Efficient synthesis of highly enantiopure β-lactam derivatives from biocatalytic transformations of amides and nitriles

Article abstract of DOI:10.1016/j.tet.2014.05.018

Catalyzed by Rhodococcus erythropolis AJ270, a nitrile hydratase and amidase containing microbial strain, under mild conditions, kinetic resolution of racemic amides provides an efficient and scalable route to highly enantioenriched R-4-carboxymethyl-β-la

Full text of DOI:10.1016/j.tet.2014.05.018

Tetrahedron xxx (2014) 1e8
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Tetrahedron
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Efficient synthesis of highly enantiopure b-lactam derivatives
from biocatalytic transformations of amides and nitriles
,
Yu-Fei Ao a, Dong-Hui Leng a, De-Xian Wang a, Liang Zhao b, Mei-Xiang Wang b
*
a Beijing National Laboratory for Molecular Sciences, CAS Key Laboratory of Molecular Recognition and Function, Institute of Chemistry,
Chinese Academy of Sciences, Beijing 100190, China
b Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology, Ministry of Education, Tsinghua University, Beijing 100084, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Catalyzed by Rhodococcus erythropolis AJ270, a nitrile hydratase and amidase containing microbial strain,
under mild conditions, kinetic resolution of racemic amides provides an efficient and scalable route to
Received 25 March 2014
Received in revised form 29 April 2014
Accepted 5 May 2014
highly enantioenriched R-4-carboxymethyl-
b
-lactams and S-4-carbamoylmethyl-b-lactams despite the
substrates contain a stereogenic center that is
b
-positioned to amide functionality. Synthetic potential of
Available online xxx
the method was demonstrated by the constructions of novel
through convenient and practical chemical transformations.
b
-lactam-fused heterocyclic compounds
Keywords:
Ó 2014 Elsevier Ltd. All rights reserved.
Rhodococcus erythropolis AJ270
Nitrile hydratase
Amidase
Kinetic resolution
4-Carboxymethyl-b-lactam
1. Introduction
therefore utilized recently to achieve enantioselective desym-
metrization of prochiral6 and meso dicarboxamides.7 Although the
amidase displays high enantioselectivity towards many types of
amides,5 the level of enantioselection of the enzyme decreases
dramatically when the stereogenic center of racemic amide sub-
Because of high biocatalytic efficiency, excellent selectivity and
environmentally benign conditions, biotransformations have be-
come important methods in organic synthesis. Biotransformations
of nitriles1 through either the nitrilase-catalyzed direct conversion
of nitriles into carboxylic acids or the nitrile hydratase-catalyzed
hydration of nitriles followed by the amidase-effected amide hy-
drolysis complement the existing asymmetric chemical and en-
zymatic methods for the synthesis of chiral carboxylic acids and
their derivatives.2 In comparison with enzymatic ester hydrolysis,
biocatalysis of nitrile and amide generates straightforwardly
valuable enantiopure amides in addition to the formation of
enantiopure carboxylic acids.1e3 One of the widely used nitrile-
hydrolyzing biocatalysts is Rhodococcus erythropolis AJ270, which
is a nitrile hydratase and amidase containing microbial strain.4 The
versatility of this whole cell biocatalyst has been shown by
enantioselective biotransformations of a large number of struc-
turally diverse racemic nitriles including functionalized nitriles
and (hetero)cyclic nitriles to produce enantioenriched carboxylic
acid and amide products.5 It has been also reported that while the
nitrile hydratase exhibits low or virtually no enantioselectivity, the
amidase is highly enantioselective.5 The amidase has been
strates moves away from
a- to b- or g-position of amido
functionality.8e12
Functionalized
construction of antibiotics.13 4-Carboxymethyl-
ample, serve as the key precursors to carbapenems.14 In addition,
functionalized -lactams are useful intermediates in organic syn-
thesis.15 Although the synthesis of 4-carboxymethyl-
-lactams and
b-lactams provide an important platform for the
b
-lactams, for ex-
b
b
their derivatives has been extensively studied, development of
simple and efficient synthetic methods for enantiopure com-
pounds is still of great importance.16 We have previously discov-
ered that biotransformations of nitriles and amides are very
powerful in the preparation of enantiopure cyclopropane-,17
oxirane-,18 aziridine-,19 azetidine-20 and azetidinone-carboxylic
acids and their amide derivatives.21 Our continuous interest in
synthetic biocatalysis of nitriles and amides led us to investigate
the enantioselective biotransformations of 4-cyanomethyl- and 4-
carbamoyl-
-positioned to cyano and amido functional groups, respectively.
We report herein highly efficient whole cell-catalyzed synthesis of
functionalized -lactam derivatives with enantiomeric excess
values up to >99.5%.
b-lactams, the substrates contain a stereogenic center
b
b
* Corresponding author. E-mail address: mxwang@iccas.ac.cn (M.-X. Wang).
0040-4020/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2014.05.018
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2
Y.-F. Ao et al. / Tetrahedron xxx (2014) 1e8
2. Results and discussion
nitrile hydration reaction was not satisfactory, we deliberately in-
creased the reaction time, envisioning the amidase-catalyzed
highly enantioselective transformation of the resulting racemic
amide 6a. To facilitate the separation of products, acid was con-
verted quantitatively into methyl ester using excess CH2N2 after
biotransformations. Although the complete hydration of nitrile 5a
was slow, an elongated reaction time (28 h) did lead to the for-
mation of amide and acid in good yields. More pleasingly, excellent
enantiomeric excess values were obtained for amide S-6a and ester
R-8a (entry 2, Table 1). Encouraged by the observation of enantio-
selective biotransformations of nitrile 5a, substrates that contain
a brominated benzyl group on nitrogen were subjected to the
whole cell-catalyzed transformations. Surprisingly, introduction of
a bromo substituent into any position of the benzene ring resulted
in the sluggish hydration reaction of cyano group. After one week’s
interaction with biocatalyst, for instance, a substantial amount of
starting nitriles with low ee values were recovered along with the
isolation of enantioenriched amide and acid products in low yields
(entries 3e5, Table 1). Noticeably, when an N-arylmethyl group was
replaced by an N-allyl moiety, biotransformations of 5i proceeded
efficiently under the identical conditions (entries 6e8, Table 1). The
catalysis of the nitrile hydratase and the amidase in tandem affor-
ded good yields and ee values of products S-6i and R-8i within 3 h
(entry 8, Table 1). Although the effect of bromine substituent on the
catalytic efficiency of the nitrile hydratase remains not clear, it is
most probably due to the change of steric bulkiness of or altered
hydrophobicity of the substrate that decrease the effective in-
teraction between the enzyme and substrates.
The starting racemic substrates were synthesized from known
compounds by means of simple and practical transformations. As
illustrated in Scheme 1, for example, 4-methoxycarbonyl-b-lac-
tames 121 were reduced efficiently with NaBH4 in the presence of
LiCl to give alcohol derivatives 2. Reaction of 2 with MsCl in the
presence of triethylamine as an acid scavenger produced in-
termediates 3 in good yields. Treatment of 3 with NaI in refluxing
acetone followed by efficient aliphatic substitution reaction with
NaCN in DMSO led to the formation of desired nitrile substrates 5.
Subsequent chemical hydration reaction furnished the target am-
ides 6.
Scheme 1. Preparation of racemic 4-cyanomethyl-b-lactams 5 and 4-carba-
moylmethyl- -lactams 6.
b
The biotransformations of racemic 1-benzyl-4-cyanomethyl-b-
lactam 5a were first examined under standard conditions.4 Thus,
nitrile 5a was incubated with Rh. erythropolis AJ270 whole cells at
30 ꢀC in neutral phosphate buffer. To increase the solubility of ni-
trile in aqueous buffer, a small amount of acetone (0.5 mL) was
added. The biocatalytic hydration of 5a proceeded effectively, with
nearly a half of the substrate being converted into amide 6a within
2 h. Unfortunately, both amide product isolated and, particularly,
the nitrile substrate recovered gave appallingly low enantiomeric
excess values (entry 1, Table 1). It should be noted that the low
enantioselectivity of the nitrile hydratase involved in the microbial
In order to synthesize enantiomeric pure
b-lactam-bearing
acetamides and acetic acids, we then turned our attention to the
biotransformation of amide taking the advantage of active and
enantioselective amidase involved within the microbial cells. To our
delight, under very mild conditions, Rh. erythropolis AJ270 dis-
played excellent activity and enantioselectivity in catalyzing the
hydrolysis of racemic 4-carbamoylmethyl-
contrast to biotransformations of racemic 1-arylmethyl-4-
cyanomethyl- -lactams 5, results summarized in Table 2 reveal
b-lactams 6. In stark
b
that all racemic amide substrates tested underwent very rapid re-
action. For example, kinetic resolution of 6a was achieved within
3.25 h (entry 1, Table 2). The presence of a bromo substituent on
benzene ring, irrespective of the substitution pattern, did not slow
down the reaction at all, with kinetic resolution of 6bec going
completion in 2 he3 h (entries 2e4, Table 2). Introduction of other
cell towards 4-cyanomethyl-b-lactam 5a was however not un-
expected. As many studies have revealed, the nitrile hydratase ex-
hibit generally very low enantioselectivity in the biotransformation
of racemic nitriles. The move of a stereogenic center from a- to b-
position within the nitrile substrates further decreases the level of
enantioselection of the enzymes.8e12 Since the enantioselectivity of
Table 1
Biotransformations of racemic 4-cyanomethyl-b
-lactams 5a
Ent.
(ꢁ)-5
Time
R-5 (%)b (ee %)c
S-6 (%)b (ee %)c
R-8 (%)b (ee %)c
1
2
3
4
5
6
7
8
5a (R¼Bn)
3.5 h
28 h
7 d
7 d
7 d
0.7 h
2 h
3 h
R-5a (40) (<3)
d
S-6a (40) (10.2)
S-6a (45) (94.4)
S-6b (29) (>99.5)
S-6c (10) (>99.5)
S-6d (17) (>99.5)
S-6i (67) (8.0)
d
5a (R¼Bn)
R-8a (43) (>99.5)
R-8b (30) (74.8)
R-8c (13) (70.6)
R-8d (38) (67.4)
R-8i (8) (>99.5)
R-8i (25) (>99.5)
R-8i (48) (75.4)
5b (R¼o-BrC6H4CH2)
5c (R¼m-BrC6H4CH2)
5d (R¼p-BrC6H4CH2)
5i (R¼Allyl)
R-5b (40) (21.0)
R-5c (75) (<3)
R-5d (40) (12.2)
R-5i (23) (8.0)
R-5i (8) (24.8)
d
5i (R¼Allyl)
S-6i (59) (43.0)
S-6i (44) (87.0)
5i (R¼Allyl)
A mixture of substrate 5 (1 mmol), Rhodococcus erythropolis AJ270 (2 g wet weight) in phosphate buffer (0.1 M 50 mL) and acetone (0.5 mL) was incubated at 30 ꢀ C. The acid
products 7 were converted directly into methyl esters 8 after biotransformations.
a
b
Isolated yield.
c
Determined by chiral HPLC analysis.
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Y.-F. Ao et al. / Tetrahedron xxx (2014) 1e8
3
Table 2
Biotransformations of racemic nitriles 6a
Ent.
Substrate
t (h)
3.25
2.5
2
3
2.5
2
2.5
6
3
33
24
S-6 (%)b (ee %)c
R-8 (%)b (ee %)c
Ed
1
2
3
4
5
6
7
8
(ꢁ)-6a (R¼Bn)
S-6a (41) (>99.5)
S-6b (44) (>99.5)
S-6c (43) (>99.5)
S-6d (47) (>99.5)
S-6e (44) (>99.5)
S-6f (46) (94.4%)
S-6g (44) (>99.5)
S-6h (46) (>99.5)
S-6i (42) (98.6)
R-8a (46) (>99.5)
R-8b (45) (>99.5)
R-8c (49) (87.6)
R-8d (46) (>99.5)
R-8e (45) (>99.5)
R-8f (44) (94.2)
R-8g (45) (>99.5)
R-8h (45) (>99.5)
R-8i (49) (89.1)
>200
>200
81
>200
>200
115
>200
>200
89
(ꢁ)-6b (R¼o-BrC6H4CH2)
(ꢁ)-6c (R¼m-BrC6H4CH2)
(ꢁ)-6d (R¼p-BrC6H4CH2)
(ꢁ)-6e (R¼o-ClC6H4CH2)
(ꢁ)-6f (R¼o-FC6H4CH2)
(ꢁ)-6g (R¼o-MeC6H4CH2)
(ꢁ)-6h (R¼o-MeOC6H4CH2)
(ꢁ)-6i (R¼Allyl)
9
10
(ꢁ)-6d (R¼p-BrC6H4CH2)
(ꢁ)-6h (R¼o-MeOC6H4CH2)
S-6d (39) (>99.5)
S-6h (45) (>99.5)
R-8d (51) (71.2)
R-8h (46) (>99.5)
29
11e
>200
Substrate 6 (1 mmol) was incubated with Rhodococcus erythropolis AJ270 (2 g wet weight) in phosphate buffer (0.1 M 50 mL) and acetone (0.5 mL) at 30 ꢀ C. The acid
products 7 were converted directly into methyl esters 8 after biotransformations.
a
b
Isolated yield.
c
Determined by chiral HPLC analysis.
d
E values were calculated based enantiomeric excess of products following a literature method.22
e
10 mmol of (ꢁ)-6h, 4 g wet weight of microbial cells were used.
substituents, such as chloro (6e), fluoro (6f) and methyl (6g) into
the benzene ring led to equally efficient conversion of amide
function group into acid within 2.5 (entries 5e7, Table 2). Only in
the case of 4-carbamoylmethyl-b-lactam 6h that contains an N-1-
(2-methoxyphenyl)methyl group, an elongated time (6 h) was re-
quired (entry 8, Table 2) due to probably the increased steric hin-
drance of the substrate. Efficient amidase-catalyzed hydrolysis was
expectedly observed for racemic 1-allyl-4-carbamoylmethyl-b-lac-
tam 6i (entry 9, Table 2). It is noteworthy that, in addition to very
fast reaction velocity, the amidase-catalyzed kinetic resolution gave
excellent enantioselectivity. This has been exemplified by high
enantioselection values (E)22 calculated from the enantiomeric ex-
cess values of resulting S-6 and R-8 of the reactions that ranged
from 81 to >200 (Table 2). In many cases, enantiopure S-4-
Fig. 1. X-ray molecular structure of S-6a.
carbamoylmethyl-b-lactams S-6 and R-4-methoxcarbonylmethyl-
b-lactams R-8 were obtained in high yields. It should be noted that
careful monitoring of the amidase-catalyzed kinetic resolution
process appeared important as a longer incubation time could cause
more than 50% conversion of an amide substrate, leading, as
a consequence, to the decrease of ee of the acid product (entry 10,
Table 2). It is also worth mentioning that although the CeN bond
of
of the ring strain, in all Rh. erythropolis AJ270-catalyzed bio-
transformations of nitriles 5 and amides 6, no -lactam ring open-
b-lactam is more reactive than that of other amides because
b
ing reaction was observed, indicating the amidase in microbial cells
acts chemo-specifically on the CeN bond of primary carboxamides.
The structures of all products were supported by spectroscopic
data and elemental analysis. To assign the absolute configuration of
the products, single crystals of amide 6a and ester 8d obtained from
biocatalysis were cultivated from the slow diffusion of petroleum
ether into ethyl acetate solution. X-ray molecular structures, which
are depicted in Fig. 1 and 2, show unambiguously the S- and R-
configuration of 6a and 8d, respectively. The formation of R-con-
figured acid and S-configured amide products indicated convinc-
ingly the R-enantioselectivity of the amidase involved in
Rhodococcus erythropolis AJ270.
Fig. 2. X-ray molecular structure of R-8d.
amidase on the structure of the substrates. While either S- or R-
enantiomers of racemic amides bearing an a-stereogentic center are
effectively transformed by the enantioselective enzyme,5 the chiral
recognition of the amidase with amides that contain a stereogenic
center b-positioned to amide function group is rather weak leading
to a dramatically decreased level of enantiocontrol.8e12 Only in the
case of racemic substrates functionalized with a carbonecarbon
unsaturated bond, do the amidase-catalyzed reactions afford high
enantioselection.23 Although further studies are badly needed to
The high enantioselectivity of the amidase towards amide sub-
strates 6 that have a
studies have revealed the dependence of enantioselectivity of the
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b-stereogenic center is astonishing. Previous
elucidate
a detailed mechanism of enantioselective amidase

4
Y.-F. Ao et al. / Tetrahedron xxx (2014) 1e8
involved in Rh. erythropolis AJ270, highly efficient and enantiose-
lective biotransformation of -lactam-bearing acetamides afore-
mentioned implies that the amidase may comprise most likely
a enantioselective binding site for -lactam moiety.
4. Experimental section
b
To a solution of reactant 1 (50 mmol) in THF (70 mL) was added
LiCl (0.1 mol, 6.05 g), NaBH4 (0.1 mol, 3.8 g) and ethanol (140 mL).
The mixture was kept stirring overnight at room temperature. The
reaction was quenched by adding water (100 mL) at 0 ꢀC, and the
mixture was extracted with ethyl acetate (3ꢂ100 mL). The com-
bined organic layer was dried over anhydrous Na2SO4. After fil-
tration and removal of solvent, crude product 2 was obtained in
80%e90% yield. Without further purification, the crude product 2
was mixed with triethylamine (0.1 mol, 13.9 mL) in DCM (200 mL)
at 0 ꢀC. Methylsulfonyl chloride (75 mmol, 5.8 mL) was added
slowly, and the resulting mixture was stirred for another one hour
at ambient temperature. After mixing with water (400 mL), or-
ganic phase was separated and washed with brine (2ꢂ100 mL)
and dried over anhydrous Na2SO4. Solvent was removed under
vacuum, and the residue was chromatographied on a silica gel
column using a mixture of petroleum ether and ethyl acetate
(v:v¼1:1) as mobile phase to give crude product 3 in 85%e95%
yield. The crude product 3 was refluxed overnight with NaI
(0.16 mol, 24.0 g) in acetone (200 mL). Water (200 mL) was added
and the mixture was extracted with DCM (3ꢂ100 mL). After drying
over anhydrous Na2SO4, organic solvent was removed under vac-
uum to afford crude product 4 in 70%e85% yields. The alkyl iodide
4 obtained was stirred with NaCN powder (0.16 mol, 7.8 g) in DMF
(80 mL) at room temperature for 24 h. Water (150 mL) was added
and the mixture was extracted with ethyl acetate (4ꢂ50 mL). The
combined organic layer was dried over anhydrous Na2SO4. After
removal of solvent under vacuum, the residue was chromato-
graphed on a silica gel column using a mixture of petroleum ether
and ethyl acetate (v:v¼1:1) as mobile phase to give pure
product 5.
b
To demonstrate the practical utility of the method, a multigram
scale biotransformation was implemented. Thus, under the iden-
tical conditions using 4 g wet weight Rh. erythropolis AJ270,
10 mmol (2.48 g) of racemic 6h were successfully resolved into
enantiopure S-6h (1.12 g, ee >99.5%) and R-7h (1.21 g, ee >99.5%)
in almost quantitative yields in 24 h (entry 11, Table 2). Coupled
with further biocatalytic hydrolysis of S-amides, S-configured acid
products were also obtained. For example, catalyzed by the
Rh. erythropolis AJ270 whole cell catalyst, enantiopure amide S-6h
underwent complete hydrolysis to afford acid S-7h without losing
enantiomeric purity (Scheme 2). It is worth emphasizing that
chemical hydrolysis of amide S-6h led to the decomposition of b-
lactam. Both amide and acid products obtained from biocatalysis
are conceivably valuable chiral intermediates in organic synthe-
sis.15 They can act as unique precursors to carbapenam antibi-
otics.14 The functional groups presented within the molecules also
provide handles for further chemical manipulations. To illustrate
the synthetic potential of the method, synthesis of b-lactam-fused
heterocycles was performed. As shown in Scheme 2, iodolactoni-
zation of S-7i that was readily prepared from biocatalytic hydro-
lysis of S-6i, gave straightforwardly a mixture of bicyclic products
9 in 89% yield with a diastereomeric ratio being 1.4:1. A b-lactam-
fused benzoazepinone product R-10 was synthesized conveniently
in a good yield from the saponification of R-8g with LiOH followed
consecutively by the reaction with SOCl2 and an intramolecular
FriedeleCrafts acylation reaction.
4.1. ( )-5a as colorless oil (4.67 g, 23.3 mmol)
1H NMR (300 MHz, CDCl3)
d 7.27e7.38 (m, 5H), 4.58
(d, J¼15.0 Hz, 1H), 4.30 (d, J¼15.0 Hz, 1H), 3.75 (ddd, J¼7.8, 5.4,
2.4 Hz, 1H), 3.21 (dd, J¼15.0, 5.1 Hz, 1H), 2.84 (dd, J¼15.0, 2.4 Hz,
1H), 2.56 (dd, J¼16.8, 5.1 Hz, 1H), 2.48 (dd, J¼17.1, 5.7 Hz, 1H); 13C
NMR (75 MHz, CDCl3)
d 165.4, 134.9, 129.1, 128.5, 128.3, 115.9, 46.9,
45.4, 42.8, 21.9; IR (KBr)
n
2251, 1751 cmꢃ1; MS (EI) m/z (%) 201
[MþH]þ, 223 [MþNa]þ. HRMS (TOF-MS-EI) Anal. Calcd for
C
12H12N2O: 200.0950 [M]þ. Found: 200.0952 [M]þ.
4.2. ( )-5b as colorless oil (3.30 g, 11.8 mmol)
1H NMR (300 MHz, CDCl3)
Scheme 2. Synthetic applications.
d
7.57 (d, J¼8.1 Hz, 1H), 7.34e7.39
(m, 2H), 7.21e7.28 (m, 1H), 4.70 (d, J¼15.0 Hz, 1H), 4.45
(d, J¼15.0 Hz,1H), 3.81 (ddd, J¼6.6, 4.8, 2.4 Hz,1H), 3.24 (dd, J¼15.0,
5.1 Hz,1H), 2.90 (dd, J¼15.0, 2.4 Hz,1H), 2.69 (dd, J¼17.1, 4.5 Hz,1H),
3. Conclusion
2.60 (dd, J¼16.8, 6.0 Hz, 1H); 13C NMR (75 MHz, CDCl3)
d 165.4,
In summary, we have shown that the amidase involved
in Rh. erythropolis AJ270 is able to catalyze enantioselective
134.2, 133.2, 131.2, 130.1, 128.3, 123.7, 115.6, 47.3, 45.3, 42.8, 21.8; IR
(KBr)
n
2251, 1754 cmꢃ1; MS (ESI) m/z (%) 301 [MþNa]þ (79Br), 303
hydrolysis of
hydratase exhibits low enzymatic activity with virtually no
enantiocontrol towards -lactam-bearing acetonitrile substrates.
b
-lactam-bearing acetamides while the nitrile
[MþNa]þ (81Br). Anal. Calcd for C12H12BrN2O: C, 51.63; H, 3.97; N,
10.04. Found: C, 51.51; H, 4.02; N, 9.92.
b
The microbial whole cell-catalyzed kinetic resolution provides
4.3. ( )-5c as colorless oil (3.60 g, 12.9 mmol)
an efficient and scalable route to highly enantioenriched R-4-
carboxymethyl-
b
-lactams and S-4-carbamoylmethyl-
b
-lactams
1H NMR (300 MHz, CDCl3)
d 7.45e7.46 (m, 2H), 7.22e7.29
under mild conditions despite the substrates contain a stereogenic
(m, 2H), 4.56 (d, J¼15.3 Hz, 1H), 4.26 (d, J¼15.3 Hz, 1H), 3.79 (ddd,
center that is
functionalized
b
b
-positioned to amide functionality. The resulting
-lactam derivatives that are not easily accessible
J¼7.8, 5.4, 2.4 Hz, 1H), 3.23 (dd, J¼15.0, 5.1 Hz, 1H), 2.86 (dd, J¼15.0,
2.1 Hz,1H), 2.59 (d, J¼5.4 Hz, 2H); 13C NMR (75 MHz, CDCl3)
d 165.4,
by other means, are useful chemical entities and their applications
are demonstrated by the constructions of novel -lactam-fused
137.4, 131.4, 131.3, 130.7, 127.0, 123.0, 115.9, 47.1, 44.6, 43.0, 21.9; IR
b
(KBr)
n
2251, 1752 cmꢃ1; MS (ESI) m/z (%) 301 [MþNa]þ (79Br), 303
heterocyclic compounds through convenient and practical chem-
ical transformations.
[MþNa]þ (81Br). Anal. Calcd for C12H12BrN2O: C, 51.63; H, 3.97; N,
10.04. Found: C, 51.59; H, 4.00; N, 9.81.
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Y.-F. Ao et al. / Tetrahedron xxx (2014) 1e8
5
4.4. ( )-5d as white solid (3.45 g, 12.4 mmol)
4.10. General procedure for the synthesis of racemic amides 6
Mp 39.0e40.0 ꢀC; 1H NMR (300 MHz, CDCl3)
d
7.49 (d, J¼8.4 Hz,
To a solution of racemic 5 (10 mmol) in ethanol (40 mL) was
added anhydrous K2CO3 powder (300 mg) while stirring. Aqueous
H2O2 (30%, 7.5 mL) was then added slowly at 0 ꢀC, and the
resulting mixture was stirred overnight. A saturated aqueous so-
lution of sodium thiosulfate (Na2S2O3) (50 mL) was added, and the
mixture was extracted with ethyl acetate (3ꢂ20 mL). Organic
phase was dried over anhydrous Na2SO4. After removal of solvent,
the residue was chromatographed on a silica gel column eluted
with a mixture of ethyl acetate and acetone (v:v¼1:1) to give pure
amide products 6.
2H), 7.20 (d, J¼8.4 Hz, 2H), 4.52 (d, J¼15.3 Hz, 1H), 4.25
(d, J¼15.3 Hz, 1H), 3.77 (ddd, J¼7.5, 5.4, 2.4 Hz, 1H), 3.19 (dd, J¼15.0,
5.1 Hz, 1H), 2.83 (dd, J¼14.7, 2.1 Hz, 1H), 2.59 (d, J¼5.4 Hz, 2H); 13
C
NMR (75 MHz, CDCl3)
d 165.3, 134.1, 130.0, 122.0, 122.6, 115.9, 46.9,
44.5, 42.7, 21.7; IR (KBr)
n
2251, 1752 cmꢃ1; MS (ESI) m/z (%) 301
[MþNa]þ (79Br), 303 [MþNa]þ (81Br). Anal. Calcd for C12H11BrN2O:
C, 51.63; H, 3.97; N, 10.04. Found: C, 51.28; H, 4.13; N, 9.85.
4.5. ( )-5e as white solid (3.14 g, 13.4 mmol)
4.10.1. (ꢁ)-6a as white solid (1.42 g, 65%). Mp 116.0e117.0 ꢀC; 1H
Mp 63.0e64.0 ꢀC; 1H NMR (300 MHz, CDCl3)
d 7.43e7.38
NMR (300 MHz, CDCl3)
d 7.27e7.37 (m, 5H), 5.35 (br s, 1H), 5.30
(m, 2H), 7.33e7.29 (m, 2H), 4.68 (d, J¼15.2 Hz, 1H), 4.42
(d, J¼15.2 Hz, 1H), 3.81e3.75 (m, 1H), 3.22 (dd, J¼14.9, 5.1 Hz, 1H),
2.88 (dd, J¼14.9, 2.2 Hz, 1H), 2.71e2.55 (m, 2H); 13C NMR (75 MHz,
(br s, 1H), 4.44 (d, J¼15.0 Hz, 1H), 4.33 (d, J¼15.0 Hz, 1H), 3.99 (ddd,
J¼8.7, 6.6, 2.1 Hz, 1H), 3.19 (dd, J¼14.7, 5.1 Hz, 1H), 2.71 (dd, J¼14.7,
2.1 Hz, 1H), 2.46 (dd, J¼15.6, 6.6 Hz, 1H), 2.37 (dd, J¼15.3, 6.9 Hz,
CDCl3)
21.8; IR (KBr)
d 165.3, 133.7, 132.5, 131.3, 129.9, 127.7, 115.6, 47.3, 42.9, 42.8,
1H); 13C NMR (75 MHz, CDCl3)
127.8, 48.4, 45.1, 42.8, 39.3; IR (KBr)
d
172.2, 167.0, 136.1, 128.8, 128.2,
3365, 3188, 1739, 1658,
n
2251, 1751 cmꢃ1; MS (ESI) m/z (%) 257 [MþNa]þ.
HRMS (FT-MS-ESI) Anal. Calcd for C12H11N2OCl: 257.0452 [MþNa]þ.
n
1622 cmꢃ1; MS (ESI) m/z (%) 219 [MþH]þ, 241 [MþNa]þ; Anal. Calcd
for C12H14N2O2: C, 66.04; H, 6.47; N, 12.84. Found: C, 66.18; H, 6.34;
N, 12.85.
Found: 257.0449 [MþNa]þ.
4.6. ( )-5f as colorless oil (4.02 g, 18.4 mmol)
4.10.2. (ꢁ)-6b as white solid (1.75 g, 59%). Mp 109.0e110.0 ꢀC; 1H
1H NMR (300 MHz, CDCl3)
d 7.37e7.30 (m, 2H), 7.20e7.07
NMR (300 MHz, CDCl3)
d
7.56 (d, J¼8.1 Hz, 1H), 7.28e7.35 (m, 2H),
(m, 2H), 4.61 (d, J¼15.2 Hz, 1H), 4.32 (d, J¼15.2 Hz, 1H), 3.81e3.75
7.15e7.20 (m, 1H), 5.77 (br s, 1H), 5.68 (br s, 1H), 4.61 (d, J¼15.6 Hz,
1H), 4.40 (d, J¼15.6 Hz, 1H), 3.96 (ddd, J¼7.8, 5.4, 2.4 Hz, 1H), 3.21
(dd, J¼14.7, 5.1 Hz, 1H), 2.76 (dd, J¼14.7, 2.1 Hz, 1H), 2.63 (dd,
J¼15.0, 5.4 Hz, 1H), 2.36 (dd, J¼15.0, 8.1 Hz, 1H); 13C NMR (75 MHz,
(m, 1H), 3.19 (dd, J¼14.9, 5.1 Hz, 1H), 2.86 (dd, J¼14.9, 2.2 Hz, 1H),
2.68e2.58 (m, 2H); 13C NMR (75 MHz, CDCl3)
d 165.2, 162.5, 159.3,
131.2, 131.2, 130.4, 130.3, 125.0, 124.9, 122.1, 121.9, 115.9, 115.6, 47.1,
42.9, 38.8, 38.7, 21.7; IR (KBr)
n
2251, 1755 cmꢃ1; MS (ESI) m/z (%)
CDCl3)
45.2, 43.2, 39.1; IR (KBr)
d 171.6, 167.0, 135.1, 133.1, 130.4, 129.6, 128.0, 123.5, 48.8,
219 [MþH]þ. HRMS (FT-MS-ESI) Anal. Calcd for C12H11N2OF:
n
3381, 3202, 1752, 1647, 1614 cmꢃ1; MS
219.0928 [MþH]þ. Found: 219.0928 [MþH]þ.
(ESI) m/z (%) 319 [MþNa]þ (79Br), 321 [MþNa]þ (81Br). Anal. Calcd
for C12H13BrN2O2: C, 48.50; H, 4.41; N, 9.43. Found: C, 48.39; H,
4.42; N, 9.11.
4.7. ( )-5g as white solid (3.42 g, 16.0 mmol)
Mp 85.0e86.0 ꢀC; 1H NMR (300 MHz, CDCl3)
d 7.26e7.20
4.10.3. (ꢁ)-6c as white solid (1.57 g, 53%). Mp 109.0e110.0 ꢀC; 1H
(m, 4H), 4.46 (dd, J¼44.9, 14.9 Hz, 2H), 3.71e3.65 (m, 1H), 3.20
NMR (300 MHz, CDCl3) d 7.40e7.43 (m, 2H), 7.18e7.26 (m, 2H), 5.38
(dd, J¼14.9, 5.1 Hz, 1H), 2.87e2.82 (m, 1H), 2.53e2.35 (m, 2H), 2.37
(br s, 1H), 5.33 (br s, 1H), 4.41 (d, J¼15.3 Hz, 1H), 4.31 (d, J¼15.3 Hz,
1H), 4.02 (ddd, J¼9.0, 6.9, 2.4 Hz, 1H), 3.20 (dd, J¼15.0, 5.1 Hz, 1H),
2.71 (dd, J¼14.7, 2.4 Hz, 1H), 2.45 (d, J¼6.6 Hz, 2H); 13C NMR
(s, 3H); 13C NMR (75 MHz, CDCl3)
d 165.1, 137.0, 132.4, 131.2, 129.6,
128.7, 126.5, 115.6, 47.0, 43.7, 42.7, 21.8, 19.2; IR (KBr)
n 2250,
1750 cmꢃ1; MS (ESI) m/z (%) 237 [MþNa]þ. HRMS (FT-MS-ESI) Anal.
(75 MHz, CDCl3)
48.5, 44.6, 42.9, 39.5; IR (KBr)
d 171.8, 167.0, 138.8, 131.2, 130.8, 130.4, 126.9, 122.7,
Calcd for
C
13H14N2O: 237.0998 [MþNa]þ. Found: 237.0996
n
3361, 3186, 1741, 1657 cmꢃ1; MS
[MþNa]þ.
(ESI) m/z (%) 319 [MþNa]þ (79Br), 321 [MþNa]þ (81Br). Anal. Calcd
for C12H13BrN2O2: C, 48.50; H, 4.41; N, 9.43. Found: C, 48.35; H,
4.48; N, 9.10.
4.8. ( )-5h as colorless oil (5.76 g, 25.0 mmol)
1H NMR (300 MHz, CDCl3)
4.10.4. (ꢁ)-6d as white solid (1.87 g, 63%). Mp 129.0e130.0 ꢀC; 1H
d
7.35e7.26 (m, 2H), 6.99e6.90
(m, 2H), 4.53 (d, J¼14.6 Hz, 1H), 4.29 (d, J¼14.6 Hz, 1H), 3.87 (s, 3H),
NMR (300 MHz, CDCl3)
d
7.45 (d, J¼8.1 Hz, 2H), 7.17 (d, J¼8.1 Hz,
3.73e3.67 (m, 1H), 3.14 (dd, J¼14.8, 5.0 Hz, 1H), 2.84e2.78 (m, 1H),
2H), 5.70 (br s, 1H), 5.61 (br s, 1H), 4.40 (d, J¼15.3 Hz, 1H), 4.25
(d, J¼15.3 Hz, 1H), 3.98 (d, J¼5.7 Hz, 1H), 3.16 (dd, J¼14.7, 5.1 Hz,
1H), 2.69 (d, J¼14.4 Hz, 1H), 2.44 (d, J¼6.6 Hz, 2H); 13C NMR
2.71e2.51 (m, 2H); 13C NMR (75 MHz, CDCl3)
d 165.2, 157.4, 130.9,
129.9, 123.0, 121.1, 115.9, 110.6, 55.4, 47.1, 42.7, 40.2, 21.6; IR (KBr)
n
2250,1750 cmꢃ1; MS (ESI) m/z (%) 253 [MþNa]þ. HRMS (FT-MS-ESI)
Anal. Calcd for C13H14N2O2: 253.0948 [MþNa]þ. Found: 253.0946
[MþNa]þ.
(75 MHz, CDCl3)
42.9, 39.6; IR (KBr)
d 171.6, 166.9, 135.5, 131.9, 130.1, 121.7, 48.3, 44.7,
n
3384, 3204, 1738, 1667, 1622 cmꢃ1; MS (ESI)
m/z (%) 319 [MþNa]þ (79Br), 321 [MþNa]þ (81Br). Anal. Calcd for
C12H13BrN2O2: C, 48.50; H, 4.41; N, 9.43. Found: C, 48.28; H, 4.37; N,
4.9. ( )-5i as colorless oil (1.81 g, 12.1 mmol)
9.24.
1H NMR (300 MHz, CDCl3)
d
5.74e5.87 (m, 1H), 5.26e5.34
4.10.5. (ꢁ)-6e as white solid (1.46 g, 58%). Mp 99.0e100.0 ꢀC; 1H
(m, 2H), 4.49 (dd, J¼15.6, 6.0 Hz, 1H), 3.89 (ddd, J¼7.5, 5.4, 2.4 Hz,
1H), 3.79 (dd, J¼15.6, 6.6 Hz, 1H), 3.21 (dd, J¼15.0, 5.1 Hz, 1H), 2.83
(dd, J¼15.0, 2.1 Hz, 1H), 2.74 (d, J¼5.4 Hz, 1H); 13C NMR (75 MHz,
NMR (300 MHz, CDCl3) d 7.39e7.35 (m, 2H), 7.29e7.24 (m, 2H),
5.39 (br s, 2H), 4.61 (d, J¼15.5 Hz, 1H), 4.42 (d, J¼15.5 Hz, 1H),
4.00e3.92 (m, 1H), 3.22 (dd, J¼14.9, 5.1 Hz, 1H), 2.75 (dd, J¼14.9,
2.2 Hz, 1H), 2.61 (dd, J¼15.2, 5.4 Hz, 1H), 2.35 (dd, J¼15.2, 8.0 Hz,
CDCl3)
d 165.2, 131.4, 119.6, 115.9, 47.1, 43.9, 42.7, 22.0; IR (KBr)
n
2250, 1750 cmꢃ1; MS (EI) m/z (%) 151 [MþH]þ, 173 [MþNa]þ.
1H); 13C NMR (75 MHz, CDCl3)
d 171.6, 167.0, 133.4, 133.4, 130.4,
HRMS (TOF-MS-EI) Anal. Calcd for C8H10N2O: 150.0793. Found:
150.0791.
129.8, 129.3, 127.3, 48.7, 43.2, 42.6, 39.1; IR (KBr) n 3341, 3195,
1736, 1668 cmꢃ1; MS (ESI) m/z (%) 275 [MþNa]þ. Anal. Calcd for
Please cite this article in press as: Ao, Y.-F.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.05.018

6
Y.-F. Ao et al. / Tetrahedron xxx (2014) 1e8
C12H13ClN2O2: C, 57.04; H, 5.19; N, 11.09. Found: C, 56.92; H, 5.18;
N, 11.19.
solution in ether (5 mL) at ꢃ15 ꢀC. The mixture was kept stirring
at room temperature for 1 h, and then mixed with water (10 mL)
and extracted with ethyl acetate (3ꢂ5 mL). The organic phase was
combined and dried over anhydrous Na2SO4. Ester products 8aed
were obtained after removal of solvent. In the case of the bio-
transformations of 6eei, the residue was column chromato-
graphed on a silica gel column eluted first with ethyl acetate to
give ester products 8eei then with ethyl acetate and acetone
(v:v¼1:1) to give additional amount of amides 6eei. Enantioen-
riched amide products have the identical spectroscopic data as
that of racemic samples.
4.10.6. (ꢁ)-6f as white solid (1.58 g, 67%). Mp 84.0e85.0 ꢀC; 1H
NMR (300 MHz, CDCl3) d 7.36e7.25 (m, 2H), 7.16e7.03 (m, 2H), 5.63
(br s, 2H), 4.54 (d, J¼15.3 Hz, 1H), 4.32 (d, J¼15.3 Hz, 1H), 3.98e3.91
(m, 1H), 3.17 (dd, J¼14.9, 5.1 Hz, 1H), 2.76e2.70 (m, 1H), 2.66e2.59
(m, 1H), 2.38 (dd, J¼15.2, 7.7 Hz, 1H); 13C NMR (75 MHz, CDCl3)
d
171.5, 166.9, 162.4, 159.1, 130.8, 130.7, 129.9, 129.7, 124.6, 124.6,
123.1, 122.9, 115.7, 115.5, 48.5, 43.1, 39.0, 38.6, 38.5; IR (KBr) n 3351,
3196, 1736, 1671 cmꢃ1; MS (ESI) m/z (%) 259 [MþNa]þ. Anal. Calcd
for C12H13FN2O2: C, 61.01; H, 5.55; N, 11.86. Found: C, 61.22; H, 5.70;
N, 11.98.
4.11.1. (S)-(ꢃ)-6a as white solid (89 mg, 41%). Mp 116.0e117.0 ꢀC;
25
[
a
]
ꢃ37.5ꢀ (c 0.5, CH3OH); ee 95.0% (HPLC analysis using a Chir-
D
4.10.7. (ꢁ)-6g as white solid (1.62 g, 70%). Mp 131.0e132.0 ꢀC; 1H
alcel ADH column). Single crystal was obtained from re-
crystallization from a mixture of petroleum ether and ethyl
acetate.
NMR (300 MHz, CDCl3) d 7.21e7.17 (m, 4H), 5.42 (br s, 1H), 5.33 (br
s, 1H), 4.40 (q, J¼15.2 Hz, 2H), 3.93e3.86 (m, 1H), 3.18 (dd, J¼14.8,
5.1 Hz, 1H), 2.76e2.70 (m, 1H), 2.47e2.25 (m, 2H), 2.34 (s, 3H); 13
NMR (75 MHz, CDCl3) 171.4, 166.7, 136.6, 133.7, 130.7, 129.2, 128.1,
C
d
4.11.2. (S)-(ꢃ)-6b as white solid (131 mg, 44%). Mp 109.0e110.0 ꢀC;
126.2, 48.6, 43.3, 43.0, 39.1, 19.1; IR (KBr)
n
3390, 3199, 1732,
[
a
]
25 ꢃ12.1ꢀ (c 0.7, CH3OH); ee>99.5% (HPLC analysis using a Chir-
D
1671 cmꢃ1; MS (ESI) m/z (%) 255 [MþNa]þ. Anal. Calcd for
alcel ADH column).
C
13H16N2O2: C, 67.22; H, 6.94; N, 12.06. Found: C, 67.20; H, 6.99; N,
12.05.
4.11.3. (S)-(ꢃ)-6c as white solid (128 mg, 43%). Mp 109.0e110.0 ꢀC;
25
[a
]
ꢃ6.7ꢀ (c 5.4, CH3OH); ee>99.5% (HPLC analysis using a Chir-
D
4.10.8. (ꢁ)-6h as white solid (1.83 g, 74%). Mp 121.0e122.0 ꢀC; 1H
alcel ODH column).
NMR (300 MHz, CDCl3)
d 7.34e7.24 (m, 2H), 7.01e6.92 (m, 2H),
4.84 (s, 2H), 4.53 (d, J¼15.0 Hz, 1H), 4.26 (d, J¼15.0 Hz, 1H),
3.89e3.83 (m, 1H), 3.87 (s, 3H), 3.07 (dd, J¼14.7, 4.9 Hz, 1H),
2.75e2.64 (m, 2H), 2.39 (dd, J¼14.6, 8.1 Hz, 1H); 13C NMR
4.11.4. (S)-(ꢃ)-6d as white solid (139 mg, 47%). Mp 129.0e130.0 ꢀC;
25
[a
]
ꢃ9.3ꢀ (c 0.7, CH3OH); ee>99.5% (HPLC analysis using a Chir-
D
alcel ADH column).
(75 MHz, CDCl3)
d 175.0, 169.4, 158.9, 131.1, 130.5, 125.0, 121.7,
111.8, 55.9, 50.3, 43.0, 41.1, 39.6; IR (KBr)
n
3350, 3195, 1732,
4.11.5. (S)-(þ)-6e as white solid (111 mg, 44%). Mp 100.0e101.0 ꢀC;
25
1669 cmꢃ1; MS (ESI) m/z (%) 271 [MþNa]þ. Anal. Calcd for
[a
]
þ4.0ꢀ (c 1.5, CH3OH); ee>99.5% (HPLC analysis using a Chir-
D
C
13H16N2O3: C, 62.89; H, 6.50; N, 11.28. Found: C, 63.00; H, 6.68;
alcel ADH column).
N, 11.44.
4.11.6. (S)-(þ)-6f as white solid (108 mg, 46%). Mp 86.0e87.0 ꢀC;
25
4.10.9. (ꢁ)-6i as white solid (0.77 g, 46%). Mp 67.0e68.0 ꢀC; 1H
[a
]
þ1.3ꢀ (c 1.5, CH3OH); ee¼94.5% (HPLC analysis using a Chir-
D
NMR (300 MHz, CDCl3)
d
5.70e5.85 (m, 1H), 5.70 (s, 1H), 5.18e5.26
alcel ADH column).
(m, 2H), 4.55 (s, 1H), 4.05 (ddd, J¼8.7, 6.9, 2.1 Hz, 1H), 3.94 (dd,
J¼15.6, 5.7 Hz, 1H), 3.71 (dd, J¼11.1, 6.6 Hz, 1H), 3.18 (dd, J¼14.7,
5.1 Hz, 1H), 2.69 (dd, J¼15.0, 2.7 Hz, 1H), 2.67 (dd, J¼15.3, 6.6 Hz,
4.11.7. (S)-(þ)-6g as white solid (102 mg, 44%). Mp 131.0e132.0 ꢀC;
25
[a
]
þ9.6ꢀ (c 1.7, CH3OH); ee>99.5% (HPLC analysis using a Chir-
D
1H), 2.47 (dd, J¼15.3, 7.2 Hz, 1H); 13C NMR (75 MHz, CDCl3)
d
172.2,
alcel ADH column).
167.0, 132.0, 118.4, 48.4, 43.7, 42.7, 39.3; IR (KBr) n 3324, 3198, 1734,
1671 cmꢃ1; MS (ESI) m/z (%) 169 [MþH]þ, 191 [MþNa]þ. HRMS (FT-
ICRMS) Anal. Calcd for C8H12N2O2: 169.0972 [MþH]þ. Found:
169.0968 [MþH]þ.
4.11.8. (S)-(ꢃ)-6h as white solid (114 mg, 46%). Mp 122.0e123.0 ꢀC;
25
[a
]
ꢃ15.3ꢀ (c 1.7, CH3OH); ee>99.5% (HPLC analysis using a Chir-
D
alcel ADH column).
4.11. General procedure for biotransformations of amides 6
and nitriles 5
4.11.9. (S)-(þ)-6i as white solid (71 mg, 42%). Mp 67.0e68.0 ꢀC;
[a
]
25 þ2.0ꢀ (c 3.0, CH3OH); ee 85.8% (HPLC analysis using a Chiralcel
D
ADH column).
In an Erlenmeyer flask (150 mL) with a screw cap a suspension
of Rh. erythropolis AJ270 cells16 (2 g wet weight) in aqueous
phosphate buffer (pH 7.0, 0.1 M, 50 mL) was activated at 30 ꢀC for
0.5 h. A solution of amides 6 or nitriles 5 (1 mmol) in acetone
(0.5 mL) was then added in one portion, and the resulting mix-
ture was incubated at 30 ꢀC with orbital shaking (200 rpm). The
reaction process was monitored using TLC method. After a period
of time (see Tables 1 and 2), the reaction was quenched by re-
moving microbial cells through a Celite pad filtration. The filtra-
tion cake was washed with water (3ꢂ15 mL) and ethyl acetate
(3ꢂ15 mL). The aqueous layer was extracted with ethyl acetate.
The combined organic phase was dried over anhydrous Na2SO4.
After removal of solvent, the residue was chromatographed on
a silica gel column using a mixture of petroleum ether and ace-
tone (v:v¼1:1) to afford pure nitriles 5 and amides 6. The aqueous
solution that contains acid product was freeze-dried, and residue
was dissolved in methanol (5 mL) and then treated with CH2N2
4.11.10. (R)-(þ)-8a as colorless oil (107 mg, 46%). [
a
]
25 þ23.3ꢀ (c 0.6,
D
benzene), ee>99.5% (HPLC analysis using a Chiralcel OJH column);
1H NMR (300 MHz, CDCl3)
d
7.25e7.37 (m, 5H), 4.50 (d, J¼15.3 Hz,
1H), 4.25 (d, J¼15.3 Hz, 1H), 3.89 (ddd, J¼8.7, 6.9, 2.4 Hz, 1H), 3.60
(s, 3H), 3.17 (dd, J¼14.7, 5.1 Hz, 1H), 2.72 (dd, J¼14.7, 2.1 Hz, 1H),
2.59 (dd, J¼16.2, 6.3 Hz, 1H), 2.48 (dd, J¼16.5, 7.2 Hz, 1H); 13C
NMR(75 MHz, CDCl3)
d 170.8, 166.6, 136.1, 128.8, 128.2, 127.7, 51.9,
47.7, 45.1, 43.0, 38.1; IR (KBr)
n
1746 cmꢃ1; MS (ESI) m/z (%) 234
[MþH]þ, 256 [MþNa]þ. HRMS (TOF-MSEI) Anal. Calcd for
C13H15NO3. 233.1052. Found: 233.1056.
4.11.11. (R)-(þ)-8b as colorless oil (140 mg, 45%). [
a]
25 þ18.0ꢀ (c 1.0,
D
diethyl ether); ee>99.5% HPLC analysis using a Chiralcel OJH col-
umn); 1H NMR (300 MHz, CDCl3)
d
7.55 (d, J¼8.1 Hz, 1H),
7.29e7.35 (m, 2H), 7.14e7.20 (m, 1H), 4.61 (d, J¼15.6 Hz, 1H), 4.38
(d, J¼15.6 Hz, 1H), 3.90 (ddd, J¼7.5, 5.1, 2.1 Hz, 1H), 3.61 (s, 3H),
Please cite this article in press as: Ao, Y.-F.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.05.018

Y.-F. Ao et al. / Tetrahedron xxx (2014) 1e8
7
3.20 (dd, J¼14.7, 5.1 Hz, 1H), 2.75 (dd, J¼14.7, 2.1 Hz, 1H), 2.71 (dd,
J¼15.9, 5.4 Hz, 1H), 2.49 (dd, J¼16.2, 8.1 Hz, 1H); 13C NMR(75 MHz,
1739 cmꢃ1; MS (ESI) m/z (%) 270 [MþNa]þ; HRMS (ESI) Anal.
Calcd for
C
14H17NO3: 270.1101 [MþNa]þ, Found: 270.1098
CDCl3)
d
170.5, 166.7, 135.0, 133.0, 130.3, 129.5, 127.9, 123.4, 51.9,
[MþNa]þ.
48.2, 45.1, 43.2, 37.8; IR (KBr)
n
1752 cmꢃ1; MS (ESI) m/z (%) 334
25
[MþNa]þ (79Br), 336 [MþNa]þ (81Br). Anal. Calcd for
4.11.17. (R)-(þ)-8h colorless oil (118 mg, 45%). [
a
]
þ3.0ꢀ (c 1.4,
D
C
13H14BrNO3: C, 50.02; H, 4.52; N, 4.49. Found: C, 51.01; H, 4.62; N,
CHCl3); ee>99.5% (HPLC analysis using a Chiralcel ODH column); 1H
4.33.
NMR (300 MHz, CDCl3) d 7.31e7.22 (m, 2H), 6.95e6.86 (m, 2H), 4.52
(d, J¼15.0 Hz, 1H), 4.23 (d, J¼15.0 Hz, 1H), 3.84e3.79 (m, 4H), 3.63
4.11.12. (R)-(þ)-8c as colorless oil (153 mg, 49%). [
a
]
25 þ3.8ꢀ (c 1.6,
(s, 3H), 3.11 (dd, J¼14.7, 5.0 Hz, 1H), 2.80e2.66 (m, 2H), 2.43 (dd,
D
diethyl ether); ee 87.6% (HPLC analysis using a Chiralcel ADH col-
J¼15.8, 8.6 Hz, 1H); 13C NMR (75 MHz, CDCl3)
d 170.8, 166.5, 157.4,
130.2, 129.3, 123.9, 120.8, 110.4, 55.3, 51.8, 48.0, 43.1, 39.7, 37.7; IR
umn); 1H NMR (300 MHz, CDCl3)
d
7.40e7.43 (m, 2H), 7.21e7.23
(m, 2H), 4.44 (d, J¼15.3 Hz, 1H), 4.25 (d, J¼15.3 Hz, 1H), 3.94 (ddd,
J¼9.0, 6.9, 2.4 Hz, 1H), 3.62 (s, 3H), 3.19 (dd, J¼14.7, 5.1 Hz, 1H), 2.73
(dd, J¼14.7, 2.4 Hz, 1H), 2.57 (d, J¼1.5 Hz, 1H), 2.55 (d, J¼0.9 Hz, 1H);
(KBr)
n
2952, 1743 cmꢃ1; MS (ESI) m/z (%) 286 [MþNa]þ. HRMS (FT-
MS-ESI) Anal. Calcd for C14H17NO4: 286.1050 [MþNa]þ. Found:
286.1047 [MþNa]þ.
13C NMR (75 MHz, CDCl3)
d 170.8, 166.7, 138.6, 131.1, 130.9, 130.4,
25
126.8, 122.7, 52.0, 48.0, 44.5, 43.0, 38.2; IR (KBr)
n
1744 cmꢃ1; MS
4.11.18. (R)-(ꢃ)-8i as colorless oil (90 mg, 49%). [
a
]
ꢃ10.3ꢀ (c 1.5,
D
(ESI) m/z (%) 334 [MþNa]þ (79Br), 336 [MþNa]þ(81Br). HRMS (TOF-
MS-EI) Anal. Calcd for C13H14BrNO3: 311.0157 (79Br), 313.0137
(81Br). Found: 311.0160 (79Br), 313.0141 (81Br).
diethyl ether); ee¼89.1% (HPLC analysis using a Chiralcel OJH
column); 1H NMR (300 MHz, CDCl3)
d 5.70e5.83 (m, 1H),
5.18e5.24 (m, 2H), 3.97 (ddd, J¼9.3, 6.9, 2.1 Hz, 1H), 3.91 (d,
J¼5.7 Hz, 1H), 3.66e3.80 (m, 1H), 3.70 (s, 3H), 3.17 (dd, J¼15.0,
5.1 Hz, 1H), 2.76 (dd, J¼16.2, 6.3 Hz, 1H), 2.70 (dd, J¼16.5, 2.1 Hz,
1H), 2.57 (dd, J¼16.2, 6.9 Hz, 1H); 13C NMR (75 MHz, CDCl3)
4.11.13. (R)-(þ)-8d as white solid (143 mg, 46%). Mp 37.0e38.0 ꢀC;
25
[
a]
þ7.6ꢀ (c 1.85, diethyl ether); ee>99.5% (HPLC analysis using
D
a Chiralcel ADH column); 1H NMR (300 MHz, CDCl3)
d
7.47
d 170.8, 166.5, 132.1, 118.4, 51.9, 47.9, 43.7, 42.9, 38.2; IR (KBr) n
(d, J¼8.4 Hz, 2H), 7.16 (d, J¼8.4 Hz, 2H), 4.44 (d, J¼15.6 Hz, 1H), 4.22
(d, J¼15.6 Hz, 1H), 3.90 (ddd, J¼8.7, 6.6, 2.1 Hz, 1H), 3.62 (s, 3H), 3.17
(dd, J¼15.0, 5.1 Hz, 1H), 2.72 (dd, J¼14.7, 2.1 Hz, 1H), 2.58 (dd,
J¼16.5, 6.6 Hz, 1H), 2.52 (dd, J¼13.2, 3.3 Hz, 1H); 13C NMR(75 MHz,
1739 cmꢃ1; MS (ESI) m/z (%) 184 [MþH]þ, 206 [MþNa]þ. HRMS
(FT-ICRMS) Anal. Calcd for C9H13NO3: 184.0968 [MþH]þ. Found:
184.0967 [MþH]þ.
CDCl3)
38.2; IR (KBr)
d
170.8, 166.7, 135.3, 131.9, 129.9, 121.7, 51.9, 47.8, 44.6, 43.0,
4.11.18.1. Procedure for multigram biotransformation of racemic
6h. Following the same aformentioned standard procedure for the
biotransformation of nitriles and amides, reaction of racemic 6h
(10 mmol, 2.48 g) gave (S)-6h (1.12 g, 45%, ee>99.5%) and (R)-8h
(1.21 g, 46%, ee>99.5%).
n
1729 cmꢃ1; MS (ESI) m/z (%) 334 [MþNa]þ(79Br),
336 [MþNa]þ(81Br). HRMS (TOF-MS-EI) Anal. Calcd for
C
13H14BrNO3: 311.0157 (79Br); 313.0137 (81Br). Found: 311.0160
(79Br); 313.0140 (81Br). X-ray quality single crystal was obtained
from recrystallization from a mixture of petroleum ether and ethyl
acetate.
4.11.18.2. Procedure for the synthesis of S-acid (S)-7h from bio-
transformation of S-amide (S)-6h. Following the general procedure
for the biotransformations of racemic nitriles and amides, (S)-6h
4.11.14. (R)-(ꢃ)-8e as colorless oil (120 mg, 45%). [
a
]
25 ꢃ13.7ꢀ (c 2.0,
D
CHCl3); ee>99.5% (HPLC analysis using a Chiralcel OJH column); 1H
(0.5 mmol, 128 mg) was converted completely within 60 h into acid
25
NMR (300 MHz, CDCl3)
d
7.39e7.33 (m, 2H), 7.29e7.24 (m, 2H), 4.62
(S)-7h as colorless oil (120 mg, 97%): [
a
]
ꢃ10.0ꢀ (c 0.7, CHCl3);
D
(d, J¼15.6 Hz, 1H), 4.39 (d, J¼15.6 Hz, 1H), 3.93e3.86 (m, 1H), 3.62
ee>99.5% (HPLC analysis of ester using a Chiralcel ADH column); 1H
(s, 3H), 3.20 (dd, J¼14.9, 5.1 Hz, 1H), 2.78e2.67 (m, 2H), 2.48 (dd,
NMR (300 MHz, CDCl3) d 7.30e7.23 (m, 2H), 6.98e6.89 (m, 2H), 4.51
J¼16.0, 7.9 Hz, 1H); 13C NMR (75 MHz, CDCl3)
d
170.5, 166.7, 133.4,
(d, J¼15.0 Hz, 1H), 4.23 (d, J¼15.0 Hz, 1H), 3.85e3.80 (m, 4H), 3.63
133.4, 130.3, 129.8, 129.3, 127.3, 51.9, 48.2, 43.2, 42.6, 37.8; IR (KBr)
(s, 3H), 3.04 (dd, J¼14.7, 5.0 Hz, 1H), 2.71e2.64 (m, 2H), 2.24 (dd,
n
2953, 1755 cmꢃ1; MS (ESI) m/z (%) 290 [MþNa]þ. HRMS (FT-MS-
J¼14.8, 9.2 Hz, 1H); 13C NMR (75 MHz, CDCl3)
d 178.4, 170.1, 159.3,
131.4, 130.7, 125.5, 122.0, 112.0, 56.2, 51.4, 43.7, 42.3, 41.1; IR (KBr) n
ESI) Anal. Calcd for C13H14NO3Cl: 290.0554 [MþNa]þ. Found:
290.0551 [MþNa]þ.
3393, 2926, 1733, 1581 cmꢃ1; MS (ESI) m/z (%) 248 [MꢃH]-. HRMS
(FT-MS-ESI) Anal. Calcd for C13H15NO4: 288.0633 [MþK]þ. Found:
288.0634 [MþK]þ.
ꢃ7.6ꢀ (c 2.0,
a] D
4.11.15. (R)-(ꢃ)-8f as colorless oil (110 mg, 44%). [
25
3); ee¼94.2% HPLC analysis using a Chiralcel ADH column); 1H
CHCl
NMR (300 MHz, CDCl3)
d
7.32e7.25 (m, 2H), 7.16e7.03 (m, 2H),
4.11.18.3. Synthesis of 9. After biotransformation of (S)-6i
(0.4 mmol, 67 mg), the acid product (S)-7i was mixed directly with
iodine (1.5 mmol, 190 mg) in acetonitrile (1.5 mL). After reaction at
room temperature for 18 h, a saturated aqueous solution of
Na2S2O3 was added, and the mixture was extracted with ethyl
acetate (3ꢂ5 mL). Organic phase was dried over anhydrous
Na2SO4. After removal of solvent under vacuum, the residue was
chromatographed on a silica gel column eluted with a mixture of
petroleum ether and ethyl acetate (v:v¼1:2) to give products 9a
and 9b.
4.55 (d, J¼15.4 Hz, 1H), 4.30 (d, J¼15.4 Hz, 1H), 3.92e3.86 (m, 1H),
3.64 (s, 3H), 3.16 (dd, J¼14.8, 5.1 Hz, 1H), 2.75e2.68 (m, 2H), 2.50
(dd, J¼16.1, 7.5 Hz, 1H); 13C NMR (75 MHz, CDCl3)
d 170.6, 166.6,
162.4, 159.1, 130.7, 130.6, 129.8, 129.7, 124.6, 124.5, 123.1, 122.9,
115.7, 115.4, 51.9, 48.1, 43.2, 38.4, 38.4, 37.8; IR (KBr)
n 2954,
1752 cmꢃ1; MS (ESI) m/z (%) 274 [MþNa]þ. HRMS (FT-MS-ESI)
Anal. Calcd for C13H14NO3F: 274.0850 [MþNa]þ. Found: 274.0847
[MþNa]þ.
25
4.11.16. (R)-2-(ꢃ)-8g as colorless oil (111 mg, 45%). [
a
]
ꢃ18.5ꢀ
9a as white solid (61 mg, 52%): mp 171.0e172.0 ꢀC; [
a]
25 ꢃ15.0ꢀ
D
D
(c 2.0, CHCl3); ee>99.5% (HPLC analysis using a Chiralcel ODH
(c 0.5, CHCl3); 1H NMR (300 MHz, CDCl3)
d
4.54e4.47 (m, 1H), 4.31
column); 1H NMR (300 MHz, CDCl3)
d
7.20e7.16 (m, 4H), 4.52
(d, J¼15.0 Hz, 1H), 3.91e3.85 (m, 1H), 3.37e3.21 (m, 3H), 3.15e3.05
(m, 2H), 2.92e2.72 (m, 2H); 13C NMR (75 MHz, CDCl3)
168.7, 164.7,
1740 cmꢃ1; MS (ESI) m/z (%)
(d, J¼15.2 Hz, 1H), 4.39 (d, J¼15.2 Hz, 1H), 3.84e3.77 (m, 1H), 3.59
(s, 3H), 3.17 (dd, J¼14.8, 5.1 Hz, 1H), 2.72 (dd, J¼14.8, 2.1 Hz, 1H),
2.56 (dd, J¼16.0, 5.4 Hz, 1H), 2.45e2.39 (m, 1H), 2.33 (s, 3H); 13C
d
78.9, 46.7, 45.3, 44.6, 41.2, 1.0; IR (KBr)
n
350 [MþNaþCH3OH]þ. HRMS (FT-MS-ESI) Anal. Calcd for
C8H10INO3: 349.9860 [MþNaþCH3OH]þ. Found: 349.9857
[MþNaþCH3OH]þ.
NMR (75 MHz, CDCl3)
d 170.5, 166.4, 136.5, 133.4, 130.7, 129.1,
128.1, 126.2, 51.8, 47.9, 43.2, 43.0, 37.8, 19.1; IR (KBr)
n 2953,
Please cite this article in press as: Ao, Y.-F.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.05.018

8
Y.-F. Ao et al. / Tetrahedron xxx (2014) 1e8
25
9b as colorless oil (44 mg, 37%): [
NMR (300 MHz, CDCl3)
a
]
þ16.7ꢀ (c 0.5, CHCl3); 1H
References and notes
D
d
4.73e4.64 (m, 1H), 4.01e3.98 (m, 1H),
3.89e3.81 (m, 1H), 3.44e3.25 (m, 5H), 2.99e2.87 (m, 2H); 13C
1. For an overview, see: Wang, M.-X. Top. Catal. 2005, 35, 117e130.
2. For reviews, see: (a) Ramteke, P. W.; Maurice, N. G.; Joseph, B.; Wadher, B. J.
Biotechnol. Appl. Biochem. 2013, 60, 459e481; (b) Prasad, S.; Bhalla, T. C. Bio-
technol. Adv. 2010, 28, 725e741; (c) Velankar, H.; Clarke, K. G.; du Preez, R.;
Cowan, D. A.; Burton, S. G. Trends Biotechnol. 2010, 28, 561e569; (d) D’Antona,
N.; Morrone, R. In Biocatalysis: Green Transformations of Nitrile Function in Green
Chemistry for Environmental Sustainability; Sharma, S. K., Mudhoo, A., Eds.; CRC:
2010.
NMR (75 MHz, CDCl3)
d 167.8, 165.8, 75.3, 45.2, 45.1, 43.6, 37.0,
0.1; IR (KBr)
n
1739 cmꢃ1; MS (ESI) m/z (%) 350 [MþNaþCH3OH]þ.
HRMS (FT-MS-ESI) Anal. Calcd for C8H10INO3: 349.9860
[MþNaþCH3OH]þ. Found: 349.9858 [MþNaþCH3OH]þ.
ꢀ
3. (a) Busto, E.; Gotor-Fernandez, V.; Gotor, V. Chem. Rev. 2011, 111, 3998e4035; (b)
4.11.18.4. Synthesis of 10. a solution of (R)-8g (0.5 mmol,
124 mg) in methanol (6 mL) was added dropwise a solution of
LiOH (1 mmol, 24 mg) in water (1.5 mL). After stirring at room
temperature overnight, dilute hydrochloric acid (1 M, 1 mL) was
added slowly. Solvent was removed under vacuum at 50 ꢀC, and
the residue was washed thoroughly with methanol (3ꢂ15 mL).
Methanol solution was collected and concentrated to give acid
product. Without further purification, the acid was mixed with
SOCl2 (0.1 mL), DMF (one drop) and dry DCM (6 mL), and the
resulting mixture was refluxed for 2 h. After removal of solvent
under vacuum, dry DCM (8 mL) and AlCl3 (2 mmol, 267 mg) were
added. The reaction mixture was stirred at room temperature for
2 h. Brine (10 mL) was added and the mixture was extracted with
DCM (3ꢂ10 mL). The combined organic layer was dried over an-
hydrous Na2SO4. Solvent was then removed and the residue was
chromatographed on a silica gel column using ethyl acetate as
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mobile phase to give pure product (R)-(ꢃ)-10 as white solid
25
(85 mg, 79%): mp 114.0e115.0 ꢀC; [
a
]
ꢃ47.5ꢀ (c 0.8, CHCl3);
D
ee¼97.1% (HPLC analysis using a Chiralcel ADH column); 1H NMR
(300 MHz, CDCl3)
d 7.42e7.39 (m, 1H), 7.29e7.21 (m, 2H), 5.00 (d,
J¼17.6 Hz, 1H), 4.26 (d, J¼17.6 Hz, 1H), 4.08e4.07 (m, 1H), 3.45 (dd,
J¼13.0, 5.9 Hz, 1H), 3.22 (dd, J¼14.9, 4.7 Hz, 1H), 3.03e2.90 (m,
2H), 2.34 (s, 3H); 13C NMR (75 MHz, CDCl3)
d 201.9, 165.8, 139.7,
135.6, 135.2, 133.4, 127.4, 127.3, 47.5, 46.9, 42.3, 42.1, 19.8; IR (KBr)
n
2925, 1751, 1682 cmꢃ1; MS (ESI) m/z (%) 238 [MþNa]þ. Anal. Calcd
for C13H13NO2: C, 72.54; H, 6.09; N, 6.51. Found: C, 72.44; H, 6.06;
N, 6.50.
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Acknowledgements
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The authors thank National Natural Science Foundation of China
(21320102002), Tsinghua University and Chinese Academy of Sci-
ences for financial Support.
20. Leng, D.-H.; Wang, D.-X.; Pan, J.; Huang, Z.-T.; Wang, M.-X. J. Org. Chem. 2009,
74, 6077e6082.
Supplementary data
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at/orgc/.
1H and 13C NMR spectra of products, X-ray molecular structures
of S-6a and R-8d (CIFs), HPLC chromatograms of products. Sup-
plementary data related to this article can be found at http://
dx.doi.org/10.1016/j.tet.2014.05.018.
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9532e9535.
Please cite this article in press as: Ao, Y.-F.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.05.018