36–7.32 (2H, m, ArH), 7.27–7.21 (3H, m, ArH), 3.89–3.81 (1H, m, CH), 2.68 (1H, quintet, J 7.1,CH), 1.48–1.40 (1H, br s, OH, exchanges with D2O), 1.27 (3H, d, J7.1, CH3), 1.23 (3H, d, J 6.3, CH3); dC (100 MHz, CDCl3) 143.5,128.6, 128.0, 126.7, 72.3, 47.9, 20.6, 17.8; m/z 150 (M+, 0.1%), 135(2), 106 (84), 105 (39), 103 (14), 91 (100), 79 (13), 78 (13), 77(19), 65 (3), 51 (5); (2S,3S)-3-phenyl-2-butanol 2o.36 10% Yield;½a�20

D ¼ �0:1 (c 0.9, EtOH),61,62 ee = 90%; dH (400 MHz, CDCl3)7.34–7.30 (2H, m, ArH), 7.25–7.19 (3H, m, ArH), 3.93–3.84 (1H,m, CH), 2.75 (1H, quintet, J 7.1, CH), 1.33 (3H, d, J 7.1, CH3), 1.10(3H, d, J 6.6, CH3); dC (100 MHz, CDCl3) 144.0, 128.4, 127.8, 126.4,72.3, 47.1, 21.0, 15.9; m/z 150 (M+, 0.1%), 135 (2), 106 (89), 105(39), 103 (12), 91 (100), 79 (12), 78 (12), 77 (18), 65 (3), 51 (5);the enantiomeric ratios of both the diasteroisomeric alcohols 2owere measured after their conversion into the corresponding 3-phenylbutan-2-yl acetates 3o:32 GC-Chirasil-DEX CB, T = 90 �C,(2S,3S)-isomer: 63.06 min, (2R,3R)-isomer: 65.33 min), (2S,3R)-iso-mer: 57.63 min, (2R,3S)-isomer: 60.86 min; mmax (neat) 3060, 3027,2960, 2926, 2853, 1733, 1603, 1493, 1450, 1371, 1257, 1240, 1105,1017, 947, 800, 700 cm�1; dH (400 MHz, CDCl3) 7.32–7.19 (5H, m,ArH), 5.09 (1H, quintet, J 6.4, CH), 2.93 (1H, quintet, J 7.0, CH),1.92 (3H, s, CH3), 1.28 (3H, d, J 7.0, CH3), 1.16 (3H, d, J 6.3, CH3);dC (100 MHz, CDCl3) 171.0, 142.9, 128.1, 128.0, 126.4, 74.3, 44.4,21.2, 17.3, 16.6.

4.5. Mosher’s esters synthesis39

Mosher’s ester analysis was used to deduce the absolute config-uration of 1-(2-fluorophenyl)propan-2-ol (2h) at the new stereo-genic center. Both diastereomeric (S)- and (R)-MTPA esters of theoptically active 1-(2-fluorophenyl)propan-2-ol (Scheme 5) 5h and4h, respectively, were prepared and analyzed (Scheme 5).

To a stirred solution of optically active 1-(2-fluorophenyl)-2-propanol 2h (10.0 mg, 65 mmol), DMAP (8.0 mg, 65 mmol), anddry Et3N (0.108 mL, 0.780 mmol) in dry dichloromethane (3 mL)at room temperature, was added (S)-MTPA-Cl (0.024 mL,0.130 mmol, 2.0 equiv). The reaction progress was monitored byTLC on silica gel (petroleum ether/EtOAc = 9:1). After completeconversion of the alcohol (15 h), the reaction mixture wasquenched by the addition of water (5 mL). The aqueous layer was


1191


extracted with two additional portions of CH2Cl2 (5 mL), and thecombined organic layers were dried (Na2SO4), filtered, and concen-trated in vacuo. The crude product mixture was purified by silica-gel chromatography (petroleum ether/ethyl acetate = 9:1) to give(2R)-1-(2-fluorophenyl)propan-2-yl 3,3,3-trifluoro-2-methoxy-2-phenylpropanoate 4h (19.5 mg, 80% Yield) as a semi-solid; (R)-MTPA ester dH (400 MHz, CDCl3) 7.43–7.30 (5H, m, ArH), 7.21–7.15 (1H, m, ArH), 7.08–6.92 (3H, m, ArH), 5.44 (1H, m, CH), 3.47(3H, s, OCH3), 2.96 (1H, dd, J 13.9, 7.7, CH), 2.89 (1H, dd, J 13.9,5.6, CH), 1.39 (3H, d, J 6.2, CH3); dC (100 MHz, CDCl3) 165.9, 161.2(d, 1JC-F 245.7), 131.6 (d, 3JC-F 4.6), 129.4, 128.5 (d, 3JC-F 7.6),128.3, 127.1, 123.9 (d, 4JC-F 3.8), 123.6 (d, 2JC-F 16.0), 123.2 (q, 1JC-

F3 288.4), 115.2 (d, 2JC-F 22.1), 73.3, 55.3, 34.8, 29.7, 19.6; d19F

(CDCl3, 376 MHz) -75.9 (s), -122.0 (m); m/z 189 (C9H8F3O+, 91%),137 (27), 136 (31), 109 (100), 105 (11), 91 (6).

4.5.1. (2S)-1-(2-Fluorophenyl)propan-2-yl 3,3,3-trifluoro-2-methoxy-2-phenylpropanoate 5h

(19.0 mg, 79% Yield) as a colorless oil; (S)-MTPA ester mmax

(neat) 3060, 2961, 2927, 2877, 1747, 1620, 1587, 1495, 1455,1383, 1261, 1169, 1120, 1107, 1018, 801, 758, 713 cm�1; dH

(400 MHz, CDCl3) 7.42–7.15 (7H, m, ArH), 7.08–7.00 (2H, m,ArH), 5.42 (1H, m, CH), 3.39 (3H, s, OCH3), 3.03–2.90 (2H, m,CH2), 1.35 (3H, d, J 6.2, CH3); dC (100 MHz, CDCl3) 166.1, 161.3 (d,1JC-F 245.7), 132.5, 132.0 (d, 3JC-F 4.6), 129.6, 128.8 (d, 3JC-F 8.4),128.5, 127.5, 124.3 (d, 4JC-F 3.8), 124.1, 123.2 (q, 1JC-F3 288.4),115.6 (d, 2JC-F 22.1), 73.9, 55.4, 35.4, 29.9, 19.7; d19F (CDCl3,376 MHz) -75.7 (s), -121.8 (m); m/z 189 (C9H8F3O+, 93%), 158 (8),137 (33), 136 (28), 109 (100), 105 (12), 91 (9).

Acknowledgements

This work was financially supported by National Project PRIN‘Stereoselezione in Sintesi Organica, Metodologie ed Applicazioni’,MiUR (Rome), and by the University of Bari. Thanks are due also toDr. Fabio Plasmati and Mrs. Luciana Volpe for their contribution toSection 4.

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