Nitrile biotransformations for the efficient synthesis of highly enantiopure 1-arylaziridine-2-carboxylic acid derivatives and their stereoselective ring-opening reactions

Article abstract of DOI:10.1021/jo062339v

Catalyzed by the Rhodococcus erythropolis AJ270 whole cell catalyst under very mild conditions, biotransformations of racemic 1-arylaziridine-2- carbonitriles proceeded efficiently and enantioselectively to produce highly enantiopure S-1-arylaziridine-2-c

Full text of DOI:10.1021/jo062339v

Nitrile Biotransformations for the Efficient Synthesis of Highly
Enantiopure 1-Arylaziridine-2-carboxylic Acid Derivatives and
Their Stereoselective Ring-Opening Reactions
Jin-Yuan Wang, De-Xian Wang, Qi-Yu Zheng, Zhi-Tang Huang, and Mei-Xiang Wang*
Beijing National Laboratory for Molecular Sciences, Laboratory of Chemical Biology, Institute of
Chemistry, Chinese Academy of Sciences, Beijing 100080, China
mxwang@iccas.ac.cn
ReceiVed NoVember 14, 2006
Catalyzed by the Rhodococcus erythropolis AJ270 whole cell catalyst under very mild conditions,
biotransformations of racemic 1-arylaziridine-2-carbonitriles proceeded efficiently and enantioselectively
to produce highly enantiopure S-1-arylaziridine-2-carboxamides and R-1-arylaziridine-2-carboxylic acids
in excellent yields. Although the nitrile hydratase exhibits no selectivity against all nitrile substrates, the
amidase is highly R-enantioselective towards 1-arylaziridine-2-carboxamides. When treated with benzyl
bromide, 1-phenylaziridine-2S-carboxamide underwent a highly regioselective and enantiospecific ring-
opening reaction to afford an almost quantitative yield of R-â-[(benzyl)phenylamino]-R-bromopropanamide
(C-2 attack) and R-R-[(benzyl)phenylamino]-â-bromopropanamide (C-3 attack) in a 10.5:1 ratio. Further
treatment of the resulting ring-opening products with an N-nucleophilic reagent such as amine and azide
led to, through most probably the aziridinium intermediate, the formation of S-R-substituted-â-[(benzyl)-
phenylamino]propanamides in good chemical yields with high enantiomeric purity.
Introduction
derivatives, are of special importance owing to their occurrence
in natural products and in synthetic pharmaceuticals and to their
versatility in the preparation of diverse chiral molecules.1-3 In
contrast to oxirane-2-carboxylic acid derivatives,4 the synthesis
of optically active aziridine-2-carboxylates still remains chal-
lenging to synthetic chemists. For example, most chiral aziridine-
2-carboxylates were prepared using either chiral starting
materials1b,3b,5 or chiral auxiliaries,1b,3b,6 and most of them are
generally time consuming or not cost effective. Although
catalytic asymmetric syntheses have been reported,3b,6f,7 they
There is a rapid increasing interest1,2 in aziridine derivatives
because of their intriguing and unique chemical and biological
properties. Among aziridine compounds, chiral aziridine-2-
carboxylic acid derivatives, a type of special amino acid
* To whom correspondence should be addressed. Phone: +86-10-62565610.
Fax: +86-10-62564723.
(1) (a) For a recent monograph, see: Aziridines and Epoxides in Organic
Synthesis; Yudin, A. K., Ed.; Wiley-VCH: New York, 2006. For recent
reviews, see: (b) Osborn, H. M.; Sweeney, J. Tetrahedron: Asymmetry
1997, 8, 1693. (c) Xu, X. E. Tetrahedron 2004, 60, 2701. (d) McCoull,
W.; Davis, F. A. Synthesis 2000, 1347.
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W. K.; Ha, H.-J. Tetrahedron: Asymmetry 2000, 11, 3283. (b) Sim, T. B.;
Kang, S. H.; Lee, K. S.; Lee, W. K. J. Org. Chem. 2003, 68, 104. (c) Yun,
J. M.; Sim, T. B.; Hahm, H. S.; Lee, W. K. J. Org. Chem. 2003, 68, 7675.
(d) Kim, Y.; Ha, H.-J.; Han, K.; Ko, S. W.; Yun, H.; Yoon, H. J.; Kim, M.
S.; Lee, W. K. Tetrahedron Lett. 2005, 46, 4407. (e) Lee, B. K.; Kim, M.
S.; Hahm, H. S.; Kim, D. S.; Lee, W. K.; Ha, H.-J. Tetrahedron 2006, 62,
8393. (f) Denolf, B.; Mangelinkx, S.; To¨rnroos, K. W.; De Kimpe, N. Org.
Lett. 2006, 8, 3129.
(3) (a) For aziridine natural products, see: Lowden, P. P. Aziridine natural
products - discovery, biological activity, and biosynthesis. In Aziridines
and Epoxides in Organic Synthesis; Yudin, A. K., Ed.; Wiley-VCH: New
York, 2006; Chapter 11. (b) For aziridinecarboxylate, see: Zhou, P.; Chen,
B.-C.; Davis, F. A. Asymmetric syntheses with aziridinecarboxylate and
aziridinephosphonate building blocks. In Aziridines and Epoxides in Organic
Synthesis; Yudin, A. K., Ed.; Wiley-VCH: New York, 2006; Chapter 3.
(4) For a useful overview of the synthesis of chiral oxirane-2-carboxylic
acid derivatives, see: Wang, M.-X.; Lin, S.-J.; Liu, C.-S.; Zheng, Q.-Y.;
Li, J.-S. J. Org. Chem. 2003, 68, 4570 and references therein.
10.1021/jo062339v CCC: $37.00 © 2007 American Chemical Society
Published on Web 02/08/2007
2040
J. Org. Chem. 2007, 72, 2040-2045

Enantiopure 1-Arylaziridine-2-carboxylic Acid DeriVatiVes
CHART 1. Nitriles Bearing a Three-Membered Ring
are limited to a narrow substrate spectrum. Recently, the lipase-
mediated kinetic resolution of racemic 1-arylaziridine-2-car-
boxylates has been shown to give in most cases disappointingly
low chemical yield and enantioselectivity.8
Biotransformations of nitriles, either through a direct conver-
sion from a nitrile to a carboxylic acid catalyzed by a nitrilase
or through the nitrile hydratase catalyzed hydration of a nitrile
followed by the amide hydrolysis catalyzed by the amidase, have
become the effective and environmentally benign methods for
the production of carboxylic acids and their amide derivatives.9
One of the well-known examples is the industrial production
of acrylamide from biocatalytic hydration of acrylonitrile.10
Recent studies have demonstrated that biotransformations of
nitriles complement the existing asymmetric chemical and
enzymatic methods for the synthesis of chiral carboxylic acids
and their derivatives.11,12 The distinct features of enzymatic
transformations of nitriles are the straightforward generation of
enantiopure amides, valuable organo-nitrogen compounds in
synthetic chemistry, in addition to the formation of enantiopure
carboxylic acids. For example, we have shown that Rhodococcus
erythropolis AJ270,13 a nitrile hydratase/amidase-containing
whole cell catalyst, is able to efficiently and enantioselectively
SCHEME 1. Biotransformations of Racemic
1-Arylaziridine-2-carbonitriles 1
transform a variety of racemic nitriles bearing an R-14 or a
â-stereocenter15 and prochiral dinitriles16 into highly enantiopure
carboxylic acids and amides. More significantly, biotransfor-
mations of nitriles bearing a three-membered ring such as
cyclopropanecarbonitriles I17 and oxiranecarbonitriles II4,18
(Chart 1) proceeded in a highly predictable manner in terms of
reaction efficiency and enantioselectivity based on the substit-
uents and configurations of the substrates. Encouraged by our
previous study, we envisioned that aziridine-2-carbonitriles III
(Chart 1), which have steric features similar to those of
cyclopropanecarbonitriles I and oxiranecarbonitriles II, might
be the suitable substrates to the microbial cell catalyst. Herein,
we report the highly efficient biotransformations of racemic
1-arylaziridine-2-carbonitriles for the synthesis of enantiopure
1-arylaziridine-2-carboxylic acids and their derivatives. The
stereoselective aziridine ring-opening reactions in the synthesis
of chiral R,â-diamino acid derivatives will also be discussed.
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Results and Discussion
We first tested the biotransformation of racemic 1-phenyl-
aziridine-2-carbonitrile 1a (Scheme 1). When incubated with
Rhodococcus erythropolis AJ270 microbial cells in phosphate
buffer with pH 7.0 at 30 °C for less than 1 h, nitrile 1a (2 mmol)
underwent efficient hydrolysis to afford excellent yields of S-1-
phenylaziridine-2-carboxamide 2a and methyl R-1-phenylaziri-
(8) Kumar, H. M. S.; Rao, M. S.; Chakravarthy, P. P.; Yadav, J. S.
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1997, 3197 and references therein.
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Soc. 2002, 124, 9024. (b) Effenberger, F.; Osswald, S. Tetrahedron:
Asymmetry 2001, 12, 279. (c) Hann, E. C.; Sigmund, A. E.; Fager, S. K.;
Cooling, F. B.; Gavagan, J. E.; Ben-Bassat, A.; Chauhan, S.; Payne, M. S.;
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87, 221.
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Lett. 2000, 41, 8549. (b) Wang, M.-X.; Liu, C.-S.; Li, J.-S. Tetrahedron:
Asymmetry 2001, 12, 3367.
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(b) Wang, M.-X.; Feng, G.-Q. New J. Chem. 2002, 1575. (c) Wang, M.-
X.; Feng, G.-Q. J. Org. Chem. 2003, 68, 621-624. (d) Wang, M.-X.; Feng,
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G.-Q.; Zheng, Q.-Y. AdV. Synth. Catal. 2003, 345, 695. (f) Wang, M.-X.;
Feng, G.-Q.; Zheng, Q.-Y. Tetrahedron: Asymmetry 2004, 15, 347.
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J. Org. Chem, Vol. 72, No. 6, 2007 2041

Wang et al.
TABLE 1. Biotransformations of Racemic
1-Arylaziridine-2-carbonitriles 1
SCHEME 2. Biocatalytic Resolution of Racemic Amide 2a
yield
ee
yield
ee
entry
R
timea
0.92 h 2a
2
(%)b (%)c
4
(%)b (%)c
Ed
83
1
2
3
4
H
48 96.2 4a 47
0.67 h 2b 48 >99.5 4b 50
0.5 h 2c 46 95.4 4c 49
5 h
1.25 h 2d 48
6f 4-MeO 0.67 h 2e
28
4-MeO 3 h 2e
8e 4-MeO 1.75 h 2e
91.0
4-F
4-Cl
4-Br
93.8 >200
87.0
67.0
53
-
82
-
2d 25 >99.5 4d 74
5e 4-Br
89.0 4d 45
96.0 4e 22
93.0
>99.5
SCHEME 3. Enantioselectivity of Amidase in the
Hydrolysis of Amides Bearing a Three-Membered Ring
7
47 >99.5 4e 46
96.0 >200
96.0 >200
48 >99.5 4e 47
9
10
11
12g
4-Me
3-Me
2-Me
H
1.25 h 2f
50 >99.5 4f
45
2h 48
2a 45 >99.5 4a 47
50
96.7 4g 50
4h 47
>99.5 >200
5 h
4 d
2 h
2g
90.4
0
85.2
79
-
90
0
a
Reaction time was optimized to the completion of nitrile conversion
and roughly 50% conversion of the amide using HPLC analysis. b Isolated
yield. c Determined by chiral HPLC analysis. d Calculated according to ref
20. Acetone (4 mL) was used as a cosolvent. Optically inactive nitrile
1h (49% yield) was recovered. g Racemic nitrile 1a (15 mmol) was incubated
with 6 g wet weight of microbial cells.
e
f
hydratase and a highly R-enantioselective amidase in Rhodo-
coccus erythropolis AJ270 cells. The excellent R-enantioselec-
tivity of the amidase was further demonstrated by the kinetic
resolution of racemic 1-phenylaziridine-2-carboxamide 2a,
which produced S-2a and R-3a with good enantiomeric purity
(Scheme 2). It is noteworthy that nitrile biotransformations can
be readily used to prepare highly enantiopure 1-phenylaziridine-
2-carboxylic acid derivatives in a gram scale (entry 12, Table
1).
dine-2-carboxylate 4a,6f the latter being obtained from the
esterification of R-1-phenylaziridine-2-carboxylic acid 3a, with
96.2% and 91.0% enantiomeric excesses, respectively (entry 1,
Table 1). The absolute configuration of product R-4a was
determined by the comparison of its optical rotation with that
of the authentic sample.6f Encouraged by this result, a number
of racemic 1-arylaziridine-2-carbonitriles 1b-h were prepared19
and subjected to the biotransformations under the identical
conditions (Scheme 1). It was found that all nitriles were rapidly
hydrated to amides under the action of the nitrile hydratase
within the cells, and the hydrolysis of the resulting amides
catalyzed by the amidase was also efficient in most cases. As
summarized in Table 1, almost all substrates reached 50% amide
transformation into the carboxylic acids within 5 h except
racemic 1-(2-methylphenyl)aziridine-2-carbonitrile 1h which
took 4 days to yield the same amount of amide 2h and acid 3h
(entry 11, Table 1). For substrates such as 1d and 1e that did
not dissolve or disperse in culture suspension, addition of
acetone as a cosolvent had a beneficial effect to accelerate their
conversions (entries 4, 5, 7, and 8, Table 1). Biotransformations
of 1-arylaziridine-2-carbonitriles proceeded with excellent enan-
tioselectivity, and the enantiomeric ratio E20 was as high as
>200 (Table 1). For example, almost all nitriles 1a-g gave
high enantiomeric excess values of the amide and acid products
irrespective of the electronic nature of the substituent on the
benzene ring. However, the substitution pattern led to a drastic
effect on the enantioselectivity of the reaction. Although the
biotransformations of 1-(p-methylphenyl)aziridine-2-carbonitrile
1f and 1-(m-methylphenyl)aziridine-2-carbonitrile 1g yielded
excellent enantiocontrol, only optically inactive amide and acid
products were produced from o-methylphenyl-substituted aziri-
dine-2-carbonitrile analogue 1h (entries 9-11, Table 1). To
understand the stereochemistry and enantioselectivity of the
enzymes involved in catalysis, the microbial transformation of
racemic 1e was quenched when half of the nitrile was
transformed. The isolation of optically inactive nitrile 1h and
the enantiopure S-amide 2h (ee 96.0%) and ester R-4h (ee >
99.5%) (entry 6, Table 1) indicated a nonselective nitrile
The outcomes of the current study are consistent with previous
observations that nitrile hydratases are a type of highly active
and less selective enzyme against a wide variety of nitrile
substrates.9 These properties of the nitrile hydratase, such as
having a broad substrate spectrum and possessing low or no
enantioselectivity, are intrinsically determined by its enzyme
structure in which there is a spacious pocket near the active
site.17,18,21 In other words, a pair of enantiomers of 1-arylaziri-
dine-2-carbonitriles, analogous to trans-configured 2-arylcyclo-
propanecarbonitriles17 and 2-aryloxiranecarbonitriles,4,18 are not
recognized or differentiated by the nitrile hydratase, and almost
identical biocatalytic hydration reactions were therefore effected.
The formation of highly enantiopure 1-arylaziridine-2R-
carboxylic acids from the biotransformations of almost all
racemic nitrile and amide substrates indicated again that the
amidase involved in Rhodococcus erythropolis AJ270 is an
enantioselective enzyme.11c Very interestingly, examination of
the stereochemistry of the amide and acid products from the
biotransformations of the corresponding nitriles and amides
containing a three-membered ring including aziridine, cyclo-
propane,17 and oxirane4,18 revealed that the amidase is able to
recognize all carboxamides with a trans-arylated three-
membered ring in the same steric sense. In other words,
irrespective of the nature of the ring, all three-membered ring-
substituted carboxamides can be kinetically resolved into the
optically active amide and acid by the amidase following the
same chiral selection model (Scheme 3). It is worth noting that
the aryl group on the nitrogen of the aziridine is most likely to
adopt a trans configuration relative to the amido group,6f though
the pyramidal inversion might take place on aziridine nitrogen.22
The almost identical chiral recognition of amidase toward
1-arylaziridine-2-carboxamides and other three-membered ring
(19) Rao, S. A.; Kumar, A.; Ila, H.; Junjappa, H. Synthesis 1981, 623.
(20) (a) Chen, C.-S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J. J. Am. Chem.
Soc. 1982, 104, 7294. (b) Faber, K.; Hoenig, H. SelectiVity; http://
www.orgc.TUGraz.at/.
(21) (a) Huang, W. J.; Jia, J.; Cummings, J.; Nelson, M.; Schneider, G.;
Lindqvist, Y. Structure 1997, 5, 691. (b) Nagashima, S.; Nakasako, M.;
Dohmae, N.; Tsujimura, M.; Takio, K.; Odaka, M.; Yohda, M.; Kamiya,
N.; Endo, I. Nat. Struct. Biol. 1998, 5, 347.
2042 J. Org. Chem., Vol. 72, No. 6, 2007

Enantiopure 1-Arylaziridine-2-carboxylic Acid DeriVatiVes
TABLE 2. Ring-Opening Reactions of Racemic 1-Phenylaziridine-2-carboxamide
entry
conditions
Nu
yield of 10 + 11 (%)a
10/11b
1
2
3
4
5
6
7
8
9
BnNH2/Cu(OTf)2/CH3CN/rt
BnNH
BnNH
BnNH
N3
N3
N3
N3
N3
N3
-
-
BnNH2/Cu(OAc)2/CH3CN/reflux
BnNH2/Ti(OiPr)4/CH3CN/reflux
NaN3/CF3CO2H/CH3CN/rt
-
-
-
trace
-
34
76
89
90
-
ndc
-
NaN3/Cu(OAc)2/CH3CN/rt
NaN3/Cu(ClO4)2/CH3CN/H2O/reflux
NaN3/BF3‚OEt2/CH3CN/reflux
TMSN3/Cu(ClO4)2/CH3CN/H2O/reflux
TMSN3/Cu(CH3CN)4PF6/CH3CN/reflux
3.1:1
1:1
3.1:1
3.1:1
a
b
1
c
Isolated yield. Determined by H NMR. Not determined.
analogues fits well with the proposal17,18 that the amidase in
Rhodococcus erythropolis AJ270 might comprise a deeply
buried and highly steric demanding active site. The hypothetic
model can also be used to explain the huge difference in reaction
efficiency and enantioselectivity between racemic 1-(2-meth-
ylphenyl)aziridine-2-carboxamide 2h and other amides 2a-g.
Compared with the amides 2a-g having a para or a meta
substituent on the benzene ring, introduction of a substituent
such as methyl on the ortho position of the benzene ring
increased the steric bulkiness of the substrate. This steric effect
might inhibit the effective interaction of 2h with the highly
sterically sensitive amidase, leading to a very sluggish and
nonenantioselective biotransformation.
SCHEME 4. Highly Regioselective and Enantiospecific
Ring-Opening Reactions of S-2a by Benzyl Bromide
As a unique type of intermediates in organic synthesis, the
ring-opening reactions of aziridines have attracted much
attention.1-3 However, the studies on the reactions of aziridine-
2-carboxylic acids and their derivatives are very limited.3b
Having had a simple and straightforward biotransformation
approach to highly enantiopure 1-arylaziridine-2-carboxylates
and carboxamides, we started to explore their ring-opening
reactions with a number of N-nucleophiles to synthesize R,â-
diamino acids and derivatives.
Initially, we examined the reaction of racemic 1-phenylaziri-
dine-2-carboxamide with benzylamine. No ring-opening prod-
ucts were observed, however, even in the presence of a Lewis
acid catalyst (entries 1-3, Table 2). Sodium azide was then
tested as a nucleophile. Only in the presence of Cu(ClO4)2 or
BF3‚OEt2 as a catalyst did azide attack the aziridine to give
ring-opening products in moderate yield (entries 4-7, Table
2). The chemical yield was then improved to 90% when TMSN3
was employed instead of NaN3 (entries 8 and 9, Table 2).
Unfortunately, the ring-opening reaction did not yield satisfac-
tory regioselectivity, forming R-azido-â-phenylaminopropana-
mide (C-2 attack product) 10 and R-phenylamino-â-azidopro-
panamide (C-3 attack product) 11 in a ratio of 1:1 to 3:1 (entries
6-9, Table 2).
benzyl bromide proceeded efficiently in refluxing acetonitrile
to give excellent yield of the ring-opening products. More
importantly, the regioselectivity of the reaction improved
significantly, with the ratio of R-bromo-â-[(benzyl)phenylamino]-
propanamide 13 to R-[(benzyl)phenylamino]-â-bromopropana-
mide 14 being as high as 10.5:1 (Scheme 4). Chiral HPLC
analysis showed that each of the products 13 and 14 was
enantiomerically pure, indicating an enantiospecific ring-opening
reaction. The ring-opening reaction was most likely to proceed
through an aziridinium intermediate 12 (Scheme 4).23,24
Having established a highly stereoselective ring-opening
reaction, we then attempted the synthesis of chiral vicinal
diamine compounds, very useful chiral intermediates in both
medicinal chemistry and asymmetric synthesis. After completion
of the ring-opening reaction of S-2a by benzyl bromide, the
reaction mixture, without isolation of 13 and 14, was treated
directly with an N-nucleophile such as benzylamine, N-methyl-
N-benzylamine, allylamine, and sodium azide. In all cases,
R-substituted â-[(benzyl)phenylamino]propanamides 15a-d
were obtained in good yield. It should be pointed out that no
desired products 15a-d were produced if the conversion of S-2a
into 13 and 14 was not effected prior to the addition of a
nucleophile. The enantiomeric excess values of the products,
however, varied from 78% to 95% depending on the nucleophile
We then envisioned that a large activation moiety on the
aziridine ring while using a larger nucleophile would improve
the regioselectivity of the ring-opening reaction of 1-phenyl-
aziridine-2-carboxamide. To test our hypothesis, benzyl bro-
mide23 was chosen to react with 2. To our delight, the reaction
between enantiopure 1-phenylaziridine-2S-carboxamide 2a and
(23) Very recently, benzyl bromide has also been used to open the
2-alkanoyloxymethyl-1-arylmentylaziridine ring. (a) D’hooghe, M.; Van
Speybroeck, V.; Waroquier, M.; De Kimpe, N. Chem. Commun. 2006, 1554.
(b) D’hooghe, M.; De Kimpe, N. Synlett 2006, 2089.
(24) The aziridinium intermediate was very recently proposed and utilized
in the synthesis of enantiopure â-amino and R,â-diamino esters from the
reaction of N,N-dibenzyl-O-methylsulfonyl serine methyl ester with nu-
cleophilic reagents. Couturier, C.; Blanchet, J.; Schlama, T.; Zhu, J. Org.
Lett. 2006, 8, 2183.
(22) For pyramidal inversion of the aziridine ring, see: March, J.
AdVanced Organic Chemistry, 4th ed.; John Wiley & Sons, Inc.: New York,
1992; pp 98-99 and references therein.
J. Org. Chem, Vol. 72, No. 6, 2007 2043

Wang et al.
TABLE 3. Synthesis of Optically Active r,â-Diamino
Carboxamides via the Benzyl Bromide Mediated Ring-Opening
Reactions of 1-Phenylaziridine-2S-carboxamide 2a
biotransformations in the synthesis of highly enantiopure
carboxylic acids and their derivatives bearing a three-membered
ring. We have also shown that 1-phenylaziridine-2-carboxamide
undergoes readily the Lewis acid catalyzed ring-opening reac-
tions with an azide, albeit in very low regioselectivity. Highly
regioselective and enantiospecific ring-opening reaction of
1-phenylaziridine-2S-carboxamide was effected with benzyl
bromide to afford a quantitative yield of R-â-[(benzyl)phenyl-
amino]-R-bromopropanamide and R-R-[(benzyl)phenylamino]-
â-bromopropanamide in a 10.5:1 ratio. The resulting ring-
opening products, when treated with an N-nucleophilic reagent
such as amine and azide, were transformed through dominantly
the aziridinium intermediate into the optically active S-R-
substituted-â-[(benzyl)phenylamino]propanamides in good yields.
entry
conditions
15 (%)a
er (%)b
1
2
3
4
PhCH2NH2, 12 h
PhCH2NHMe, 12 h
CH2dCHCH2NH2, 24 h
NaN3, 48 h
15a (87)
15b (89)
15c (83)
15d (78)
97.5:2.5
95.6:4.4
92.0:8.0
89.2:10.8
Experimental Section
General Procedure for the Biotransformations of Nitriles or
Amides. To an Erlenmeyer flask (150 mL) with a screw cap were
added Rhodococcus erythropolis AJ270 cells13,14a (2 g wet weight)
and potassium phosphate buffer (0.1 M, pH 7.0, 50 mL), and the
resting cells were activated at 30 °C for 0.5 h with orbital shaking.
Racemic nitriles or amide (2 mmol) were added in one portion to
the flask, and the mixture was incubated at 30 °C using an orbital
shaker (200 rpm). The reaction, monitored by TLC and HPLC, was
quenched after a specified period of time (see Table 1) by removing
the biomass through a Celite pad filtration. The resulting aqueous
solution was extracted with ethyl acetate. After drying (MgSO4)
and removing the solvent under a vacuum, the residue of the organic
phase was chromatographed on a silica gel column using a mixture
of petroleum ether and ethyl acetate as the mobile phase to give
pure amide product 2. The aqueous phase was freeze-dried (-50
to -60 °C), and the residue was treated with CH2N2 in ether below
-15 °C. After filtration, ether was then removed from the solution
under a vacuum and the residue was subjected to silica gel column
chromatography using a mixture of petroleum ether and ethyl
acetate as the mobile phase to give pure methyl ester 4. All products
were fully characterized by spectroscopic data and microanalyses
(see Supporting Information). The absolute configurations of 4a
and 17 were determined by comparing the directions of their optical
rotations with that of the authentic samples,6f,25 and the absolute
configurations of other esters 4 were assigned assuming that the
introduction of a substituent on the benzene ring did not change
the direction of optical rotation. Enantiomeric excess values were
obtained from chiral HPLC analysis (see Supporting Information).
a
b
Isolated yield. Determined by chiral HPLC analysis.
SCHEME 5. Conversion of 15 into Optically Active
2R-Amino-3-(phenylamino)propanoic Acid 17
employed (Table 3). Most surprisingly, by comparison of the
stereochemistry of aziridine S-2a, the eutomer of all products
15 had the opposite configuration at the C-2 position, which
was determined by chemical conversion of 15 into the known
2R-amino-3-(phenylamino)propanoic acid 1725 (Scheme 5). This
suggested that the reaction did not proceed through SN2
nucleophilic replacement of the 2-bromo substituent of 13. If
the nucleophile attacked at C-2 of 13, configuration inversion
would occur to yield 2S-amino-3-(phenylamino)propanoic acid,
a product retaining the absolute configuration of starting
aziridine S-2a. The reaction between a nucleophile and 13 and
14 proceeded mainly via an aziridinium intermediate 12 which
was generated in situ from the neighboring amino participation.24
Observation of partial racemization of products 15 was most
probably due to the competitive direct SN2 substitution reaction
pathway taking place on 13. This competitive reaction route
became more noticeable when a sterically less-hindered nu-
cleophile such as allylamine or sodium azide was applied, which
was exemplified by the isolation of less enantiopure products
15c and 15d (Table 3).
Biotransformations of racemic 1-phenylaziridine-2-carbonitrile
1a gave S-(-)-1-phenylaziridine-2-carboxamide (2a) and methyl
R-(+)-1-phenylaziridine-2-carboxylate (4a). 2a: white solid; mp
146-147 °C [lit.26 mp 145-146 °C (racemic)]; [R]25 -204° (c
D
1.0, CHCl3); ee 96.2% (Chiral HPLC analysis); 1H NMR (300 MHz,
CDCl3, TMS) δ 7.24-7.30 (m, 2H), 6.98-7.07 (m, 3H), 6.50 (br,
1H), 5.89 (br, 1H), 2.72 (dd, J ) 3.0, 6.9 Hz, 1H), 2.47 (dd, J )
1.2, 3.0 Hz, 1H), 2.36 (dd, J ) 1.2, 6.9 Hz, 1H); IR (KBr) ν 3321.8,
Conclusion
3158.8 (CONH2), 1678.7 cm-1 (CdO). 4a: oil; [R]25 +165° (c
D
1.5, CH2Cl2); ee 91.0% (Chiral HPLC analysis) [lit.8 S-enantiomer
In summary, we have demonstrated that biotransformations
of racemic 1-arylaziridine-2-carbonitriles, catalyzed by the
Rhodococcus erythropolis AJ270 whole cell catalyst under very
mild conditions, lead to an efficient synthesis of highly
enantiopure S-1-arylaziridine-2-carboxamides and R-1-arylaziri-
dine-2-carboxylic acids. Although the nitrile hydratase exhibits
no selectivity against all nitrile substrates, the amidase is highly
R-enantioselective toward 1-arylaziridine-2-carboxamides. The
results have expanded further application of nitrile and amide
[R]25 -173.2° (c 0.25, CHCl3); ee 84%]; H NMR (300 MHz,
1
D
CDCl3, TMS) δ 6.99-7.03 (m, 5H), 3.80 (s, 3H), 2.80 (dd, J )
3.0, 6.0 Hz, 1H), 2.67 (dd, J ) 1.2, 3.0 Hz, 1H), 2.31 (dd, J ) 1.2,
6.0 Hz, 1H); IR (KBr) ν 1747.2 cm-1 (CdO). Kinetic resolution
of racemic amide 2a yielded S-(-)-2a in 54% yield with 98.2% ee
and R-(+)-4a in 45% yield with 89.0% ee. Biotransformation of
15 mmol of racemic nitrile 1a using 6 g wet weight whole cells
yielded S-(-)-2a in 45% yield (1.1 g) with >99.5% ee and R-(+)-
4a in 47% yield (1.2 g) with 85.2% ee.
(25) Goda, Y.; Suzuki, J.; Maitani, T.; Yoshihira, K.; Takeda, M.;
Uchiyama, M. Chem. Pharm. Bull. 1992, 40, 2236.
(26) Kotera, K.; Motomura, M.; Miyazaki, S.; Okada, T.; Matsukawa,
Y. Tetrahedron 1968, 24, 1727.
2044 J. Org. Chem., Vol. 72, No. 6, 2007

Enantiopure 1-Arylaziridine-2-carboxylic Acid DeriVatiVes
Ring-Opening Reaction of 1-Phenylaziridine-2-carboxamide
with NaN3 and TMSN3. A mixture of (()-1-phenylaziridine-2-
carboxamide 2a (0.5 mmol) and Lewis acid (0.1 mmol) in a specific
solvent (10 mL) (Table 2) was stirred for 10 min at room
temperature. A nucleophilic reagent was added, and the resulting
mixture was kept stirring under conditions specified in Table 2.
After completion of the reaction, which was monitored by TLC,
water was added and extracted with ether. The organic phase was
dried with anhydrous MgSO4, and the solvent was removed under
a vacuum. The residue was chromatographed on a silica gel column
eluting with a mixture of petroleum ether and ethyl acetate to give
a mixture of 10 and 11. Products 10 and 11 were inseparable from
column chromatography, and their ratio was determined roughly
mL) was refluxed for 11 h until the starting 2a was consumed,
which was monitored by TLC. After cooling to room temperature,
a nucleophilic reagent (2 mmol) was added and the resulting mixture
was refluxed for another period of time (Table 3). The solvent was
removed under a vacuum, and the residue was chromatographed
on a silica gel column eluting with a mixture of petroleum ether
and ethyl acetate to give pure R,â-diamino carboxamides 15a-d.
R-(-)-3-[(Benzyl)phenylamino]-2-(benzylamino)propana-
mide (15a): white solid; mp 110-111 °C; [R]25 -6.5° (c 0.93
D
1
CH2Cl2); ee 95.0% (Chiral HPLC analysis); H NMR (300 MHz,
CDCl3, TMS) δ 7.13-7.29 (m, 12H), 7.08 (br, 1H), 6.78-6.87
(m, 3H), 5.41 (br, 1H), 4.51 (d, J ) 16.9 Hz, 1H), 4.44 (d, J )
16.9 Hz, 1H), 3.70-3.83 (m, 2H), 3.46-3.56 (m, 3H), 1.61 (br,
1H); 13C NMR (75 MHz, CDCl3, TMS) δ 176.1, 148.8, 139.3,
138.2, 129.4, 128.7, 128.5, 128.0, 127.3, 127.1, 126.9, 118.2, 114.2,
61.1, 55.9, 54.8, 52.7; IR (KBr) ν 3343.6, 3317.0 (CONH2), 3181.0
(N-H), 1680.7 cm-1 (CdO); MS (ESI) m/z (%) 359.9 (100) [M
+ H+], 382.2 (6) [M + Na+]. Anal. Calcd for C23H25N3O: C, 76.85;
H, 7.01; N, 11.69. Found: C, 76.48; H, 7.06; N, 11.45.
1
by an H NMR spectrum.
(()-2-Azido-3-(phenylamino)propanamide (10) and 3-Azido-
2-(phenylamino)propanamide (11) Were Obtained in a 3.1:1
1
Ratio. H NMR (300 MHz, CDCl3, TMS) (10) δ 7.17-7.24 (m,
2H), 6.63-6.83 (m, 3H), 6.54 (br, 1H), 6.47 (br, 1H), 4.18-4.22
(dd, J ) 4.5, 7.2 Hz, 1H), 4.15 (br, 1H), 3.75 (dd, J ) 4.5, 13.6
Hz, 1H), 3.45 (dd, J ) 7.3, 14.0 Hz, 1H); (11) δ 7.17-7.24 (m,
0.65H), 6.63-6.83 (m, 0.97H), 6.54 (br, 0.32H), 6.47 (br, 0.32H),
4.37 (br, 0.32H), 3.91-3.96 (m, 0.32H), 3.83-3.89 (m, 0.32H),
3.68-3.72 (m, 0.32H); 13C NMR (75 MHz, CDCl3, TMS) (10) δ
171.2, 146.6, 129.5, 118.6, 113.3, 62.6, 46.5; (11) δ 173.9, 145.9,
129.6, 119.7, 113.9, 58.1, 52.4.
Conversion of 15 into R-(-)-2-Amino-3-(phenylamino)pro-
panoic Acid 17. Under atmospheric hydrogen, a mixture of R-(-)-
3-[(benzyl)phenylamino]-2-(benzylamino)propanamide 15a (0.5
mmol) and Pd/C (10 mg) in methanol was stirred at room
temperature for 4 h. After removal of Pd/C through Celite pad
filtration and removal of solvent under a vacuum, the residue was
heated with hydrochloric acid (6 N) at 80 °C for 6 h. The solvent
was removed, and the residue was subjected to a cationic exchange
resin column (Dowex, 50 × 8) followed by a reversed-phase ODS
column (35-70 µm) to give pure R-(-)-2-amino-3-(phenylamino)-
Ring-Opening Reaction of S-1-Phenylaziridine-2-carboxam-
ide with Benzyl Bromide. A mixture of S-(-)-1-phenylaziridine-
2-carboxamide 2a (0.5 mmol) and benzyl bromide (0.6 mmol) in
acetonitrile (10 mL) was refluxed for 11 h until the starting 2a
was consumed, which was monitored by TLC. The solvent was
removed under a vacuum, and the residue was chromatographed
on a silica gel column eluting with a mixture of petroleum ether
and ethyl acetate to give a mixture of R-3-[(benzyl)phenylamino]-
2-bromopropanamide 13 and R-2-[(benzyl)phenylamino]-3-bro-
mopropanamide 14 in a 10.5:1 ratio. Chiral HPLC analysis showed
propanoic acid 17: mp 134-136 °C; [R]25 -8.9° (c 0.45 H2O/
D
CH3CN/TFA ) 90:10:0.1); ee 85.7% (Chiral HPLC analysis) [lit.25
1
S-enantiomer: [R]25 +2.5° (H2O/CH3CN/TFA ) 90:10:0.1]; H
D
NMR (300 MHz, D2O, TMS) δ 7.22-7.27 (m, 2H), 6.77-6.82
(m, 3H), 3.91 (dd, J ) 4.2, 7.6 Hz, 1H), 3.65 (dd, J ) 3.9, 12.0
Hz, 1H), 3.52 (dd, J ) 8.0, 12.1 Hz, 1H), 2.43 (s, 3H); IR (KBr)
ν 3422.1, 1636.3 cm-1 (CdO). When using R-(-)-2-azido-3-
[(benzyl)phenylamino]propanamide 15d as starting material, the
reaction gave 17 with an ee of 63.8%.
1
that both 13 and 14 were of >99.5% ee. H NMR (300 MHz,
CDCl3, TMS) (13) δ 7.15-7.33 (m, 7H), 6.73-6.78 (m, 3H), 6.06
(br, 1H), 5.56 (br, 1H), 4.72(s, 2H), 4.54 (t, J ) 7.0 Hz, 1H), 4.30
(dd, J ) 7.3, 15.4 Hz, 1H), 3.91 (dd, J ) 6.1, 15.4 Hz, 1H); (14)
δ 7.15-7.33 (m, 0.67H), 6.78-6.90 (m, 0.29H), 6.06 (br, 0.09H),
5.54 (br, 0.09H), 4.72 (s, 0.20H), 4.52-4.57 (m, 0.09H), 4.10 (dd,
Acknowledgment. We thank the Ministry of Science and
Technology (2003CB716005, 2006CB806106), the National
Natural Science Foundation of China, and the Chinese Academy
of Sciences for financial support.
J ) 5.2, 11.0 Hz, 0.09H), 3.73 (dd, J ) 8.3, 10.8 Hz, 0.09H); 13
C
NMR (75 MHz, CDCl3, TMS) δ 170.3, 147.0, 138.2, 129.5, 128.6,
126.9, 117.7, 112.8, 55.8, 55.1, 45.0. Anal. Calcd for C16H17-
BrN2O: C, 57.41; H, 5.10; N, 8.41. Found: C, 57.42; H, 5.12; N,
8.20.
Supporting Information Available: Preparation of starting
nitriles and their spectroscopic data, full characterization of products,
1H and 13C NMR spectra of 2, 4, 10, 11, and 13-17, and HPLC
analysis of all chiral products. This material is available free of
charge via the Internet at http://pubs.acs.org.
General Procedure for the One-Pot Synthesis of Optically
Active R,â-Diamino Amides 15 from S-1-Phenylaziridine-2-
carboxamide. A mixture of S-(-)-1-phenylaziridine-2-carboxamide
2a (0.5 mmol) and benzyl bromide (0.6 mmol) in acetonitrile (10
JO062339V
J. Org. Chem, Vol. 72, No. 6, 2007 2045