Enantioselective biotransformations of racemic α-substituted phenylacetonitriles and phenylacetamides using Rhodococcus sp. AJ270

Article abstract of DOI:10.1016/S0957-4166(00)00025-2

Rhodococcus sp. AJ270 is an efficient whole-cell system able to catalyze the stereoselective conversions of racemic α-substituted phenylacetonitriles and amides under very mild conditions into enantiopure carboxylic acids and derivatives. The nitrile hydratase involved generally has a broad substrate spectrum against phenylacetonitriles irrespective of the electronic nature of the α-substituent while the amidase is very sensitive to both the electronic and steric factors of the substituent of amides. The overall enantioselectivity of nitrile hydrolysis is mainly determined by the combination of selectivities of nitrile hydratase and of amidase, with the latter being a major contributor. The amidase has high S-enantiocontrol against amides while the nitrile hydratase exhibits low R-selectivity against nitriles. The scope and limitations of this enantioselective biotransformation process are discussed. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0957-4166(00)00025-2

Tetrahedron: Asymmetry 11 (2000) 1123±1135
Enantioselective biotransformations of racemic a-substituted
phenylacetonitriles and phenylacetamides using
Rhodococcus sp. AJ270
Mei-Xiang Wang,a,* Gang Lu,a Gai-Jiao Ji,a Zhi-Tang Huang,a
Otto Meth-Cohnb and John Colbyb
aCenter for Molecular Science, Institute of Chemistry, The Chinese Academy of Sciences, Beijing 100080, China
bSchool of Health Sciences, Sunderland University, Sunderland ST1 3SD, UK
Received 6 December 1999; accepted 4 January 2000
Abstract
Rhodococcus sp. AJ270 is an ecient whole-cell system able to catalyze the stereoselective conversions of
racemic a-substituted phenylacetonitriles and amides under very mild conditions into enantiopure
carboxylic acids and derivatives. The nitrile hydratase involved generally has a broad substrate spectrum
against phenylacetonitriles irrespective of the electronic nature of the a-substituent while the amidase is
very sensitive to both the electronic and steric factors of the substituent of amides. The overall enantio-
selectivity of nitrile hydrolysis is mainly determined by the combination of selectivities of nitrile hydratase
and of amidase, with the latter being a major contributor. The amidase has high S-enantiocontrol against
amides while the nitrile hydratase exhibits low R-selectivity against nitriles. The scope and limitations of
this enantioselective biotransformation process are discussed. # 2000 Elsevier Science Ltd. All rights
reserved.
1. Introduction
The past decade has seen tremendous development of biotransformations using microorgan-
isms and isolated enzymes in organic synthesis. Since most of the reactions are highly chemo-,
regio- and stereoselective and are performed under very mild conditions, biotransformations o€er
many opportunities for the syntheses of compounds which are sometimes not readily obtainable
by `conventional' chemical means.1±3 For example, lipases and esterases have been routinely used
for the kinetic resolution of racemic esters and alcohols and for the preparation of homochiral
molecules from prochiral and meso diesters and diols.2±4 In contrast to these extensively investi-
gated hydrolytic enzymes, nitrile-hydrolyzing enzymes in organic synthesis have remained largely
* Corresponding author. Fax: 86-10-62569564; e-mail: wangmx@public2.east.net.cn
0957-4166/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(00)00025-2

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M.-X. Wang et al. / Tetrahedron: Asymmetry 11 (2000) 1123±1135
unexplored until recently,5,6 despite the fact that nitriles are very important compounds7 and the
bioconversion of nitriles into the corresponding carboxylic acids has been known for decades.8
Enzyme-catalyzed hydrolyses of nitriles have been shown to proceed by either direct trans-
formation to carboxylic acids with a nitrilase9 or a nitrile hydratase-catalyzed formation of amides
which are further hydrolyzed to the acids by an amidase10 (Scheme 1). A range of nitrile-hydro-
lyzing micro-organisms has been isolated11 and some nitrilases, nitrile hydratases and amidases
have also been puri®ed.12 The mechanisms of enzymatic hydrolysis of nitriles using nitrilase and
nitrile hydratase have been proposed, and for the latter the hydration stage is believed to involve
complexion of the nitrile function to a transition metal such as iron or cobalt.12 It is particularly
worth mentioning that the microbial hydration of acrylonitrile is currently being applied in Japan
to produce tens of thousands of tonnes of acrylamide per year.13
Scheme 1.
In our previous studies,11,12 it has been demonstrated that Rhodococcus sp. AJ270 is a powerful
and robust nitrile hydratase/amidase-containing micro-organism. Compared with other micro-
organisms reported recently, it has a very broad activity against almost all types of nitriles
including aromatic, heterocyclic and aliphatic ones, and both amides and acids can be produced
in high yields from appropriate nitriles.11 We have also shown that this novel biocatalyst dis-
played excellent regioselectivity in hydrolyzing aromatic dinitriles and a variety of aliphatic di-
nitriles bearing a suitably placed second chelating moiety.12 In order to further explore its potential
in organic synthesis, and also to understand the mechanisms of enzymatic hydrolyses of nitriles
and amides, we have conducted a systematic study of stereoselective biotransformations by
examining the e€ect of substrate structures on both the stereoselectivity and reactivity of the
reactions. Here we wish to report the enantioselective biotransformations of a-substituted
phenylacetonitriles and amides using Rhodococcus sp. AJ270, a convenient and ecient synthesis
of some enantiopure a-arylalkanoic acids and derivatives of pharmaceutical interest.14
Several studies of enantioselective hydrolysis have been reported and most of them have
focused on a-arylpropionitriles, which is not surprising since their hydrolytic product a-aryl-
propionic acids are related to the non-steroidal anti-in¯ammatory drugs.14 Thus, using Rhodococcus
rhodochrous IFO 15564 (formerly classi®ed as Rhodococcus butanica ATCC21197), a micro-
organism containing nitrilase, nitrile hydratase and amidase, Kakeya and co-workers15 and
others16 obtained good enantiomeric excesses of both amide and acid with opposite con®guration
from the hydrolysis of nitrile. Turner's group17 used an immobilized whole-cell Rhodococcus
SP361, a nitrile hydratase/amidase system developed by the Novo Industry of Denmark, and
found that the amidase involved is highly S-selective towards amide substrates while nitrile
hydration is non-selective or poorly R- or S-selective depending upon the substituent attached to
the para position of the phenyl ring of the a-arylpropionitrile. Similar stereoselectivity has also
been observed with other nitrile hydratase/amidase containing micro-organisms, including

M.-X. Wang et al. / Tetrahedron: Asymmetry 11 (2000) 1123±1135
1125
Rhodococcus equi TG328,18 Rhodococcus sp. C3II,16,19 Rhodococcus erythropolis MP50,16,19
Agrobacterium tumefaciens d320 and Pseudomonas putida NRRL-18668,21 and with nitrilase-con-
taining micro-organisms such as Rhodococcus NCIMB 1121622 and Acinetobacter sp. AK226.23
Hydrolyses of other racemic substrates such as cyanohydrins24±27 and their O-protected deriva-
tives28, and amino nitriles29,30 have been reported to give a range of enantioselectivities varying
with the micro-organisms used. It is interesting to note that, catalyzed by Pseudomonas sp.
B21C9,31 2-(4-chlorophenyl)-3-methylbutyronitrile was kinetically resolved into the correspond-
ing R-amide and S-acid while Pseudomonas putida NRRL-1866821 has been shown very recently
to have excellent S-nitrile hydratase selectivity, yielding the S-amide although it exhibited S- and
R-selectivities of amidase and nitrile hydratase, respectively, against a-arylpropionitriles and a-
arylpropionamides.
2. Results
2.1. Enantioselective biotransformations of nitriles
To understand the scope and limitations of the Rhodococcus sp. AJ270 in enantioselective
hydrolyses of nitriles and amides, we ®rst tested a number of racemic a-substituted phenylaceto-
nitriles 1a±g (Scheme 2). The reaction was carried out conveniently by using whole cells (2 g wet
weight) at 30ꢀC in an aqueous phosphate bu€er (pH 7.0). The con®gurations of the amides 2 and
acids 3 were obtained by comparing the direction of speci®c rotations with those of authentic
samples. Since optically active a-methylthiophenylacetic acid 3g and its amide 2g have not been
reported in the literature, we assigned their con®gurations on the basis of speci®c rotation
assuming that the correlationship between the speci®c rotation and absolute con®guration for a-
methoxyphenylacetic acid holds similar to that for a-methylthiophenylacetic acid.32 The con-
®gurations of the recovered nitriles 1c and 1e were determined by measuring the optical rotations
of the corresponding amides which were obtained from the chemical hydrolysis although partial
racemerization occurred during the course of chemical transformation.
Scheme 2.
The results summarized in Table 1 demonstrate clearly that Rhodococcus sp. AJ270 can
hydrolyze phenylacetonitriles bearing a variety of substituents at the a-position. The reaction
outcome, however, was strongly in¯uenced by the nature of the a-substituent, both steric and
electronic factors dramatically a€ecting the reactivity and, more importantly, the enantio-
selectivity (Table 1). For the nitriles, having a sterically small group such as methyl 1a and ethyl
1b, the biotransformation was very ecient (Table 1, entries 1±4). E€ective hydrolysis was also

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M.-X. Wang et al. / Tetrahedron: Asymmetry 11 (2000) 1123±1135
Table 1
Enantioselective biotransformations of nitriles
observed when a-isopropylphenylacetonitrile 1d was used as a substrate (Table 1, entry 7). Only
when a large and conformationally ¯exible group such as n-propyl and n-butyl was introduced did
the hydrolysis of 1c and 1e proceed slowly (Table 1, entries 6 and 8). Introduction of a polar
moiety such as a methoxy or methylthio group instead of an alkyl at the a-position had no e€ect
on the rate of hydration, both 1f and 1g being transformed into the corresponding amides e€ectively.
Surprisingly, however, only a small amount of a-methythiophenylacetic acid 3g was obtained
after a long incubation time. In the case of a-methoxyphenylacetonitrile 1f, no corresponding
acid at all was isolated although prolonged reaction time caused a decrease in chemical yield of
amide 2f (Table 1, entries 9±11).
Although the electronic nature of the a-substituent of nitrile 1 did not greatly in¯uence the
eciency of biohydrolysis of nitrile, interestingly it led to a totally di€erent stereochemistry.
For example, a-alkylphenylacetonitriles 1a, 1b and 1d underwent highly stereoselective

M.-X. Wang et al. / Tetrahedron: Asymmetry 11 (2000) 1123±1135
1127
biotransformations to give optically active R-amides and S-acids with enantiomeric excesses
higher than 99% (Table 1, entries 1, 4 and 7). Slow and incomplete biotransformations of the
sterically more bulky nitriles 1c and 1e gave rise predominantly to S-enantiomers of the
corresponding acids 3c and 3e, albeit in very low chemical yields. However, It is noteworthy
that both amides 2c and 2e and untransformed nitriles 1c and 1e isolated from the reaction were
also S-enantiomers in excess although the optical yields were low (Table 1, entries 5, 6 and 8).
This seems very strange, as an enantioselective nitrile hydratase should give amide and nitrile with
opposite con®gurations. The same S-enantioselectivity of amidase was observed for the bio-
transformation of methylthiophenylacetonitrile 1g, which gave a 96% enantiomeric excess of acid
3g (Table 1, entry 11). In sharp contrast, no stereoselectivity at all was found for the reaction of
a-methoxyphenylacetonitrile 1f (Table 1, entries 9 and 10). It was considered that a phenyl-
acetonitrile substrate substituted at the a-position by an even more polar group might lead to high
and reversed enantioselectivity. We, therefore, examined the biotransformations of a-acetoxyphenyl-
acetonitrile 1h (O-acetylmandelonitrile) and a-benzoyloxyphenylacetonitrile 1i (O-benzoyl-
mandelonitrile) under the same reaction conditions. The reaction of 1h was rapid and ecient
while 1i could not be fully transformed, with more than half being recovered after 6 days' reaction.
Unfortunately, in both cases, no corresponding amide and acid were obtained and, instead,
S-mandelic acid in ca. 30% ee was isolated along with benzoic acid.
2.2. Enantioselective biotransformations of amides
To shed further light on these stereoselective reactions some racemic amides were prepared and
subjected to the Rhodococcus sp. AJ270-catalyzed hydrolysis (Scheme 3). The results listed in
Table 2 show high activity and enantioselectivity of amidase against some a-alkylphenylacet-
amides. For example, both the unchanged amide 2b and the product acid 3b were obtained in
excellent chemical yields and enantiomeric excesses if the reaction was quenched at the right stage
(Table 2, entry 4). As with the nitrile hydratase, the amidase was also sensitive to steric hindrance.
The presence of a larger alkyl group such as n-propyl and n-butyl within the substrate therefore
greatly inhibited the hydrolysis, with only a small proportion of amide being transformed after
more than ten days (Table 2, entries 5±8). More noticeably, however, the enantioselectivity was
almost lost for amide 2e after prolonged incubation (Table 2, entries 7 and 8). This result is sur-
prising since the biotransformation using the corresponding nitrile 1e as a substrate a€orded the
acid 3e in an enantiomeric excess of 98% (Table 1, entry 8). It is very important to note that in all
cases the amide obtained was R-enantiomer in excess, the stereochemistry being di€erent from
that of the reaction using nitriles as the substrate (Table 1, entries 5, 6 and 8).
Scheme 3.

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M.-X. Wang et al. / Tetrahedron: Asymmetry 11 (2000) 1123±1135
Table 2
Enantioselective biotransformations of amides
3. Discussion
The rapid and ecient conversion of most of the nitriles tested in this study into the corresponding
amides indicates clearly that the nitrile hydratase involved in Rhodococcus sp. AJ270 has a
broader substrate spectrum irrespective of the electronic nature of the substituent of the nitriles.
This is in agreement with the conclusion we reached from our previous studies.11 In contrast to
the nitrile hydratase, the amidase of Rhodococcus sp. AJ270, however, shows both electronic
and steric limitations. The presence of a polar a-substituent such as methoxy or methylthio in the
molecule strongly inhibits the hydrolysis of amides. The introduction of a bulky and ¯exible
alkyl group including n-propyl and n-butyl also has a detrimental e€ect towards the amidase
action.
The results of the biotransformations of nitriles and of amides have shown strong evidence that
the enantioselectivity of the biotransformations of nitriles is the outcome of a combination of
selectivities of the nitrile hydratase and of the amidase, with the latter being a major and impor-
tant contributor. The formation of the S-carboxylic acid in almost all cases either from the bio-
transformation of nitrile or from the biotransformation of amide demonstrated the strict S-
enantioselectivity of the amidase. The results obtained also suggest that the amidase in
Rhodococcus sp. AJ270 seems to have the ability to recognize substituents of di€erent sizes and of
marginally di€erent polarities. The replacement of the alkyl substituent such as methyl and ethyl

M.-X. Wang et al. / Tetrahedron: Asymmetry 11 (2000) 1123±1135
1129
by a polar methoxy or methylthio, for instance, led to a mismatch in the recognition between the
amidase and substrate, and the hydrolysis of amides 2f and 2g, therefore, was less e€ective.
Increase in the length of the alkyl substituent to more than three methylenes, on the other hand,
also resulted greatly in the inhibition of amidase function. It should be noted that the reason for
the huge di€erence in the enantioselective formation of S-a-n-butylphenylacetic acid 3e between
using nitrile and using amide as the substrate (Table 1, entry 8 and Table 2, entries 7 and 8) is not
clear. One may suggest that a speci®c racemase against the acid was stimulated under the reaction
conditions when using the amide as the substrate. It may also be due to the low solubility of the
amide 2e in the aqueous bu€er, which leads to only a very limited amount of both enantiomers of
amide in solution available to the amidase. We did ®nd that when a very small amount of nitrile
or amide was fed to the organism under the same conditions, the conversion to acid was rapid
and usually went to completion with the formation of racemic acid. To prepare optically active
carboxylic acids using Rhodococcus sp. AJ270, the advantage of employing nitriles as starting
materials is apparent.
The diculty remains at this stage in de®ning the stereoselectivity of nitrile hydratase. The
isolation of both S-nitrile and S-amide from racemic substrates 1c and 1e makes rationalization
even more dicult. Nevertheless, although the nitrile hydratase also su€ers from steric limita-
tions, it has a relatively broader substrate spectrum against both enantiomers of nitriles tested
and therefore the enantioselective control appears less e€ective than amidase. The low enantio-
meric excess of the S-nitriles 1c and 1e recovered from the reaction showed that the nitrile
hydratase in Rhodococcus sp. AJ270 has poor R-selectivity against these racemic nitriles. It should
be noted that if the enantioselectivities of the biotransformation of nitrile were a€ected only by
the combination of a highly selective S-amidase and a low R-selective nitrile hydratase, the amide
formed during the course of the reaction would be R-con®gured. The fact that an excess of S-
amide was obtained in these cases suggested the involvement of a racemase that most probably
acted speci®cally against a-n-propyl- and a-n-butylphenylacetamides. The proposal of a speci®c
amide racemase rather than a nitrile racemase was further supported by the observation of an
extremely poor enantiomeric excess of amide 2c from the biotransformation of racemic amide 2c
while the acid 3c was obtained in excellent enantiopurity (Table 2, entries 5 and 6).
The isolation of mandelic acid 5 and benzoic acid from the hydrolysis of acylated mandelo-
nitriles revealed for the ®rst time that Rhodococcus sp. AJ270 has esterase activity.24 The rapid
hydrolysis of a relatively bulky acylated mandelonitrile 1h also strongly suggested that ester
hydrolysis precedes nitrile attack, since we have already established that bulky a-substituents
inhibit nitrile and amide conversion. As a consequence, the interaction of a-acetoxy-
phenylacetonitrile 1h with Rhodococcus sp. AJ270 ®rst resulted in the hydrolysis of the ester to
a€ord mandelonitrile 4, which was then converted into mandelic acid 5 by the action of nitrile
hydratase and amidase. The isolation of a small amount of benzoic acid (<10%) from this
reaction provided further evidence of the formation of mandelonitrile, as mandelonitrile co-
existed in an equilibrium with HCN and benzaldehyde and the latter was oxidized by air to form
benzoic acid (Scheme 4). Due to the steric hindrance of the phenyl group, the ester hydrolysis of
O-benzoylmandelonitrile 1i was less e€ective and the resulting mandelonitrile underwent similar
hydrolysis. The fact that the chemical yield of benzoic acid was slightly higher than theoretical in
this case again indicated that oxidation of benzaldehyde occurred during the course of the reac-
tion. It is hard to determine at this stage whether it is the nitrile hydratase/amidase or the esterase
that leads to the low enantioselectivity of the reaction. It is noteworthy that the esterase involved
in Rhodococcus sp. AJ270 seems somewhat speci®c against esters derived from mandelonitrile,

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M.-X. Wang et al. / Tetrahedron: Asymmetry 11 (2000) 1123±1135
Scheme 4.
especially O-acetylmandelonitrile 1h, as our earlier study showed that methyl benzoate was not
attacked by the organism.11 Both the speci®city and stereoselectivity of the esterase of Rhodococcus
sp. AJ270 await further study.
4. Conclusion
Rhodococcus sp. AJ270 is an ecient biocatalytic system, very useful for the preparation of
enantiopure carboxylic acids and derivatives through stereoselective hydrolyses of racemic a-
substituted phenylacetonitriles and amides. The nitrile hydratase involved generally has a broad
substrate spectrum against phenylacetonitriles irrespective of the electronic nature of the a-sub-
stituent while the amidase is very sensitive to both electronic and steric factors of the substituent
of amides. The overall enantioselectivity of nitrile hydrolysis is mainly determined by the combi-
nation of selectivities of nitrile hydratase and of amidase, with the latter being a major con-
tributor. The amidase has high S-enantiocontrol against amides while the nitrile hydratase
exhibits lower R-selectivity against nitriles. Rhodococcus sp. AJ270 may probably contain an
amide racemase acting speci®cally on a-n-propyl- and a-n-butylphenylacetamides. The bio-
transformations of these amides and the corresponding nitriles therefore led to very low enan-
tiomeric excesses of R- or even S-amide. Rhodococcus sp. AJ270 has been shown to have speci®c
esterase activity against acylated mandelonitriles, especially O-acetylmandelonitrile.
5. Experimental
5.1. General
Both melting points, which were determined using a Reichert Ko¯er hot-stage apparatus, and
boiling points are uncorrected. IR spectra were obtained on a Perkin±Elmer 782 instrument as
liquid ®lms or KBr discs. NMR spectra were recorded on Varian Unity 200 and Bruker AM 300
spectrometers. Chemical shifts are reported in ppm and coupling constants are given in hertz.
Mass spectra were measured on an AEI MS-50 mass spectrometer and microanalyses were carried
out by the Analytical Laboratory of the Institute.

M.-X. Wang et al. / Tetrahedron: Asymmetry 11 (2000) 1123±1135
1131
Polarimetry was carried out using an Optical Activity AA-10R polarimeter and the measure-
ments were made at the sodium D-line with a 5 cm pathlength cell. Concentrations (c) are given
in g/100 ml. The enantiomeric excess (ee) values of all products were obtained by means of chiral
HPLC and chiral GC analyses using racemic samples as references. The enantiomeric excesses of
all amides were obtained with a Shimadzu LC-10AVP HPLC system using a Chiracel OD column
at a ¯ow rate of 0.8 ml/min, with 2-propanol:hexane:H3PO4 [90:10:0.1] as the mobile phase. The
enantiomeric excesses of a-methoxy-, a-methylthio- and a-hydroxyphenylacetic acids were ana-
lyzed using the same chiral HPLC conditions but at a ¯ow rate of 0.4 ml/min. The chiral analysis
of nitriles was carried using a Chiral BSA-RP column with phosphate bu€er (0.1 M, pH
7.4):acetonitrile:1-butanol [88:10:2] as the mobile phase at a ¯ow rate of 1 ml/min. To obtain
good chiral separation, a-alkylphenylacetic acids were converted to their methyl esters using
diazomethane.33 The methyl esters of a-methyl- and a-ethylphenylacetic acids were well separated
on a Chiracel OJ column with hexane:2-propanol [180:1] as the solvent at a ¯ow rate of 0.8 and
0.4 ml/min, respectively. The separations of enantiomers of methyl a-n-propylphenylacetic acetate
and of methyl a-n-butylphenylacetic acetate were achieved using a chiral GC CP-Chirasil-Dex CB
column at the column temperature of 110 and 115ꢀC, respectively.
Except for 1a and 1b, which were purchased from Aldrich Chemical Co., nitriles 1c±e,34 1f,35
1g,36 1h37 and 1i37 were prepared according to the literature. Racemic amides38 and acids39 were
obtained from chemical hydrolysis of nitriles following the literature methods.
5.2. General procedure for the biotransformations of nitriles and amides
To an Erlenmeyer ¯ask (250 ml) with a screw cap was added Rhodococcus sp. AJ270 cells (2 g
wet weight) and potassium phosphate bu€er (0.1 M, pH 7.0, 50 ml) and the resting cells were
activated at 30ꢀC for 0.5 h with orbital shaking. Nitriles (3 mmol) or amides (2 mmol) were added
in one portion to the ¯ask and the mixture was incubated at 30ꢀC using an orbital shaker (200
rpm). The reaction, monitored by TLC, was quenched after a period of time (see Tables 1 and 2)
by removing the biomass through a Celite pad ®ltration. The resulting aqueous solution was
basi®ed to pH 12 with aqueous NaOH (2 M). Extraction with diethyl ether gave, after drying
(MgSO4) and concentration, the amide and unconverted nitrile. Separation of amide and nitrile
was e€ected by column chromatography. The aqueous solution was then acidi®ed using aqueous
HCl (2 M) to pH 2 and extracted with diethyl ether. Acid was obtained after removal of the sol-
vent. All products were characterized by their spectra data and comparison of the melting
points and optical rotary power with the known compounds, which are listed below, or by full
characterization.
5.2.1. Enzymic hydrolysis of (‹)-2-phenylpropionitrile 1a
25
D
(R)-( )-2-Phenylpropionamide 2a: 10 h (42%) ‰ꢀŠ
56.8 (c 4.31, CHCl3), ee >99%; 13.5 h
25
D
25
D
(36%) ‰ꢀŠ
53.4 (c 4.31, CHCl3) [lit.40 ‰ꢀŠ +46 (EtOH), (S)-(+)-2-phenylpropionamide], ee
>99% (chiral HPLC). Mp: 90±92ꢀC [lit.40 101±102ꢀC, (S)-(+)-2-phenylpropionamide]; ꢁmax
1
(KBr)/cm 3345, 3170 (NH2), 1650 (CˆO); ꢂH 7.23±7.38 (m, 5H, ArH), 5.81 (br s, 1H, NHH),
25
5.37 (br s, 1H, NHH), 3.59 (q, J=7.0, 1H, CH), 1.52 (d, J=7.0, 3H, CH3).
(S)-(+)-2-Phenylpropanoic acid 3a: 10 h (48%) ‰ꢀŠ +61.8 (c 4.27, CHCl3), ee 90%; 13.5 h
D
25
D
20
D
(58%) ‰ꢀŠ +57.8 (c 5.52, CHCl3) [lit.40 ‰ꢀŠ +81.1 (EtOH)], ee 67% (chiral HPLC on the
1
corresponding methyl ester). Oil; ꢁmax (®lm)/cm 2870±3113 (COOH), 1700 (CˆO); ꢂH 8.32 (br
s, 1H, COOH), 7.20±7.40 (m, 5H, ArH), 3.72 (q, J=7.2, 1H, CH), 1.51 d, J=7.2, 3H, CH3).

1132
M.-X. Wang et al. / Tetrahedron: Asymmetry 11 (2000) 1123±1135
5.2.2. Enzymic hydrolysis of (‹)-2-phenylbutyronitrile 1b
25
D
(R)-( )-2-Phenylbutyramide 2b: 70 h (58%) ‰ꢀŠ
23.2 (c 12.3, CHCl3), ee 35%; 96 h (34%)
25
D
24
D
‰ꢀŠ
56.5 (c 6.99, CHCl3) [lit.40 ‰ꢀŠ +57 (EtOH) (S)-(+)-2-phenylbutyramide], ee 96% (chiral
HPLC). Mp: 74±76 C [lit.40 80.5±81ꢀC, (S)-(+)-2-phenylbutyramide]; ꢁmax (KBr)/cm 3375,
ꢀ
1
3170 (NH2), 1649 (CˆO); ꢂH 7.20±7.39 (m, 5H, ArH), 5.99 (br s, 1H, NHH), 5.49 (br s, 1H,
25
NHH), 3.27 (t, J=7.8, 1H, CH), 1.67±2.26 (m, 2H, CH2), 0.88 (t, J=7.4, 3H, CH3).
(S)-(+)-2-Phenylbutyric acid 3b: 70 h (39%) ‰ꢀŠ +61.3 (c 4.97, CHCl3), ee >99%; 96 h
D
25
D
25
D
(40%) ‰ꢀŠ +63.7 (c 5.48 CHCl3) [lit.40 ‰ꢀŠ +78.5 (EtOH)], ee >99% (chiral HPLC on the
1
corresponding methyl ester). Oil; ꢁmax (®lm)/cm 2865±3110 (COOH), 1690 (CˆO); ꢂH 9.85 (br
s, 1H, COOH), 7.25±7.36 (m, 5H, ArH), 3.45 (t, J=7.8, 1H, CH), 1.70±2.21 (m, 2H, CH2), 0.91
(t, J=7.4, 3H, CH3).
5.2.3. Enzymic hydrolysis of (‹)-2-phenylvaleronitrile 1c34
25
D
(S)-(+)-2-Phenylvaleramide 2c: 150 h (27%) ‰ꢀŠ +39.9 (c 5.80, CHCl3), ee 41%; 214 h (40%)
25
D
25
D
‰ꢀŠ 22.8 (c 6.86, CHCl3) [lit.41 ‰ꢀŠ
50 (EtOH) (R)-( )-2-phenylvaleramide], ee 13% (chiral
HPLC). Mp: 78±79ꢀC [lit.41 106±108ꢀC, (R)-( )-2-phenylvaleramide]; ꢁmax (KBr)/cm 3420,
3200 (NH2), 1651 (CˆO); ꢂH 7.20±7.40 (m, 5H, ArH), 5.70 (br s, 1H, NHH), 5.41 (br s, 1H,
NHH), 3.39 (t, J=6.8, 1H, CH), 1.67±2.21 (m, 2H, CH2), 1.13±1.40 (m, 2H, CH2), 0.90 (t, J=7.0,
1
3H, CH3).
25
D
(S)-(+)-2-Phenylvaleric acid 3c: 150 h (8%) ‰ꢀŠ +55.0 (c 1.09, CHCl3), ee >99%; 214 h
25
D
25
D
(13%) ‰ꢀŠ +56.9 (c 1.96, CHCl3) [lit.41 ‰ꢀŠ +63.4 (c 0.9, EtOH)], ee >99% (chiral GC on the
1
corresponding methyl ester). Oil; ꢁmax (®lm)/cm 2864±3095 (COOH), 1710 (CˆO); ꢂH 7.33 (s,
5H, ArH), 3.57 (t, J=7.6, 1H, CH), 1.64±2.18 (m, 2H, CH2), 1.11±1.40 (m, 2H, CH2), 0.91 (t,
J=7.2, 3H, CH3).
25
D
(S)-(+)-2-Phenylvaleronitrile 1c:34 150 h (55%) ‰ꢀŠ +13.4 (c 3.84, CHCl3), ee 24%; 214 h
25
(33%) ‰ꢀŠ +21.2 (c 2.5, CHCl3), ee 28% (chiral HPLC). Oil; ꢁmax (®lm)/cm 2230 (CN); ꢂH
1
D
7.30±7.42 (m, 5H, ArH), 3.78 (t, J=6.4, 1H, CH), 1.41±2.04 (m, 4H, CH2CH2), 0.97 (t, J=7.2,
3H, CH3).
5.2.4. Enzymic hydrolysis of (‹)-3-methyl-2-phenybutyronitrile 1d34
25
D
24
(R)-( )-3-Methyl-2-phenylbutyramide 2d: 120 h (47%) ‰ꢀŠ
72.9 (c 1.32, CHCl3) [lit.40 ‰ꢀŠ
+56 (EtOH), (S)-(+)-3-methyl-2-phenylbutyramide], ee >99% (chiral HPLC). Mp: 110±112ꢀDC
[lit.40 141±142ꢀC, (S)-(+)-3-methyl-2-phenylbutyramide]; ꢁmax (KBr)/cm 3395, 3180 (NH2),
1
1641 (CˆO); ꢂH 7.34 (s, 5H, ArH), 6.90 (br s, 1H NHH), 5.85 (br s, 1H, NHH), 2.99 (d, J=10.6,
1H, CH), 2.39±2.42 (m, 1H, CH), 1.10 (d, J=6.3, 3H, CH3), 0.75 (d, J=6.3, 3H, CH3).
25
D
25
D
(S)-(+)-3-Methyl-2-phenylbutyric acid 3d: 120 h (46%) ‰ꢀŠ +60 (c 1.95, CHCl3) [lit.42 ‰ꢀŠ
+60.5 (CHCl3)], ee >99% (chiral HPLC on the corresponding methyl ester). Mp: 50±51ꢀC (lit.42
49.5±50.5ꢀC); ꢁmax (®lm)/cm 2860±3100 (COOH), 1705 (CˆO); ꢂH 7.31±7.39 (m, 5H, ArH),
1
7.11 (br s, 1H, COOH), 3.20 (d, J=10.6, 1H, CH), 2.32±2.45 (m, 1H, CH), 1.13 (d, J=6.5, 3H,
CH3), 0.76 (d, J=6.6, 3H, CH3).
5.2.5. Enzymic hydrolysis of (‹)-2-phenylhexanenitrile 1e34
25
25
D
(S)-(+)-2-Phenylhexanamide 2e: 300 h (34%) ‰ꢀŠ +20.8 (c 4.87, CHCl3) [lit.41 ‰ꢀŠ +41.9
D
ꢀ
(EtOH)], ee 20% (chiral HPLC). Mp: 98±99 C (lit.41 92±93ꢀC); ꢁmax (KBr)/cm 3370, 3180
(NH2), 1650 (CˆO); ꢂH 7.30 (s, 5H, ArH), 5.83 (br s, 1H, NHH), 5.42 (br s, 1H, NHH), 3.36 (t,
J=7.0, 1H, CH), 1.67±2.22 (m, 2H, CH2), 1.14±1.40 (m, 4H, CH2CH2), 0.86 (t, J=7.0, 3H, CH3).
1

M.-X. Wang et al. / Tetrahedron: Asymmetry 11 (2000) 1123±1135
1133
25
D
25
D
(S)-(+)-2-Phenylhexanoic acid 3e: 300 h (23%) ‰ꢀŠ +51.1 (c 4.34, CHCl3) [lit.41 ‰ꢀŠ +62.4
(c 0.967, EtOH)], ee 98% (chiral GC on the corresponding methyl ester); ꢁmax (KBr)/cm 1 2860±
3100 (COOH), 1705 (CˆO); ꢂH 8.62 (br s, 1H, COOH), 7.30 (s, 5H, ArH), 3.52 (t, J=7.6, 1H,
CH), 1.66±2.15 (m, 2H, CH2), 1.17±1.36 (m, 4H, CH2CH2), 0.86 (t, J=7.2, 3H, CH3).
25
D
(S)-(+)-2-Phenylhexanenitrile 1e:34 300 h (36%) ‰ꢀŠ +32.6 (c 5.41, CHCl3), ee 36% (chiral
HPLC); ꢁmax (KBr)/cm 1 2239 (CN); ꢂH 7.29±7.37 (m, 5H, ArH), 3.76 (t, J=6.8, 1H, CH), 1.76±
1.99 (m, 2H, CH2), 1.26±1.57 (m, 4H, CH2CH2), 0.91 (t, J=7.4, 3H, CH3).
5.2.6. Enzymic hydrolysis of (‹)-2-methoxy-2-phenylacetonitrile 1f 35
25
D
(‹)-2-Methoxy-2-phenylacetamide 2f: 37 h (74%) ‰ꢀŠ 0 (c 3.04, CHCl3), ee 0%. 49 h (56%)
25
‰ꢀŠ 0 (c 9.51, CHCl3), ee 0% (chiral HPLC). Mp: 111±112ꢀC (lit.43 112±114ꢀC); ꢁmax (KBr)/
D
1
cm 3450, 3175 (NH2), 1690, 1650 (CˆO); ꢂH 7.31±7.41 (m, 5H, ArH), 6.62 (br s, 1H, NHH),
6.40 (br s, 1H, NHH), 4.59 (s, 1H, CH), 3.36 (s, 3H, OCH3).
5.2.7. Enzymic hydrolysis of (‹)-2-methylthio-2-phenylacetonitrile 1g36
( )-2-Methylthio-2-phenylacetamide 2g: a solution of 2-methylthio-2-phenylacetonitrile 1g (2
mmol, 326 mg) in acetone (3 ml) was added to the suspension of Rhodococcus sp. AJ270 in bu€er
and the mixture was incubated for 120 h to give, after puri®cation, ( )-2-methylthio-2-phenyl-
25
D
acetamide 2g (64%) ‰ꢀŠ
26.0 (c 4.16, CHCl3), ee 15% (chiral HPLC). Mp: 141ꢀC. Found: C,
1
59.36; H, 6.04; N, 7.73. C9H11NOS requires: C, 59.64; H, 6.12; N, 7.73; ꢁmax (KBr)/cm 3390,
3175 (NH2), 1653 (CˆO); ꢂH 7.30±7.45 (m, 5H, ArH), 6.60 (br s, 1H, NHH), 5.91 (br s, 1H,
25
NHH), 4.48 (s, 1H, CH), 2.17 (s, 3H, SCH3); m/z (EI) 181 (M+, 3%), 137 (100).
(+)-2-Methylthio-2-phenylacetic acid 3g: 120 h (15%) ‰ꢀŠ +113.7 (c 2.34, CHCl3), ee 96%
D
(chiral HPLC). Mp: 73±76ꢀC (lit.44 81ꢀC); ꢁmax (KBr)/cm 1 2875±3063 (COOH), 1706 (CˆO); ꢂH
9.84 (br s, 1H, COOH), 7.32±7.48 (m, 5H, ArH), 4.51 (s, 1H, CH), 2.13 (s, 3H, SCH3).
5.2.8. Enzymic hydrolysis of (‹)-2-acetoxy-2-phenylacetonitrile 1h37
(‹)-2-Acetoxy-2-phenylacetonitrile 1h (420 mg, 2.4 mmol) yielded, after 62 h, (S)-(+)-
25
D
mandelic acid 5 (208 mg, 57%) and benzoic acid (10 mg, 3%). (S)-(+)-Mandelic acid ‰ꢀŠ +17.0
25
(c 3.43, CH3OH) [lit.43 ‰ꢀŠ +156.6 (H2O)], ee 29% (chiral HPLC). Mp: 115±118ꢀC (lit.43 133±
D
138ꢀC); ꢁmax (KBr)/cm 3400 (OH), 2630±3090 (COOH), 1719 (sh), 1710 (CˆO); ꢂH 7.36±7.47
1
(m, 5H, ArH), 5.36 (br s, 2H, OH, COOH), 5.27 (s, 1H, CH).
5.2.9. Enzymic hydrolysis of (‹)-2-benzoyloxy-2-phenylacetonitrile 1i37
(‹)-2-Benzoyloxy-2-phenylacetonitrile 1i (420 mg, 1.77 mmol) yielded, after 96 h, (S)-(+)-
25
D
acid (168 mg, 78%). (‹)-2-Benzoyloxy-2-phenylacetonitrile (153 mg, 34%) was also recovered.
mandelic acid 5 (50 mg, 19%) ‰ꢀŠ +32.7 (c 0.80, CH3OH), ee 35% (chiral HPLC) and benzoic
25
1
‰ꢀŠ 0 (c 2.56, CH3OH); ꢁmax (KBr)/cm 1730, 1724 (CˆO); ꢂH 7.47±8.11 (m, 10H, ArH), 6.70
D
(s, 1H, CH).
5.2.10. Enzymic hydrolysis of (‹)-2-phenylpropionamide 2a
25
D
58.0 (c 2.16, CHCl3), ee >99% (chiral HPLC).
(R)-( )-2-Phenylpropionamide 2a: 8 h (44%) ‰ꢀŠ
58.0 (c 3.32, CHCl3), ee >99%; 27 h
25
D
(22%) ‰ꢀŠ
25
D
(S)-(+)-2-Phenylpropanoic acid 3a: 8 h (51%) ‰ꢀŠ +47.6 (c 4.60, CHCl3), ee 56%;
25
27 h (78%) ‰ꢀŠ +20.5 (c 5.13, CHCl3), ee 49% (chiral HPLC on the corresponding methyl
D
ester).

1134
M.-X. Wang et al. / Tetrahedron: Asymmetry 11 (2000) 1123±1135
5.2.11. Enzymic hydrolysis of (‹)-2-phenybutyramide 2b
(R)-( )-2-Phenylbutyramide 2b: 48 h (72%) ‰ꢀŠ
25
D
67.1 (c 8.02, CHCl3), ee >99% (chiral HPLC).
23.8 (c 4.95, CHCl3), ee 29%; 120 h (47%)
25
D
‰ꢀŠ
25
D
(S)-(+)-2-Phenylbutyric acid 3b: 48 h (20%) ‰ꢀŠ +70.2 (c 3.79, CHCl3), ee >99%; 120 h
25
(51%) ‰ꢀŠ +68.5 (c 10.4, CHCl3), ee >99% (chiral HPLC on the corresponding methyl ester).
D
5.2.12. Enzymic hydrolysis of (‹)-2-phenylvaleramide 2c
25
D
(R)-( )-2-Phenylvaleramide 2c: 256 h (89%) ‰ꢀŠ
2.6 (c 5.90, CHCl3), ee 15%; 325 h (84%)
50 (EtOH), (R)-( )-2-phenylvaleramide] ee 20% (chiral
25
25
D
‰ꢀŠ
7.5 (c 13.3, CHCl3) [lit.41 ‰ꢀŠ
D
HPLC).
25
D
(S)-(+)-2-Phenylvaleric acid 3c: 256 h (8%) ‰ꢀŠ +58.2 (c 0.74, CHCl3), ee 95%; 325 h (15%)
25
‰ꢀŠ +58.8 (c 1.45, CHCl3), ee >99% (chiral GC on the corresponding methyl ester).
D
5.2.13. Enzymic hydrolysis of (‹)-2-phenylhexanamide 2e
25
D
(R)-( )-2-Phenylhexanamide 2e: 256 h (60%) ‰ꢀŠ
3.6 (c 4.89, CHCl3), ee 3%; 325 h (57%)
25
25
D
‰ꢀŠ
4.7 (c 4.75, CHCl3) [lit.41 ‰ꢀŠ +41.9 (EtOH), (S)-(+)-2-phenylhexanamide], ee 6% (chiral
25
D
HPLC).
(S)-(+)-2-Phenylhexanoic acid 3e: 256 h (27%) ‰ꢀŠ +8.2 (c 2.45, CHCl3), ee 12%; 325 h
D
25
D
(25%) ‰ꢀŠ +8.1 (c 2.88, CHCl3), ee 14% (chiral GC on the corresponding methyl ester).
Acknowledgements
This work is supported by the National Natural Science Foundation of China and the Chinese
Academy of Sciences. We thank Professors Q.-H. Fan at the Institute of Chemistry and H.-F.
Zou at the Dalian Institute of Chemical Physics for their assistance in analyses of chiral com-
pounds. M.-X. Wang and O. Meth-Cohn also thank The Royal Society, London, for ®nancial
support for a Joint Research Project between Chinese and UK groups.
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