Nitrile biotransformation for highly enantioselective synthesis of 3-substituted 2,2-dimethylcyclopropanecarboxylic acids and amides

Article abstract of DOI:10.1021/jo026490q

Biotransformations of differently configured 2,2-dimethyl-3-substitued-cyclopropanecarbonitriles were studied using a nitrile hydratase/amidase-containing Rhodococcus sp. AJ270 whole-cell catalyst under very mild conditions. Although all of the cis-3-aryl-2,2-dimethylcyclopropanecarbonitriles appeared inert toward the biocatalyst, a number of racemic trans-isomers efficiently underwent a highly enantioselective hydrolysis to produce (+)-(1R,3R)-3-aryl-2,2-dimethylcyclopropanecarboxylic acids and (-)-(1S,3S)-3-aryl-2,2-dimethylcyclopropanecarboxamides in high yields with excellent enantiomeric excesses in most cases. The overall enantioselectivity of the biotransformations of nitriles originated from the combined effects of 1R-enantioselective nitrile hydratase and amidase, with the later being a dominant factor. The influence of the substrates on both reaction efficiency and enantioselectivity was discussed in terms of steric and electronic effects. Coupled with chemical transformations, biotransformations of nitriles provided convenient syntheses of optically pure geminally dimethyl-substituted cyclopropanecarboxylic acids and amides, including chrysanthemic acids, in both enantiomeric forms.

Full text of DOI:10.1021/jo026490q

few drawbacks. Whereas the first two approaches usually
require lengthy multistep reactions and often give low
overall yields, the last one often produces a mixture of
two diastereomers and the control of both diastereo-
selectivity and enantioselectivity appears difficult. En-
zymatic methods have also been explored; however, they
are mainly limited to lipase- or esterase-catalyzed7 hy-
drolysis of esters with only modest enantioselectivity. It
was also reported that, in Lonza, the amidase was
successfully utilized to resolve 2,2-dimethylcyclopro-
panecarboxamide.8
Biotransformations of nitriles,9 through either a direct
nitrilase-catalyzed conversion of a nitrile to a carboxylic
acid10 or a nitrile hydratase-catalyzed hydration of a
nitrile followed by the hydrolysis of amide to acid by the
action of the amidase,11 have been demonstrated as being
unique and environmentally benign methods for the
synthesis of chiral carboxylic acids and their amide
derivatives because of the excellent selectivity and very
mild reaction conditions. Our earlier work has shown that
Rhodococcus sp. AJ 270, a robust nitrile hydratase/
amidase-containing microorganism,12 was able to hydro-
lyze a wide range of structurally diverse mono-13 and
dinitriles14 with excellent chemo- and regioselectivieties.
Very recently, we have found that Rhodococcus sp. AJ 270
could efficiently and enantioselectively catalyze the hy-
drolysis of a number of racemic nitriles and prochiral
dinitriles to produce enantiopure carboxylic acids and/
or amides in high yields.15 Our continuous interest in
understanding reaction scope and applications of both
nitrile hydratase and amidase involved in Rhodococcus
Nitr ile Biotr a n sfor m a tion for High ly
En a n tioselective Syn th esis of 3-Su bstitu ted
2,2-Dim eth ylcyclop r op a n eca r boxylic Acid s
a n d Am id es
Mei-Xiang Wang* and Guo-Qiang Feng
Laboratory of Chemical Biology, Center for Molecular
Science, Institute of Chemistry, Chinese Academy of
Sciences, Beijing 100080, China
mxwang@infoc3.icas.ac.cn
Received September 28, 2002
Abstr a ct: Biotransformations of differently configured 2,2-
dimethyl-3-substitued-cyclopropanecarbonitriles were stud-
ied using a nitrile hydratase/amidase-containing Rhodococ-
cus sp. AJ 270 whole-cell catalyst under very mild conditions.
Although all of the cis-3-aryl-2,2-dimethylcyclopropanecar-
bonitriles appeared inert toward the biocatalyst, a number
of racemic trans-isomers efficiently underwent a highly
enantioselective hydrolysis to produce (+)-(1R,3R)-3-aryl-
2,2-dimethylcyclopropanecarboxylic acids and (-)-(1S,3S)-
3-aryl-2,2-dimethylcyclopropanecarboxamides in high yields
with excellent enantiomeric excesses in most cases. The
overall enantioselectivity of the biotransformations of nitriles
originated from the combined effects of 1R-enantioselective
nitrile hydratase and amidase, with the later being a
dominant factor. The influence of the substrates on both
reaction efficiency and enantioselectivity was discussed in
terms of steric and electronic effects. Coupled with chemical
transformations, biotransformations of nitriles provided
convenient syntheses of optically pure geminally dimethyl-
substituted cyclopropanecarboxylic acids and amides, in-
cluding chrysanthemic acids, in both enantiomeric forms.
(6) (a) Doyle, M. P.; McKervey, M. A.; Ye, T. Modern Catalytic
Methods for Organic Synthesis with Diazo Compounds: From Cyclo-
propanes to Ylides; Wiley: New York, 1998; pp 163-288. (b) Aratani,
T. Pure Appl. Chem. 1985, 57, 1839.
(7) (a) Bao, A. B.; Rehman, H.; Krishnakumari, B.; Yadav, J . S.
Tetrahedron Lett. 1994, 35, 2611. (b) Yadav, J . S.; Rao, A. B.; Reddy,
Y. R.; Reddy, K. V. R. Tetrahedron: Asymmetry 1997, 8, 2291. (c)
Nanda, S.; Rao, A. B.; Yadav, J . S. Tetrahedron Lett. 1999, 40, 5905.
(d) Schneider, M.; Engel, N.; Boensmann, H. Angew. Chem., Int. Ed.
Engl. 1984, 23, 64.
The chiral geminally dimethyl-substituted cyclopropyl
group occurs in natural products1 and synthetic phar-
maceuticals2 and agrochemicals.3 Construction of opti-
cally active geminally dimethyl-substituted cyclopropyl
structure, a remaining challenging task in organic syn-
thesis, has been developed via (i) multistep synthesis
starting from chiral substrates (chiral pool),4,5 (ii) asym-
metric synthesis utilizing a chiral auxiliary,4,5 and (iii)
catalytic asymmetric cyclopropanation of olefins with
diazo compounds.6 However, each method suffers from a
(8) Stinton, S. C. Chem. Eng. News 1994, 72, 38.
(9) For recent reviews, see: (a) Sugai, T.; Yamazaki, T.; Yokoyama,
M.; Ohta, H. Biosci. Biotechnol. Biochem. 1997, 61, 1419. (b) Crosby,
J .; Moiliet, J .; Parratt, J . S.; Turner, N. J . J . Chem. Soc., Perkin Trans.
1 1994, 1679.
(10) (a) Harper, D. B. Biochem. Soc. Trans. 1976, 4, 502. (b) Harper,
D. B. Biochem. J . 1977, 165, 309. (c) Kobayashi, M.; Shimizu, S. FEMS
Microbiol. Lett. 1994, 120, 217.
(11) (a) Asano, Y.; Tani, Y.; Yamada, H. Agric. Biol. Chem. 1980,
44, 2251. (b) Asano, Y.; Fujishiro, K.; Tani, Y.; Yamada, H. Agric. Biol.
Chem. 1982, 46, 1165. (c) Asano, Y.; Tachibana, M.; Tani, Y.; Yamada,
H. Agric. Biol. Chem. 1982, 46, 1175.
(12) Blakey, A. J .; Colby, J .; Williams, E.; O’Reilly, C. FEMS
Microbiol. Lett. 1995, 129, 57.
(13) (a) Meth-Cohn, O.; Wang, M.-X. Tetrahedron Lett. 1995, 36,
9561. (b) Meth-Cohn, O.; Wang, M.-X. J . Chem. Soc., Perkin Trans. 1
1997, 1099.
(14) (a) Meth-Cohn, O.; Wang, M.-X. Chem. Commun. 1997, 1041.
(b) Meth-Cohn, O.; Wang, M.-X. J . Chem. Soc., Perkin Trans. 1 1997,
3197.
(15) (a) Wang, M.-X.; Lu, G.; J i, G. J .; Huang, Z.-T. Meth-Cohn, O.;
Colby, J . Tetrahedron: Asymmetry 2000, 11, 1123. (b) Wang, M.-X.;
Li, J .-J .; J i, G.-J .; Li, J .-S. J . Mol. Catal. B: Enzym. 2001, 14, 77. (c)
Wang, M.-X.; Lin, S.-J . Tetrahedron Lett. 2001, 42, 6501. (d), Wang,
M.-X.; Lin, S.-J . J . Org. Chem. 2002, 67, 6542. (e) Wang, M.-X.; Zhao,
S.-M. Tetrahedron Lett. 2002, 43, 6617. (f) Wang, M.-X.; Zhao, S.-M.
Tetrahedron: Asymmetry 2002, 13, 1695. (g) Wang, M.-X.; Liu, C.-S.;
Li, J .-S. Tetrahedron: Asymmetry 2001, 12, 3367.
* To whom correspondence should be addressed. Phone: +8610-
62554628. Fax: +8610-62569564.
(1) (a) Gerwick, W. H.; Proteau, P. J .; Nagle, D. G.; Hamel, E.;
Blokhin, A.; Slate, D. L. J . Org. Chem. 1994, 59, 1243. (b) Graham, D.
W.; Ashton, W. T.; Barash, L.; Brown, J . E.; Brown, R. D.; Canning, L.
F.; Chen, A.; Springer, J . P.; Rogers, E. F. J . Med. Chem. 1987, 30, 4.
(c) Zheng, G.-C.; Hatano, M.; Ishitsuka, M. O.; Kusumi, T.; Kakisawa,
H. Tetrahedron Lett. 1990, 31, 2617. (d) Zheng, G.-C.; Ichikawa, A.;
Ishitsuka, M. O.; Kusumi, T.; Yamamoto, H.; Kakisawa, H. J . Org.
Chem. 1990, 55, 3677.
(2) For a recent example, see: Ahmad, S.; Doweyko, L. M.; Dugar,
S.; Grazier, N.; Ngu, K.; Wu, S. C.; Yost, K. J .; Chen, B.-C.; Gougoutas,
J . Z.; DiMarco, J . D.; Lan, S.-J .; Gavin, B. J .; Chen, A. Y.; Dorso, C.
R.; Serafino, R.; Kirby, M.; Atwal, K. S. J . Med. Chem. 2001, 44, 3302.
(3) (a) Tombo, G. M. R.; Bellus, D. Angew. Chem., Int. Ed. Engl.
1991, 30, 1193. (b) Arlt, D.; J autelat, M.; Lantzsch, R. Angew. Chem.,
Int. Ed. Engl. 1981, 20, 703.
(4) Salaun, J . Chem. Rev. 1989, 89, 1247.
(5) Donaldson, W. A. Tetrahedron 2001, 57, 8589.
10.1021/jo026490q CCC: $25.00 © 2003 American Chemical Society
Published on Web 12/05/2002
J . Org. Chem. 2003, 68, 621-624
621

TABLE 1. Biotr a n sfor m a tion s of tr a n s-(()-3-Ar yl-2,2-d im eth ylcyclop r op a n eca r bon itr iles 1a
(1S,3S)-amide 2 (1R,3R)-acid 3
yieldb (%) eec (%)
(1S,3S)-nitrile 1
entry
substrate
Ar
conc (mmol)
time
yieldb (%)
eec (%)
yieldb (%)
eec (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
1a
1a
1a
1a
1b
1b
1c
1c
1d
1d
1e
1e
1f
C6H5
C6H5
C6H5
C6H5
4-F-C6H4
4-F-C6H4
4-Cl-C6H4
4-Cl-C6H4
3-Cl-C6H4
3-Cl-C6H4
2-Cl-C6H4
2-Cl-C6H4
4-MeO-C6H4
4-MeO-C6H4
4-Me-C6H4
4-Me-C6H4
2-Me-C6H4
2-Me-C6H4
2-Me-C6H4
1
1
1
0.5
1
0.5
1
0.5
1
0.5
1
0.5
1
1
1
1
1
1
0.5
4 h
12 h
33 h
30 h
40 h
65 h
80 h
44 h
33 h
58 h
7 d
53
32
29
48
31
43
29
38
30
45
33
74
36
45
10
tr
33d
59
>99
>99
>99
>99
>99
95
50
>99
14
15
93
>99
>99
6
>99
40
18
16
99
>99
>99
43
50
46
49
52
54
59
33
52
14
20
40
51
49
53
14
26
14
>99
99
>99
82
76
56
58
91
80
12
86
9
10
tr
29
83
0
>99
43
15
7 d
>99
16 h
23 h
36 h
96 h
3 d
96 (>99)e
10
1f
91
81
84
66
74
1g
1g
1h
1h
1h
33
30
36
24
83
81
17
4
30
41
83
51
25
19
7 d
6 d
>99
a
Throughout the works in this Table, 2 g wet weight of Rhodococcus sp. AJ 270 cells were used in 50 mL of phosphate buffer, and
reaction conditions were not optimized. Isolated yield. c Determined by chiral HPLC analysis. (+)-(1R,3R)-2a was obtained as favorable
b
d
enantiomer. e After recrystallization.
SCHEME 1. Bioca ta lytic Hyd r olysis of Ra cem ic
tr a n s-2,2-Dim eth yl-3-a r ylcyclop r op a n eca r bon itr iles
1
with a lower substrate concentration, enantiopure amide
2a and acid 3a were prepared in almost quantitative
yield from biotransformation of 1a (entry 4).
To test the generality of the reaction and also to
understand the substituent effect on the reaction, a
number of racemic trans-3-aryl-2,2-dimethylcyclopro-
panecarbonitriles 1b-h were synthesized17 and subjected
to Rhodococcus sp. AJ 270. We have found that both the
nature of and the substitution pattern of the substituent
on the benzene ring showed intriguing effects on the
conversion rate and enantioselectivity of nitrile hydration
and amide hydrolysis reactions. Almost all nitriles bear-
ing a para- or a meta-substituted aromatic group under-
went efficient hydration on the cyano group and consecu-
tive hydrolysis of amide to afford the corresponding (-)-
(1S,3S) amides and (+)-(1R,3R) acids in excellent yields
with good to excellent enantiomeric excesses (entries
5-10, 13 and 14). The presence of a 4-methoxy group
seemed to facilitate the reaction leading to higher enan-
tioselectivity (entries 13 and 14). Surprisingly, however,
the introduction of a 4-methyl group resulted in sluggish
nitrile hydration reaction, and the amide hydrolysis
exceeded the nitrile hydration step. As a consequence,
biotransformation of 1g led to good yields of acid (+)-
(1R,3R)-3g and nitrile (-)-(1S,3S)-1g together with only
a small or trace amount of amide (-)-(1S,3S)-2g (entries
15 and16). In the case of 3-(2-chloro- or 2-methylphenyl)-
2,2-dimethylcyclopropanecarbonitriles 1e and 1h , the
biocatalytic hydrolysis proceeded very slowly. When the
substrate concentration was halved and the hydration
of the cyano group completed, the enantiopure acid was
obtained, although the yield was less than 20% (entries
12 and 19). It should also be noted that the nitrile
recovered from the reaction showed dramatically varied
enantiomeric excesses depending upon the structure of
the substrates. For the ortho-substituted and para-chloro-
sp. AJ 270 and in synthesizing enantiopure cyclopropane
derivatives16 led us to undertake the current study of
biotransformation of 2,2-dimethyl-3-substituted-cyclo-
propanecarbonitriles. It is also hoped that the use of
differently substituted and configurated cyclopropanecar-
bonitriles and amides as the substrates would allow us
to probe the steric feature of the active site of the nitrile
hydratase and amidase.
We first examined biotransformation of racemic trans-
2,2-dimethyl-3-phenylcyclopropanecarbonitrile 1a . Cata-
lyzed by Rhodococcus sp. AJ 270 cells under very mild
conditions, nitrile (()-1a underwent enantioselective
hydrolysis to produce optically active products (Scheme
1). As summarized in Table 1, when the hydration of
nitrile 1a was terminated around 60% conversion, (-)-
(1S,3S)-1a was obtained in excellent yield (40%) with
excellent enantiomeric purity (99% ee). In addition,
amide (+)-(1R,3R)-2a was isolated in 53% yield with
enantiomeric excess of 33% (entry 1). The isolation of
nitrile (-)-(1S,3S)-1a and the formation of amide (+)-
(1R,3R)-2a in the initial period of incubation suggest the
nitrile hydratase is 1R-enantioselective toward 2,2-
dimethyl-3-phenylcyclopropanecarbonitrile. With the in-
crease of incubation time, the conversion of nitrile
hydration went to completion and the subsequent enan-
tioselective hydrolysis of amide took place effectively to
give optically active amide (-)-(1S,3S)-2a and acid (+)-
(1R,3R)-3a (entries 2 and 3). Under controlled conditions
(16) (a) Wang, M.-X.; Feng, G.-Q. Tetrahedron Lett. 2000, 41, 6501.
(b) Feng, G.-Q.; Wang, M.-X Chin. J . Chem. 2001, 19, 113. (c) Wang,
M.-X.; Feng, G.-Q. New J . Chem. 2002, 26, 1575.
(17) (a) Babler, J . H.; Spina, K. P. Tetrahedron Lett. 1985, 26, 1923.
(b) Babler, J . H.; Ill, E. US patent 4,567,265, 1986.
622 J . Org. Chem., Vol. 68, No. 2, 2003

SCHEME 2. Kin etic Resolu tion of Ra cem ic tr a n s-
2,2-Dim eth yl-3-a r ylcyclop r op a n eca r boxa m id es 2
SCHEME 3. Biotr a n sfor m a tion of Ra cem ic
Ch r ysa n th em ic Nitr iles a n d Am id es
TABLE 2. Bioca ta lytic Kin etic Resolu tion of
tr a n s-3-Ar yl-2,2-d im eth ylcyclop r op a n eca r boxa m id es
(1S,3S)-amide (1R,3R)-acid
2
3
sub-
entry strate
yieldb
(%)
eec
(%)
yieldb eec
Ar
timea
(%)
(%)
the spacious pocket of the enantioselective nitrile hy-
dratase while the amidase comprised a relatively deep-
buried and size-limited enentioselective active site. The
outcomes of the current study further support the
hypotheses of action of both the nitrile hydratase and the
amidase. Compared to trans-2-arylcyclopropanecarboni-
triles and trans-2-arylcyclopropanecarboxamides, trans-
2,2-dimethyl-3-arylcyclopropanecarbonitriles 1 and trans-
2,2-dimethyl-3-arylcyclopropanecarboxamides 2 have
increased steric hindrance around the cyano and amido
group, respectively. Therefore, the interactions of these
geminally dimethylated cyclopropanecarbonitrile and
amide substrates with the active sites of the nitrile
hydratase and amidase, respectively, should be slower
but more stereoselective than their 2-arylcyclopropan-
ecarbonitrile and amide analogues. These were indeed
exemplified by the observation of slower reaction rate
with much increased enantioselectivity for both nitrile
hydration and amide hydrolysis in comparison to the
biotransformation of racemic trans-2-arylcyclopropane-
carbonitriles. Introduction of geminally substituted di-
methyl groups into the cyclopropane skeleton further
increased the bulkiness of already sterically crowded cis-
configurated 2-arylcyclopropanecarbonitrile and amide
molecules, and as a consequence the enzymatic reaction
was completely inhibited.
On the basis of the results obtained, it can be concluded
that the efficient and highly enantioselective biotrans-
formation of cyclopropanecarbonitriles and amides is
predominantly governed by the steric effect of both
substituents attached to the 2- and 3-positions of cyclo-
propane ring. In other words, the reactivity and en-
antioselectivity of enzymatic hydrolysis of cyclopropan-
ecarbonitriles and amides can be regulated by tuning the
substituents either on the 2- or 3-position or on both
positions of the three-membered ring. This may also allow
us to envisage an efficient and highly enantioselective
hydrolysis of 2,2-dimethylcyclopropanecarbonitrile or
amide bearing a 3-substituent sterically smaller than a
phenyl group. To test the validity of this predication and
to prepare optically active chrysanthemic acids,3 the
hydrolysis of chrysanthmic nitriles was studied.
1
2
3
4
5
6
7
8
9
2a
2b
2c
2d
2e
2f
2g
2h
2h
C6H5
73 h
52 h
48 h
84 h
6 d
46
40
30
44
85
45
49
70
68
>99
>99
67
52
57
65
49
13
52
47
26
28
92
67
31
74
80
88
57
95
>99
4-F-C6H4
4-Cl-C6H4
3-Cl-C6H4
2-Cl-C6H4
91
<5
>99
80
39
42
4-MeO-C6H4 25 h
4-Me-C6H4
2-Me-C6H4
2-Me-C6H4
44 h
7 d
7 dd
a
Racemic amides 2 (0.5 mmol) were incubated with Rhodococ-
cus sp. AJ 270 (2 g wet weight) in phosphate buffer. b Isolated yield.
c Determined by chiral HPLC analysis. 2 mL of methanol was
d
added.
and methoxy-substituted substrates, the nitriles recov-
ered were almost optically inactive (entries 7, 11, 13, and
18).
To shed further light on the stereochemistry of the
biotransformation of nitriles 1, racemic trans-3-aryl-2,2-
dimethylcyclopropanecarboxamides 2 were prepared18
and their biohydrolysis was investigated. Under identical
catalytic conditions, amide (()-2 underwent a kinetic
resolution process to give enantiomerically enriched
amide (-)-(1S,3S)-2 and acid (+)-(1R,3R)-3 (Scheme 2).
As illustrated in Table 2, the similar effects of the
substituent and substitution pattern on the reactivity and
enantioselectivity of the amidase, which were found from
biotransformation of nitriles 1 (Table 1), were observed.
It should be addressed that the relatively lower enan-
tiomeric excesses obtained for (+)-(1S,3S)-2 and (-)-
(1R,3R)-3 from kinetic resolution of (()-2 suggested that
the enantioselectivity of the biotransformation of nitriles
1 originated from the combined action of (1R)-enantio-
selective nitrile hydratase and (1R)-enantioselective ami-
dase, with the later being a dominant one.
Encouraged by the abovementioned results, we then
tested the biotransformation of racemic cis-2,2-dimethyl-
3-arylcyclopropanecarbonitriles 4.17 In sharp contrast to
an effective biotransformation of racemic trans-2,2-dim-
ethyl-3-arylcyclopropanecarbonitriles 1, no hydrolysis of
nitriles 4 was observed under the same conditions. Even
a longer period (7 days) of interaction with Rhodococcus
sp. AJ 270 led to only the recovery of the starting nitriles.
In our previous study16 we have found that biotrans-
formations of trans-2-arylcyclopropanecarbonitriles pro-
ceeded rapidly with modest enantiocontrol whereas cis-
2-arylcyclopropanenitriles underwent very slow biohydroly-
sis but with excellent enantioselection. We have proposed
that a readily reachable reactive site be embedded within
As we expected, all chrysanthemic nitriles19 5 and 8
tested underwent effective biohydrolysis to produce opti-
cally active chrysanthemic acids 7 and 10 and amides 6
and 9 (Scheme 3). In the case of racemic trans-configu-
rated nitriles 5 and amides 6, the biotransformations
(19) (a) VanCantfort, C. K.; Coates, R. M. J . Org. Chem. 1981, 46,
4331. (b) Campbell, I. G. M.; Harper, S. H. J . Chem. Soc. 1945, 283.
(18) Hall, J . H.; Gisler, M. J . Org. Chem. 1976, 41, 3679.
J . Org. Chem, Vol. 68, No. 2, 2003 623

TABLE 3. Biotr a n sfor m a tion of Ch r ysa n th em ic Nitr iles
a n d Am id es
the configuration and the nature of the substituents. The
presence of a 3-substituent smaller than an aryl group,
either transoid or cisoid to the cyano or amido functional
moiety with the molecule, facilitated the biohydrolysis
with excellent enantioselectivity, which has been exem-
plified by the efficient preparation of highly enantiopure
chrysanthemic acids from the biotransformations of
chrysanthemic nitriles and amides. Coupled with facile
chemical transformations, this biotransformation process
provided effective and convenient syntheses of optically
geminally dimethyl-substituted cyclopropanecarboxylic
acids and amides in both enantiomeric forms.
sub-
entry strate
time
6 or 8
7 or 9
EWG
R
(h)a (yield,b eec)
(yield,b eec)
1
2
3
4
5
6
7
8
(()-5a CN
(()-5b CN
(()-6a CONH2 Me 73
48
Me 82
6a (48, >99) 7a (49, >99)
6b (45, >99) 7b (53, 81)
6a (48, >99) 7a (51, 97)
6b (47, >99) 7b (48, 89)
Cl
46
(()-6b CONH2 Cl
(()-8a CN
(()-8b CN
Me 65d 9a (12, >99) 10a (48, >99)
Cl
46e 9b (29, >99) 10b (47, >99)
(()-9a CONH2 Me 66
9a (48, >99) 10a (49, 97)
9b (48, >99) 10b (47, 95)
(()-9b CONH2 Cl
48
a
Racemic nitriles or amides (0.5 mmol) were incubated with
Rhodococcus sp. AJ 270 (2 g wet weight) in phosphate buffer.
b
Isolated yield (%). c Determined by chiral HPLC analysis (%).
Exp er im en ta l Section 20
d
25% of (-)-nitrile 8a were recovered. e 9% of nitrile 8b were
recovered.
All racemic nitriles 1,17 4,17 5,19 and 819 and amides 2,18 6,19
and 919 were prepared following the literature methods. The
configurations of optically active products were determined by
conversion into known compounds followed by comparison of
their sign of optical rotation with that of authentic samples.21
All enantiomeric excess values were obtained from HPLC
analysis with use of Chiralcel OD and OJ and Chiralpak AD
columns (see Supporting Information).
SCHEME 4. Ch em ica l Tr a n sfor m a tion s of Am id es
a n d Acid s
Gen er a l P r oced u r e for Biotr a n sfor m a tion s of Nitr iles
a n d Am id es. To an Erlenmeyer flask (100 mL) with a screw
cap were added Rhodococcus sp. AJ 270 cells12,13 (2 g wet weight)
and potassium phosphate buffer (0.1 M, 50 mL), and the resting
cells were activated at 30 °C for 30 min with orbital shaking.
Racemic nitriles or amides were added in one potion to the flask,
and the mixture was incubated at 30 °C with the use of an orbital
shaker (200 rpm). The reaction, monitored by TLC, was quenched
after a specified period of time (see Tables 1-3) by removing
the biomass through a Celite pad filtration. The resulting
aqueous solution was basified to pH 12 with aqueous NaOH (2
M). Extraction with ethyl acetate gave, after drying and
concentration, the amides and unconverted nitriles. Separation
of amide and nitrile was effected by column chromatography.
The aqueous solution was then acidified using aqueous HCl (2
M) to pH 2 and extracted with ethyl acetate. Acid was obtained
after removal of the solvent. All products were characterized by
their spectra data and comparison of the melting points and
optical rotary power with that of the known compounds or by
full characterization (see Supporting Information).
were very efficient and highly enantiopure products were
afforded in quantitative yields (entries 1-4 in Table 3).
Though less efficient than their trans-isomers, the cis-
configurated nitrile 8 and amide 9 analogues also un-
derwent high bioconversion to yield almost enantiopure
acids 10 and amides 9 (entries 5-8 in Table 3). It is
interesting to point out that the nitriles and amides
containing a geminal dichlorovinyl moiety appeared to
be more suitable substrates than those bearing a geminal
dimethylvinyl substituent, indicating again that the
enzymes involved in cells are mainly size selective.
To further show the utility of biotransformations of
nitriles in the preparation of both antipodes of enanti-
omers, acids (+)-(1R,3R)-3a and -7a and amides (-)-
(1S,3S)-2a and -6a were converted in excellent yields into
amides (+)-(1R,3R)-2a and -6a and acids (-)-(1S,3S)-3a
and -7a , respectively, using conventional chemical means.
No racemization was observed during the functional
group transformations (Scheme 4).
Ack n ow led gm en t . This work was supported by
the Major Basic Research Development Program
(2000077506), Ministry of Science and Technology (Grant
2002CCA0310), the National Natural Science Founda-
tion of China, and the Chinese Academy of Sciences.
M.X.W. thanks O. Meth-Cohn and J . Colby for discus-
sion.
In conclusion, we have shown that Rhodococcus sp.
AJ 270 cells effectively catalyzed the hydrolysis of racemic
trans-3-aryl-2,2-dimethylcyclopropanecarbonitriles under
very mild conditions to form (1R,3R)-2,2-dimethyl-3-
arylcyclopropanecarboxylic acids and (1S,3S)-2,2-diemthyl-
3-arylcyclopropanecarboxamides in excellent yields with
high enantiomeric excesses. The overall enantioselectivity
of biotransformations of nitriles originated from the
combined effects of a higher 1R-enantioselective amidase
and a 1R-enantioselective nitrile hydratase involved in
microbial cells. We have also demonstrated that the
catalytic efficiency and enantioselectivity of both nitrile
hydratase and amidase were strongly determined by both
Su p p or tin g In for m a tion Ava ila ble: Preparation of ra-
cemic nitriles and amides; details of characterization of
nitriles, amides, and acids; results of biocatalytic kinetic
resolution of amides; chemical transformations of acids and
amides; and HPLC analysis of amides, acids, and nitriles. This
material is available free of charge via the Internet at
http://pubs.acs.org.
J O026490Q
(20) General experiment information was given in ref 15a.
(21) (a) Kohmoto, S.; Kasimura, H.; Nishio, T.; Lida, I.; Kishikawa,
K.; Yamamoto, M.; Yamada, K. J . Chem. Soc., Perkin Trans. 2 1994,
1565. (b) Farkas, J .; Kourim, P.; Sorm, F. Collect. Czech. Chem.
Commun. 1959, 24, 2460.
624 J . Org. Chem., Vol. 68, No. 2, 2003