Highly efficient and enantioselective biotransformations of β-lactam carbonitriles and carboxamides and their synthetic applications

Article abstract of DOI:10.1039/c0ob00198h

Catalyzed by Rhodococcus erythropolis AJ270, a nitrile hydratase and amidase containing microbial whole cell catalyst, a number of racemic 1-arylmethyl- and 1-allyl-4-oxoazetidine-2-carbonitriles and carboxamides underwent efficient transformations under very mild conditions to produce enantiopure functionalized S-amide and R-acid products in excellent yields. While the nitrile hydratase showed good enzyme activity but virtually no enantioselectivity, the amidase displayed high R-enantioselectivity against almost all amide substrates tested. The synthetic applications of the resulting functionalized chiral β-lactam derivatives were demonstrated by the facile preparation of β-lactam-fused heterocyclic compounds.

Full text of DOI:10.1039/c0ob00198h

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Highly efficient and enantioselective biotransformations of b-lactam
carbonitriles and carboxamides and their synthetic applications†‡
Dong-Hui Leng,a De-Xian Wang,a Zhi-Tang Huanga and Mei-Xiang Wang*a,b
Received 2nd June 2010, Accepted 8th July 2010
DOI: 10.1039/c0ob00198h
Catalyzed by Rhodococcus erythropolis AJ270, a nitrile hydratase and amidase containing microbial
whole cell catalyst, a number of racemic 1-arylmethyl- and 1-allyl-4-oxoazetidine-2-carbonitriles and
carboxamides underwent efficient transformations under very mild conditions to produce enantiopure
functionalized S-amide and R-acid products in excellent yields. While the nitrile hydratase showed good
enzyme activity but virtually no enantioselectivity, the amidase displayed high R-enantioselectivity
against almost all amide substrates tested. The synthetic applications of the resulting functionalized
chiral b-lactam derivatives were demonstrated by the facile preparation of b-lactam-fused heterocyclic
compounds.
been isolated,9 and the microbial transformation of nitriles has
Introduction
become the most effective and environmentally benign method for
the production of carboxamides and carboxylic acids.10 One of the
best-known examples is the industrial production of acrylamide
from the biocatalytic hydration of acrylonitrile.11 More remark-
ably, owing to the enantioselectivity of the enzymes, the bio-
transformation of nitriles has been demonstrated to complement
the existing asymmetric chemical and enzymatic methods for the
synthesis of chiral carboxylic acids and their amide derivatives.10,12
One of the distinct advantages of enzymatic transformation of
nitriles is 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 transform a variety of
racemic nitriles into highly enantiopure carboxylic acids and
amides.12 Using the highly enantioselective nitrile biotransforma-
tion approach, many structurally diverse acids and amides that
contain a three-membered ring such as cyclopropane,14 epoxide15
and aziridine16 have been synthesized. Very recently, we17 have
discovered that the simple four-membered heterocyclic nitriles,
azetidine-2-carbonitriles, are converted efficiently into enantiop-
ure azetidine-2-carboxylic acids and azetidine-2-carboxamides via
biocatalysis. Our interests in functionalized b-lactam compounds
and in exploration of nitrile biotransformation in organic synthesis
led us to undertake the current study. Herein, we report the
biotransformation of 1-alkyl-4-oxoazetidine-2-carbonitriles and
2-carboxamimdes, a highly efficient method for the preparation of
enantiopure b-lactam derivatives. Preliminary applications of the
resulting b-lactam products in synthesis are also demonstrated.
Chiral b-lactam constitutes the core structure of b-lactam antibi-
otics that are extensively used in a clinical setting.1 Amoxicillin,
for example, remained one of the most prescribed antibiotics
in the United States, with approximately 49.5 million prescrip-
tions filled in 2009.2 Chiral b-lactam compounds are also very
useful intermediates in organic synthesis, and they provide a
versatile platform for the preparation of various chiral organic
compounds.1,3 Enantiomerically pure b-lactam compounds are
synthesized mainly from various intramolecular cyclizations of
chiral starting materials and 2+2 cycloaddition reactions employ-
ing the chiral auxiliary.4 While the lipase-catalyzed hydrolysis of
cis-3-acetyloxy-4-t-butyl-2-azetidinone gives enantiopure 3R-cis-
3-acetyloxy-4-t-butyl-2-azetidinone in a good yield,5 biocatalytic
reductions of a-keto-b-lactams with yeast reductases produce a-
hydroxy-b-lactams with re- and si-face selectivity.6 Recently, the
N-heterocyclic carbene catalyzed asymmetric Staudinger reaction
has provided another attractive approach to the preparation of
chiral b-lactam derivatives.7 Because of the paramount importance
of b-lactams in chemistry, biology and medicine, it is highly desir-
able to establish general and efficient methods for the synthesis of
diverse b-lactam compounds of excellent enantiopurity.
One of the biocatalytic nitrile degradation pathways involves the
nitrile hydratase-catalyzed hydration of a nitrile followed by the
amide hydrolysis catalyzed by the amidase.8 A large number of the
microorganisms containing nitrile hydratases and amidases have
aBeijing National Laboratory for Molecular Sciences, CAS Key Laboratory
of Molecular Recognition and Function, Institute of Chemistry, Chinese
Academy of Sciences, Beijing, 100190, China
bThe Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical
Biology (Ministry of Education), Department of Chemistry, Tsinghua
University, Beijing, 100084, China. E-mail: wangmx@mail.tsinghua.edu.cn;
Tel: +8610-62796761
Results and discussion
† This paper is part of an Organic & Biomolecular Chemistry web theme
issue on chemical biology
We initiated our study by examining the biotransformations of
racemic 1-benzyl-4-oxoazetidin-2-carbonitrile 1a.18 The reaction
was conducted in neutral aqueous potassium phosphate buffer at
30 ◦C. Since the substrate 1a is highly viscous and does not disperse
well in the aqueous buffer, a very small amount of acetone was used
as a co-solvent. To facilitate the isolation of product, acid 3a was
‡ Electronic supplementary information (ESI) available: Synthesis and
characterization of all racemic nitrile, amide and ester compounds,
characterization and 1H and 13C spectra of all products, and X-ray
structures of S-2e and 17. CCDC reference numbers 779903 and 779904.
For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/c0ob00198h
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Table 1 Enantioselective biotransformations of racemic nitriles 1a
Table 2 Biocatalytic kinetic resolution of racemic b-cyano-b-lactams 1a
Entry 1 Ar
t/min S-1 (%)b S-1 (ee)c R-2 (%)b R-2 (ee)c
1
2
3
4
5
6
7
1a C6H5
1b 4-F-C6H4
120
70
34
59
45
59
44
89
99
NDd
NDd
NDd
1.2
61
40
53
39
56
10
—
4.3
Entry 1 Ar
t/h S-2 (%)b S-2 (ee)c R-4 (%)b R-4 (ee)c Ed
11.4
12.6
2.2
19.2
9.8
1c 4-Me-C6H4 120
1
1a C6H5
3.5 45
8.75 45
10 39
>99.5 49
97.6 48
>99.5 47
>99.5 40
>99.5 21
>99.5 47
>99.5 29
>99.5 >200
>99.5 >200
>99.5 >200
>99.5 >200
>99.5 >200
>99.5 >200
>99.5 >200
1d 4-Br-C6H4
1e 3-Br-C6H4
1f 2-Br-C6H4
180
70
1 d
2e
3
1a C6H5
NDd
NDd
NDd
1b 4-F-C6H4
1c 4-Me-C6H4 17.5 50
4
1g 4-MeO-C6H4 7 d
—
5f
6
1d 4-Br-C6H4
1e 3-Br-C6H4
1f 2-Br-C6H4
72 23
13 45
192 30
a Nitrile (2 mmol) was incubated with Rhodococcus erythropolis AJ270 cells
(2 g wet weight) in potassium phosphate buffer (0.1 M, pH 7.0) at 30 ◦C.
Acetone (1 mL) was used as a co-solvent. b Isolated yield. c Determined by
chiral HPLC analysis. d Not determined.
7g
8h
1g 4-MeO-C6H4 192
—
—
—
—
—
a Nitrile (1 mmol) was incubated with Rhodococcus erythropolis AJ270
cells (2 g wet weight) in potassium phosphate buffer (0.1 M, pH 7.0, 50
mL) at 30 ◦C. Acetone (0.5 mL) was used as a co-solvent. b Isolated yield.
c Determined by chiral HPLC analysis. d Calculated following a literature
method.20 e Nitrile (2 mmol) was used. f Nitrile S-1d (41%, ee 5.2%) was
recovered. g Nitrile S-1f (41%, ee 13.2%) was recovered. h Nitrile 1g (99%)
was recovered.
of all racemic nitrile substrates 1a–f produced enantiomerically
pure S-amide and R-acid products with an enantiomeric ratio E20
being higher than 200 (entries 1 to 7, Table 1). This suggested
that the formation of enantiopure amide and acid products from
nitrile biotransformations was not affected by the nature of the
substituent on the benzene ring or its substitution pattern. On the
other hand, however, recovered nitriles after 50% conversion gave
very low enantiomeric excess values, with ee of S-1d and S-1f being
only 5.2% and 13.2%, respectively (entries 5 and 7), indicating
low enantioselectivity of the nitrile hydratase against these nitrile
substrates. It might be worth noting that the microbial cell catalyst
did not effect the hydrolytic ring opening reaction of b-lactam even
with a very long incubation time (entries 7 and 8, Table 1). The
amidase within Rhodococcus erythropolis AJ270 therefore acted
specifically on a carbamoyl group rather than a b-lactam moiety.
To gain a deep insight into the catalytic efficiency and stereo-
chemistry of the nitrile hydratase and the amidase of Rhodococcus
erythropolis AJ270, kinetic resolution of nitrile hydration and
of amide hydrolysis was investigated. As demonstrated by the
results in Table 2, except for 1f and 1g, all racemic nitriles
tested underwent rapid biocatalytic hydration reaction, with
50% conversion being completed within 3 h. In all cases, the
enantiomeric excess values of S-nitriles and R-amides isolated
were extremely low, ranging from 1.2% to 19.2%. This indicated
clearly that the nitrile hydratase in Rhodococcus erythropolis AJ270
shows extremely low R-enantioselectivity against 1-arylmethyl-4-
oxoazetidin-2-carbonitrile substrates. The results are consistent
with previous observations12 that the nitrile hydratases are gener-
ally a type of highly active but less enantioselective enzymes against
a variety of nitrile substrates. These characteristics of the nitrile
hydrates, such as having a broad substrate spectrum and possessing
low or no enantioselectivity, are intrinsically determined by the
enzyme structure21 in which there is a spacious pocket around
active site. In other words, a pair of enantiomers of 1-arylmethyl-
4-oxoazetidin-2-carbonitriles were hardly differentiated by the
nitrile hydratase, leading almost identical biocatalytic hydration
reactions.
converted into its methyl ester using CH2N2 (Table 1). It was found
that, hydrolysis of 1, catalyzed by Rhodococcus erythropolis AJ270
whole cell catalyst,19 proceeded very efficiently and enantiose-
lectively. Enantiopure 1-benzyl-4-oxoazetidine-2-carboxamide S-
2a and methyl 1-benzyl-4-oxoazetidine-2-carboxylate R-4a were
obtained in good yield within a short period of time (entries 1 and
2, Table 1). To study the effect of the substituent of benzyl on the
reaction, racemic nitrile analogs 1b–g were prepared according
to a literature procedure18 and subjected to biotransformations.
As summarized in Table 1, the efficiency of biotransformations
was strongly governed by the nature of the substituent and
its substitution pattern. While 4-fluoro (1b) and 4-methyl (1c)
substituted nitrile substrates underwent efficient reactions to
afford high yields of amide and acid products in 10 h and 17.5
h, respectively (entries 3 and 4), slow nitrile hydration reaction
was observed for 4-bromo substituted analog 1d. Along with
the isolation of amide S-2d (23% yield) and ester R-4d (21%
yield), for example, nearly 50% of the starting nitrile 1d was
recovered after 3 days’ incubation with Rhodococcus erythropolis
AJ270 whole cells (entry 5, Table 1). The presence of a methoxy
group at the para-position caused complete inhibition of nitrile
hydration reaction of 1g. After 8 days, intact starting substrate was
recovered almost quantitatively (entry 8, Table 1). Interestingly,
moving the bromo substituent from the para- to the meta-position
led to the acceleration of biotransformations, as substrate 1e was
biocatalytically transformed into enantiopure S-amide S-2e and
R-acid R-3e in excellent yields in 13 h (entry 6, Table 1). However,
further moving the bromo substituent to the ortho-position had
a detrimental effect. This was exemplified by the isolation of
41% of 1f after 8 days’ interaction with the biocatalyst (entry
7, Table 1). It is important to note that, except for nitrile 1g,
which was inert to the whole cell biocatalyst, biotransformations
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Table 3 Biocatalytic kinetic resolution of racemic b-carbamoyl-b-
lactams 2a
double bond on the biocatalysis, biotransformation of 1-propyl-
oxoazetidine-2-carbonitrile was also examined. To our delight,
under the identical biocatalytic conditions, nitrile ( )-5 underwent
very rapid hydration reaction to form amide ( )-6, which was
efficiently resolved into enantioenriched R-1-allyl-4-oxoazetidine-
2-carboxamide S-6 (ee > 99.5%) and, after esterification, methyl
S-1-allyl-4-oxoazetidine-2-carboxylate R-7 (ee 94.0%) in excellent
yields (Scheme 2 and ESI‡). When the carbon–carbon double
bond was reduced, a slightly slow biotransformation of racemic
substrate 8 was observed. Noticeably, however, the enantiomeric
excess values of the amide S-9 and ester R-10 were only moderate
(Scheme 1), indicating a dramatic decrease of enantioselectiv-
ity of the amidase against racemic 1-propyl-4-oxoazetidine-2-
carboxamide. The intriguing effect of a carbon–carbon unsatu-
rated bond on the increase of reaction rate and the enhancement
of the enantioselectivity of the amidase has been noted in our
previous study.22 This somewhat general and interesting feature of
the amidase can serve as a guideline in the design of substrates in
order to achieve efficient and enantioselective biocatalysis.
Entry 2 Ar
t/h S-2 (%)b S-2 (ee)c R-4 (%)b R-4 (ee)c Ed
1
2
3
2d 4-Br-C6H4
2f 2-Br-C6H4
22 49
28 45
2g 4-MeO-C6H4 20 46
>99.5 49
>99.5 46
>99.5 45
>99.5 >200
>99.5 >200
92.0
125
a Amide (1 mmol) was incubated with Rhodococcus erythropolis AJ270 cells
(2 g wet weight) in potassium phosphate buffer (0.1 M, pH 7.0) at 30 ◦C.
Methanol (3 mL) was used as a co-solvent. b Isolated yield. c Determined
by chiral HPLC analysis. d Calculated following a literature method.20
In stark contrast to the nitrile hydratase which catalyzed a
virtually non-enantioselective nitrile hydration reaction, the ami-
dase within Rhodococcus erythropolis AJ270 displayed excellent
enantioselection. Biocatalytic kinetic resolution of racemic amides
2d, 2f and 2g afforded excellent yields of the corresponding
S-amides and R-acids with ee values ranging from 92.0% to
>99.5% (Table 3). The outcomes are also in agreement with the
previous observations12 that the amidase within the Rhodococcus
erythropolis AJ270 microbial cell is a highly enantioselective
enzyme. It can be concluded that the overall enantioselectivity
of the sequential nitrile biotransformations tabulated in Table 1
stems from the combined effect of a virtually non-enantioselective
nitrile hydratase and a high R-enantioselective amidase.
The previous studies12 have revealed that the amidase is a more
demanding enzyme than the nitrile hydratase in terms of the
steric effect of the substrates. If the nitrile with a sterically bulky
substituent does not act as a good substrate of the nitrile hydratase,
the corresponding amide is hardly accepted by the amidase. For
instance, while 2,6-dichlorobenzonitrile is a fairly good substrate
for the nitrile hydratase within Rhodococcus erythropolis AJ270,
the amidase does not at all catalyze the hydrolysis of 2,6-
dichlorobenzamide.9 In the current study, however, the amidase
efficiently transformed amides bearing a 4-bromo- (2d), 2-bromo-
(2f) or 4-methoxy- (2g) substituent on the benzene ring of
the substrate, whereas their nitrile counterparts 1d, 1f and 1g
were difficult substrates for the nitrile hydratase. Although the
mechanism of interaction between the amidase and substrate
awaits further investigation, enantioselective biotransformations
of b-lactam carbonitriles and the corresponding amides ensure
efficient preparation of functionalized b-lactam derivatives of high
enantiomeric purity.
Scheme 1 Enantioselective biotransformations of racemic nitriles 5
and 8.
Encouraged by the efficient and highly enantioselective bio-
transformations of 1-arylmethyl-4-oxoazetidien-2-carbonitriles
( )-1 and -carboxamides ( )-2, we then investigated the reaction of
racemic 1-allyl-4-oxoazetidine-2-carbonitrile ( )-5 and its carbox-
amide ( )-6, for the synthesis of enantiopure polyfunctionalized b-
lactam derivatives. To understand the effect of the carbon–carbon
Scheme 2 Chemical transformation of R-4a into R-2a and of S-6 into
S-9.
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The structures of all products were established on the basis of
their spectroscopic data and microanalysis results. To determine
the absolute configurations of the products, a single crystal of
biotransformed amide product 2e (entry 6, Table 1), a heavy atom-
containing compound, was cultivated from the slow evaporation
of its solution in a mixture of petroleum ether and ethyl acetate
(3 : 1). As illustrated in Fig. 1, X-ray diffraction analysis revealed
unambiguously that the chiral center of the b-lactam ring of 2e is
S-configured. The absolute configuration of esters 4 was obtained
by the comparison of the optical rotation of biotransformed amide
S-2a ([a]2D5 -24◦ (c 0.5, CHCl3)) with that of R-2a ([a]25 +28◦ (c
D
0.5, CHCl3)), which was obtained from the chemical aminolysis
of R-4a (Scheme 2). Catalytic hydrogenation of the double
bond of amide S-6 (ee >99.5%) gave 1-propyl-4-oxoazetidien-2-
carboxamide S-9 (ee >99.5%, [a]2D5 -80◦ (c 0.75, H2O)) (Scheme 2)
that has the same direction of the optical rotation as that of
amide S-9 (ee 57.0%, [a]D25 -32◦ (c 0.75, H2O)) obtained directly
from biotransformations of nitrile, showing clearly that 9 has
the same absolute configuration as 6 (Scheme 1). The amidase
therefore exhibited the same enantio-preference for both 1-allyl-
and 1-propyl-4-oxoazetidine-2-carboxamides. The absolute R-
configuration of product 7 was then determined by the X-ray
crystallography of its derivative (vide infra).
Fig. 1 X-Ray structure of S-2e.
Scheme 3 Synthesis of b-lactam-fused heterocyclic compounds.
b-Lactam products obtained from enantioselective nitrile
and amide biotransformations are valuable chiral intermedi-
ates in organic synthesis. Their transformations into a wide
variety of chiral compounds are conceivable according to pre-
vious studies.1b,3,4 To demonstrate their synthetic usefulness,
we attempted the synthesis of b-lactam-fused heterocycles that
have potential pharmaceutical applications. As depicted in
Scheme 3, CuI-catalyzed intramolecular cross coupling reaction23
of S-1-(2-bromophenyl)methyl)-4-oxoazetidine-2-carboxamide S-
2g produced S-1,10a-dihydroazeto[1,2-a]benzo[e][1,4]diazepine-
2,10(4H,9H)-dione 11 in a good yield, albeit with partial
racemization. Reduction of the ester group of R-4f and R-7
with NaBH4 led to the formation of alcohols 12 and 14 in
almost quantitative yields. Catalyzed by Pd(OAc)2 with a di-
tert-butylphosphinobinaphthyl ligand under basic condition,24
carbon–oxygen bond forming reaction was effected through
intramolecular cross coupling reaction of 12 to furnish R-10,10a-
dihydro-1H-azeto[2,1-c]benzo[f ][1,4]oxazepin-2(4H)-one 13 in
65% yield. Intramolecular iodoetherification25 of 14 afforded
3S,7R-3-iodo-5-oxo-1-azabicyclo[5.2.0]nonan-9-one 15 in 71%
yield (Scheme 3). The structure of 15 was determined with X-ray
crystallography (Fig. 2). Absolute S-configuration at 3-position
Fig. 2 X-Ray structure of 15.
of the lactam ring revealed the R-enantioselectivity of the ami-
dase against 1-allyl-4-oxoazetidine-2-carboxamide (Scheme 1). To
the best of our knowledge, the types of b-lactam-fused seven-
membered heterocyclic compounds 11, 13 and 15 have not been
reported. Not only do they stand as appealing scaffolds for
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medicinal chemistry, they are also amenable to further chemical
manipulations.
NMR (300 MHz, CDCl3) d 7.23–7.28 (m, 2H), 7.02–7.08 (m, 2H),
5.70 (s, 1H), 5.56 (s, 1H), 4.66 (d, J = 14.7 Hz, 1H), 4.18 (d,
J = 15.0 Hz, 1H), 3.84 (dd, J = 2.7 Hz, 5.7 Hz, 1H), 3.26 (dd, J =
5.7 Hz, 14.7 Hz, 1H), 3.02 (dd, J = 2.4 Hz, 14.7 Hz, 1H); 13C NMR
(75 MHz, d6-DMSO) d 171.2, 166.1, 161.5 (d, J = 241.5 Hz, C-F),
132.2 (d, J = 3.0 Hz, C-F), 130.0 (d, J = 8.3 Hz, C-F), 115.3 (d,
J = 21.0 Hz, C-F), 50.6, 44.1, 41.6; MS (ESI) m/z (%) 223 [M+H]+
(10), 245 [M+Na]+ (100). Anal. Calcd. for C11H11FN2O2: C, 59.45;
H, 4.99; N, 12.61. Found: C, 59.11; H, 4.95; N, 12.50.
Conclusion
In summary, we have showed that biotransformations of racemic
1-arylmethyl- and 1-allyl-4-oxoazetidine-2-carbonitriles and car-
boxamides using Rhodococcus erythropolis AJ270, a nitrile hy-
dratase and amidase containing microbial whole cell catalyst,
under very mild conditions provided an efficient and high yielding
method for the preparation of S-4-oxoazetidine-2-carboxamides
and R-4-oxoazetidine-2-carboxylic acids with ee values up to
>99.5%. The overall excellent enantioselectivity of nitrile biotrans-
formations originated from a combined effect of a virtually non-
enantioselective nitrile hydratase and a highly R-enantioselective
amidase involved in Rhodococcus erythropolis AJ270. We have
demonstrated that the resulting enantiopure b-lactam derivatives
were useful chiral intermediates in organic synthesis, and they were
readily converted into the b-lactam-fused heterocyclic compounds.
(S)-(-)-1-(4-Methylbenzyl)-4-oxoazetidine-2-carboxamide (2c).
Solid; mp 197.0–198.0 ◦C; IR (KBr) n 3365, 3189, 1741, 1662 cm-1;
[a]2D5 -32◦ (c 0.125, CHCl3); ee > 99.5% (chiral HPLC analysis);
1H NMR (300 MHz, CDCl3) d 7.16 (s, 4H), 5.73 (s, 1H), 5.58 (s,
1H), 4.61 (d, J = 14.7 Hz, 1H), 4.18 (d, J = 15.0 Hz, 1H), 3.84 (dd,
J = 2.4 Hz, 5.4 Hz, 1H), 3.25 (dd, J = 6.0 Hz, 14.7 Hz, 1H), 3.00
(dd, J = 1.8 Hz, 14.7 Hz, 1H), 2.34 (s, 3H); 13C NMR (75 MHz,
d6-DMSO) d 171.2, 166.0, 136.5, 132.8, 129.1, 127.9, 50.3, 44.4,
41.6, 20.6; MS (ESI) m/z (%) 219 [M+H]+ (5), 241 [M+Na]+ (100).
Anal. Calcd. for C12H14N2O2: C, 66.04; H, 6.47; N, 12.84. Found:
C, 65.95; H, 6.42; N, 13.00.
Experimental section
(S)-(-)-1-(4-Bromob◦enzyl)-4-oxoazetidine-2-carboxamide (2d).
Solid; mp 179.0–180.0 C; IR (KBr) n 3367, 3188, 1741, 1662 cm-1;
[a]2D5 -53◦ (c 0.15, CHCl3); ee > 99.5% (chiral HPLC analysis); 1H
NMR (300 MHz, CDCl3) d 7.49 (d, J = 8.1 Hz, 2H), 7.15 (d,
J = 7.8 Hz, 2H), 5.80 (s, 2H), 4.65 (d, J = 15.0 Hz, 1H), 4.14 (d,
J = 15.0 Hz, 1H), 3.84 (dd, J = 3.0 Hz, 5.1 Hz, 1H), 3.26 (dd,
J = 5.7 Hz, 14.7 Hz, 1H), 3.02 (d, J = 14.7 Hz, 1H); 13C NMR
(75 MHz, d6-DMSO) d 171.1, 166.2, 135.4, 131.4, 130.2, 120.5,
50.7, 44.2, 41.6; MS (ESI) m/z (%) 305 [M+Na]+, 307 [M+2+Na]+.
Anal. Calcd. for C11H11BrN2O2: C, 46.66; H, 3.92; N, 9.89. Found:
C, 46.44; H, 3.97; N, 9.70.
General procedure for the biotransformations of nitriles and amides
To an Erlenmeyer flask (150 mL) with a screw cap were added
Rhodococcus erythropolis AJ270 cells19 (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 dissolved in acetone or racemic amide dissolved
in methanol (see Tables 1–3) 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 or HPLC,
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 extracted with ethyl acetate. After drying
(Na2SO4) 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 and recovered nitrile 1. The
aqueous phase was freeze-dried (-50 to -60 ◦C), and the residue
was treated with CH2N2 in ether below -15 ◦C. When esterification
was finished, the reaction was quenched by addition of some water
and then extracted with ethyl acetate. After drying (Na2SO4) and
removing the solvent under a vacuum, the pure methyl ester 4 was
obtained. All new compounds were fully characterized as follows.
(S)-(-)-1-(3-Bromob◦enzyl)-4-oxoazetidine-2-carboxamide (2e).
Solid; mp 210.0–211.0 C; IR (KBr) n 3344, 3182, 1742, 1680 cm-1;
[a]2D5 -20◦ (c 0.2, CHCl3); ee > 99.5% (chiral HPLC analysis); 1H
NMR (300 MHz, CDCl3) d 7.43–7.47 (m, 2H), 7.20–7.26 (m, 2H),
5.65 (s, 1H), 5.48 (s, 1H), 4.69 (d, J = 15.0 Hz, 1H), 4.15 (d,
J = 15.3 Hz, 1H), 3.88 (dd, J = 2.4 Hz, 5.4 Hz, 1H), 3.29 (dd, J =
5.7 Hz, 14.7 Hz, 1H), 3.04 (dd, J = 2.1 Hz, 14.7 Hz, 1H); 13C NMR
(75 MHz, d6-DMSO) d 171.1, 166.3, 138.9, 130.7, 130.5, 130.2,
126.9, 121.8, 50.8, 44.2, 41.7; MS (ESI) m/z (%) 283 [M+H]+ (11),
285 [M+H+2]+ (10), 305 [M+Na]+ (100), 307 [M+Na+2]+ (94).
Anal. Calcd. for C11H11BrN2O2: C, 46.66; H, 3.92; N, 9.89. Found:
C, 46.70; H, 3.96; N, 9.72. A single crystal of S-2e was obtained
from slow evaporation of its solution in a mixture of petroleum
ether and ethyl acetate (3 : 1).
(S)-(-)-1-Benzyl-4-oxoazetidine-2-carboxamide (2a). Solid;
mp 178.0–179.0 ◦C; IR (KBr) n 3367, 3190, 1741, 1643 cm-1; [a]2D5
-24◦ (c 0.5, CHCl3); ee > 99.5% (chiral HPLC analysis); 1H NMR
(300 MHz, CDCl3) d 7.26–7.37 (m, 5H), 5.73 (s, 1H), 5.60 (s, 1H),
4.67 (d, J = 14.7 Hz, 1H), 4.21 (d, J = 15.0 Hz, 1H), 3.86 (dd, J =
2.4 Hz, 5.4 Hz, 1H), 3.27 (dd, J = 5.7 Hz, 14.7 Hz, 1H), 3.01 (d,
J = 14.7 Hz, 1H); 13C NMR (75 MHz, d6-DMSO) d 171.2, 166.1,
135.9, 128.6, 127.9, 127.4, 50.5, 44.7, 41.6; MS (ESI) m/z (%) 205
[M+H]+, 227 [M+Na]+. Anal. Calcd. for C11H12N2O2: C, 64.69;
H, 5.92; N, 13.72. Found: C, 64.46 H, 5.94; N, 13.73.
(S)-(-)-1-(2-Bromob◦enzyl)-4-oxoazetidine-2-carboxamide (2f).
Solid; mp 185.0–186.0 C; IR(KBr) n 3378, 3202, 1752, 1678 cm-1;
[a]2D5 -48◦ (c 0.125, CHCl3); ee > 99.5% (chiral HPLC analysis);
1H NMR (300 MHz, CDCl3) d 7.58 (d, J = 8.1 Hz, 1H), 7.30–7.38
(m, 1H), 7.17–7.26 (m, 3H), 5.82 (s, 1H), 5.63 (s, 1H), 4.76 (d,
J = 14.7 Hz, 1H), 4.42 (d, J = 15.0 Hz, 1H), 3.90 (dd, J = 2.4 Hz,
5.4 Hz, 1H), 3.28 (dd, J = 5.7 Hz, 14.7 Hz, 1H), 3.02 (dd, J = 2.1 Hz,
14.7 Hz, 1H); 13C NMR (75 MHz, CDCl3) d 171.9, 166.7, 134.2,
133.3, 131.4, 130.2, 128.3, 124.1, 52.1, 46.4, 43.3; MS (ESI) m/z
(%) 283 [M+H]+ (11), 285 [M+H+2]+ (10), 305 [M+Na]+ (100),
307 [M+Na]+ (94). Anal. Calcd. for C11H11BrN2O2: C, 46.66; H,
3.92; N, 9.89. Found: C, 46.64; H, 3.82; N, 9.52.
(S)-(-)-1-(4-Fluorob◦enzyl)-4-oxoazetidine-2-carboxamide (2b).
Solid; mp 178.0–179.0 C; IR (KBr) n 3369, 3192, 1741, 1640 cm-1;
[a]2D5 -27◦ (c 0.15, CHCl3); ee > 99.5% (chiral HPLC analysis); 1H
4740 | Org. Biomol. Chem., 2010, 8, 4736–4743
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(S)-(-)-1-(4-Methoxybenzyl)-4-oxoazetidine-2-carboxamide
(2g). Solid; mp 186.0–187.0 ◦C; IR(KBr) n 3370, 3193, 1737,
1640 cm-1; [a]D25 -48◦ (c 0.125, CHCl3); ee > 99.5% (chiral HPLC
ee > 99.5% (chiral HPLC analysis); 1H NMR (300 MHz, CDCl3)
d 7.40–7.43 (m, 2H), 7.17–7.27 (m, 2H), 4.69 (d, J = 15.0 Hz,
1H), 4.18 (d, J = 15.0 Hz, 1H), 3.98 (dd, J = 2.7 Hz, 5.7 Hz, 1H),
3.72 (s, 3H), 3.25 (dd, J = 5.7 Hz, 14.7 Hz, 1H), 3.07 (dd, J =
2.4 Hz, 14.7 Hz, 1H); 13C NMR (75 MHz, CDCl3) d 170.6, 165.8,
137.2, 131.4, 131.1, 130.4, 127.1, 122.8, 52.5, 50.1, 45.1, 42.1; MS
(EI) m/z (%) 297 [M]+ (3), 299 [M+2]+ (3), 238 (9), 240 (9), 210
(14), 212 (14), 169(100), 171(94). Anal. Calcd. for C12H12BrNO3:
297.0001 [M]+, 298.9980 [M+2]+. Found: 297.0005 [M]+, 298.9984
[M+2]+.
1
analysis); H NMR (300 MHz, CDCl3) d 7.19 (d, J = 8.4 Hz,
2H), 6.88 (d, J = 8.7 Hz, 2H), 5.78 (s, 1H), 5.67 (s, 1H), 4.59 (d,
J = 14.7 Hz, 1H), 4.17 (d, J = 14.7 Hz, 1H), 3.82–3.84 (m, 1H),
3.80 (s, 3H), 3.24 (dd, J = 5.7 Hz, 14.7 Hz, 1H), 2.99 (dd, J =
1.8 Hz, 14.7 Hz, 1H); 13C NMR (75 MHz, d6-DMSO) d 171.3,
166.0, 158.6, 129.3, 127.7, 114.0, 55.0, 50.1, 44.1, 41.5; MS (ESI)
m/z (%) 235 [M+H]+ (3), 257 [M+Na]+ (100). Anal. Calcd. for
C12H14N2O3:C, 61.53; H, 6.02; N, 11.96; Found: C, 61.57; H, 6.05;
N, 11.90.
(R)-(+)-Methyl 1-(2-bromobenzyl)-4-oxoazetidine-2-carboxy-
late (4f). Oil; IR (KBr) n 1764 cm-1; [a]D25 +16◦ (c 0.5, CHCl3);
ee > 99.5% (chiral HPLC analysis); 1H NMR (300 MHz, CDCl3)
d 7.57 (d, J = 8.1 Hz, 1H), 7.28–7.31 (m, 2H), 7.15–7.21 (m,
1H), 4.79 (d, J = 15.0 Hz, 1H), 4.41 (d, J = 15.0 Hz, 1H), 4.00
(dd, J = 2.4 Hz, 5.7 Hz, 1H), 3.71 (s, 3H), 3.24 (dd, J = 5.7 Hz,
14.7 Hz, 1H), 3.02 (dd, J = 2.1 Hz, 14.7 Hz, 1H); 13C NMR
(75 MHz, CDCl3) d 170.8, 165.6, 134.3, 133.1, 131.1, 129.7, 127.9,
123.9, 52.4, 50.6, 45.9, 42.3; MS (ESI) m/z (%) 298 [M+H]+,
300 [M+H+2]+, 320 [M+Na]+, 322 [M+Na+2]+. Anal. Calcd. for
C12H13BrNO3: 298.0079 [M+H]+, 300.0080 [M+H+2]+, Found:
298.0090 [M+H]+, 300.0071 [M+H+2]+.
(R)-(+)-Methyl 1-benzyl-4-oxoazetidine-2-carboxylate (4a).
Oil; IR (KBr) n 1761 cm-1; [a]D25 +28◦ (c 0.5, CHCl3); ee > 99.5%
(chiral HPLC analysis); 1H NMR (300 MHz, CDCl3) d 7.14–7.29
(m, 5H), 4.65 (d, J = 15.0 Hz, 1H), 4.10 (d, J = 15.0 Hz, 1H), 3.86
(dd, J = 2.4 Hz, 5.7 Hz, 1H), 3.60 (s, 3H), 3.11 (dd, J = 5.7 Hz,
14.4 Hz, 1H), 2.94 (dd, J = 2.1 Hz, 14.4 Hz, 1H); 13C NMR
(75 MHz, CDCl3) d 170.8, 165.7, 134.8, 128.8, 128.5, 127.9, 52.4,
49.9, 45.7, 41.9; MS (EI) m/z (%) 219 [M]+ (3), 191 (17), 160 (12),
132 (22), 91 (100). Anal. Calcd. for C12H13NO3: 219.0895, Found:
219.0899.
(R)-(+)-Methyl 1-(4-fluorobenzyl)-4-oxoazetidine-2-carboxylate
(4b). Oil; IR(KBr) n 1760 cm-1; [a]D25 +28◦ (c 0.5, CHCl3); ee
> 99.5% (chiral HPLC analysis); 1H NMR (300 MHz, CDCl3) d
7.20–7.27 (m, 2H), 7.00–7.06 (m, 2H), 4.69 (d, J = 15.0 Hz, 1H),
4.19 (d, J = 15.0 Hz, 1H), 3.94 (dd, J = 2.7 Hz, 5.7 Hz, 1H), 3.71
(s, 3H), 3.21 (dd, J = 5.7 Hz, 14.7 Hz, 1H), 3.04 (dd, J = 2.4 Hz,
14.7 Hz, 1H); 13C NMR (75 MHz, CDCl3) d 170.7, 165.7, 162.4
(d, J = 244.5 Hz, C-F), 130.7 (d, J = 3.8 Hz, C-F), 130.3 (d, J =
8.3 Hz, C-F), 115.7 (d, J = 21.8 Hz, C-F), 52.4, 50.0, 45.0, 41.9;
MS (EI) m/z (%) 237 [M]+ (3), 209 (17), 150 (19), 109 (100). Anal.
Calcd. for C12H12NO3F: 237.0801. Found: 237.0803.
(R)-(+)-Methyl 1-(4-methoxybenzyl)-4-oxoazetidine-2-carbo-
xylate (4g). Oil; IR (KBr) n 1759 cm-1; [a]D25 +24◦ (c 0.5, CHCl3);
ee 92.0% (chiral HPLC analysis); H NMR (300 MHz, CDCl3)
1
d 7.11 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 8.7 Hz, 2H), 4.68 (d, J =
14.7 Hz, 1H), 4.12 (d, J = 14.7 Hz, 1H), 3.91 (dd, J = 2.7 Hz,
5.7 Hz, 1H), 3.80 (s, 3H), 3.70 (s, 3H), 3.18 (dd, J = 5.7 Hz,
14.4 Hz, 1H), 3.01 (dd, J = 2.2 Hz, 14.3 Hz, 1H); 13C NMR
(75 MHz, CDCl3) d 170.8, 165.6, 159.3, 129.9, 126.8, 114.2, 55.2,
52.3, 49.7, 45.0, 41.7; MS (EI) m/z (%) 249 [M]+ (6), 221 (7), 190
(5), 162 (17), 121 (100). Anal. Calcd. for C13H15NO4: 249.1001.
Found: 249.1004.
(R)-(+)-Methyl 1-(4-methylbenzyl)-4-oxoazetidine-2-carboxy-
late (4c). Oil; IR (KBr) n 1762 cm-1; [a]D25 +20◦ (c 0.5, CHCl3);
ee > 99.5% (chiral HPLC analysis); 1H NMR (300 MHz, CDCl3)
d 7.10–7.17 (m, 4H), 4.72 (d, J = 15.0 Hz, 1H), 4.12 (d, J =
14.7 Hz, 1H), 3.92 (dd, J = 2.7 Hz, 5.7 Hz, 1H), 3.70 (s, 3H), 3.19
(dd, J = 5.4 Hz, 14.4 Hz, 1H), 3.02 (dd, J = 1.8 Hz, 14.7 Hz, 1H),
2.33 (s, 3H); 13C NMR (75 MHz, CDCl3) d 170.8, 165.7, 137.7,
131.7, 129.5, 128.5, 52.4, 49.8, 45.4, 41.8, 21.1; MS (ESI) m/z (%)
233 [M]+ (2), 205 (16), 190 (9), 174 (9), 146 (14), 105 (100). Anal.
Calcd. for C13H15NO3: 233.1052. Found: 233.1055.
(S)-(-)-1-Allyl-4-oxoazetidine-2-carboxamide (6). Solid; mp
◦
100.0–101.0 C; IR (KBr) n 3351, 3181, 1742, 1679 cm-1; [a]D25
-28◦ (c 0.5, H2O); ee > 99.5% (chiral HPLC analysis); 1H NMR
(300 MHz, d6-DMSO) d 7.69 (s, 1H), 7.26 (s, 1H), 5.70–5.83 (m,
1H), 5.13–5.21 (m, 2H), 3.92–3.97 (m, 2H), 3.50 (dd, J = 6.6 Hz,
15.9 Hz, 1H), 3.11 (dd, J = 5.4 Hz, 14.1 Hz, 1H), 2.70 (dd, J =
1.8 Hz, 14.4 Hz, 1H); 13C NMR (75 MHz, D2O) d 174.8, 170.2,
130.4, 119.3, 51.5, 44.6, 41.0; MS (ESI) m/z (%) 155 [M+H]+.
Anal. Calcd. for C7H10N2O2:C, 54.54; H, 6.54; N, 18.17; Found:
C, 54.63; H, 6.30; N, 18.05.
(R)-(+)-Methyl 1-(4-bromob◦enzyl)-4-oxoazetidine-2-carboxy-
late (4d). Solid; mp 60.0–61.0 C; IR (KBr) n 1762 cm-1; [a]D25
+21◦ (c 0.3, CHCl3); ee > 99.5% (chiral HPLC analysis); 1H
NMR (300 MHz, CDCl3) d 7.48 (d, J = 8.4 Hz, 2H), 7.13 (d,
J = 8.4 Hz, 2H), 4.67 (d, J = 15.3 Hz, 1H), 4.17 (d, J = 15.0 Hz,
1H), 3.94 (dd, J = 2.4 Hz, 5.4 Hz, 1H), 3.71 (s, 3H), 3.22 (dd,
J = 5.7 Hz, 14.7 Hz, 1H), 3.05 (dd, J = 2.1 Hz, 14.4 Hz, 1H);
13C NMR (75 MHz, CDCl3) d 170.6, 165.7, 133.9, 132.0, 130.2,
122.0, 52.5, 50.0, 45.1, 42.0; MS (ESI) m/z (%) 298 [M+H]+, 300
[M+H+2]+. Anal. Calcd. for C12H12BrNO3: C, 48.34; H, 4.06; N,
4.70. Found: C, 48.54; H, 4.19; N, 4.57.
(R)-(+)-Methyl 1-allyl-4-oxoazetidine-2-carboxylate (7). Oil;
IR (KBr) n 1762 cm-1; [a]D25 +34◦ (c 0.7, CHCl3); ee 94.0% (chiral
HPLC analysis); H NMR (300 MHz, CDCl3) d 5.70–5.83 (m,
1H), 5.19–5.24 (m, 2H), 4.09–4.15 (m, 2H), 3.78 (s, 3H), 3.71 (dd,
J = 6.9 Hz, 15.3 Hz, 1H), 3.23 (dd, J = 5.7 Hz, 14.4 Hz, 1H), 3.00
(dd, J = 2.1 Hz, 14.4 Hz, 1H); 13C NMR (75 MHz, CDCl3) d 171.0,
165.7, 131.1, 119.2, 52.4, 50.2, 44.3, 41.9; MS (ESI) m/z (%) 170
[M+H]+. Anal. Calcd. for C8H11NO3: 192.06294 [M+Na]+; Found:
192.06311 [M+Na]+.
1
(S)-(-)-4-Oxo-1-propylazetidine-2-carboxamide (9). Solid; mp
85.0–86.0 ◦C; IR (KBr) n 3359, 3183, 1741, 1678 cm-1; [a]D25 -80◦
(c 0.75, H2O); ee > 99.5% (chiral HPLC analysis); 1H NMR
(300 MHz, D2O) d 4.17 (dd, J = 1.7, 5.0 Hz, 1H), 3.11–3.26 (m,
(R)-(+)-Methyl 1-(3-bromobenzyl)-4-oxoazetidine-2-carboxy-
late (4e). Oil; IR (KBr) n 1761 cm-1; [a]D25 +32◦ (c 0.5, CHCl3);
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2H), 2.87–2.96 (m, 1H), 2.79 (d, J = 14.8 Hz, 1H), 1.33–1.50 (m,
2H), 0.75 (t, J = 7.2 Hz, 3H); 13C NMR (75 MHz, D2O) d 174.8,
170.4, 51.5, 43.6, 40.7, 20.0, 10.7; MS (ESI) m/z (%) 157 [M+H]+
(5), 179 [M+Na]+ (100). Anal. Calcd. for C7H12N2O2: C, 53.83; H,
7.74; N, 17.94; Found: C, 53.65; H, 7.69; N, 17.72.
(EI) m/z (%) 270 [M+H]+ (8), 272 [M+H+2]+ (7), 238 (9), 240 (6),
190 (100), 169 (39), 171 (35), 132 (60), 107 (54). Anal. Calcd. for
C11H12BrNO2: C, 48.91; H, 4.48; N, 5.19; Found: C, 49.03; H, 4.48;
N, 5.03.
Compound 13 was prepared from the Pd-catalyzed intramolec-
ular coupling reaction of 12 following a literature procedure24
Compound 13 was obtained as a white solid; mp 79.0–80.0 ◦C;
IR(KBr) n 1759 cm-1; [a]D25 +57◦ (c 1.4, CHCl3); ee > 99.5% (chiral
(R)-(+)-Methyl 4-oxo-1-propylazetidine-2-carboxylate (10).
Oil; IR (KBr) n 1759 cm-1; [a]D25 +96◦ (c 0.25, CHCl3); ee 45.2%
(chiral HPLC analysis); 1H NMR (300 MHz, CDCl3) d 4.11 (dd,
J = 2.4, 5.4 Hz, 1H), 3.80 (s, 3H), 3.34–3.44 (m, 1H), 3.22 (dd, J =
5.4, 14.4 Hz, 1H), 3.03–3.13 (m, 1H), 2.99 (dd, J = 2.1 14.4 Hz,
1H), 1.48–1.69 (m, 2H), 0.92 (t, J = 7.2 Hz, 3H); 13C NMR
(75 MHz, CDCl3) d 171.1, 166.0, 52.5, 50.6, 43.4, 41.8, 20.8, 11.5;
MS (ESI) m/z (%) 172 [M+H]+. Anal. Calcd. for C8H13NO3:
171.0895 [M]+; Found: 171.0897 [M]+.
1
HPLC analysis); H NMR (300 MHz, CDCl3) d 7.16–7.23 (m,
2H), 7.05–7.09 (m, 2H), 4.73 (d, J = 15.8 Hz, 1H), 4.60 (dd, J =
3.6, 12.0 Hz, 1H), 4.20 (d, J = 15.8 Hz, 1H), 4.01–4.06 (m, 1H),
3.64 (dd, J = 9.6, 12.0 Hz, 1H), 3.10 (dd, J = 4.4, 14.7 Hz, 1H), 2.57
(dd, J = 1.8, 14.6 Hz, 1H); 13C NMR (75 MHz, CDCl3) d 165.0,
159.3, 130.3, 129.1, 129.0, 124.5, 122.5, 76.3, 51.7, 44.1, 40.0; MS
(ESI) m/z (%) 190 [M+H]+ (65), 212 [M+Na]+ (100). Anal. Calcd.
for C11H11NO2: 189.0790; Found: 189.0793.
Synthesis of 11
Synthesis of 15
Under argon protection, a mixture of S-2g (40 mg, 0.14 mmol),
CuI (0.056 mmol, 11 mg), N,N-dimethylglycine hydrochloride
(0.112 mmol, 16 mg), Cs2CO3 (0.28 mmol, 92 mg) and dry 1,4-
dioxane (5 mL) was refluxed for 20 h. After cooling, ethyl acetate
(15 mL) was added and the resulting mixture was filtered through
a short silica gel (100–200 mesh) pad. The filtrate was concentrated
and the residue was filtered with a silica gel column (100–200 mesh)
and washed with ethyl acetate to give a crude product of 11. The
second silica gel (200–300 mesh) column chromatography eluted
with a mixture of petroleum ether and ethyl acetate (1 : 1) gave
pure product 11 (23 mg, 84%) as white solids; mp 166.0–167.0 ◦C;
IR (KBr) n 3203, 1762, 1660 cm-1; [a]D25 +241.5◦ (c 0.265, CHCl3);
ee 79.6% (chiral HPLC analysis); 1H NMR (300 MHz, CDCl3) d
8.59 (s, 1H), 7.36–7.41 (m, 1H), 7.21–7.31 (m, 2H), 7.11–7.14 (m,
1H), 4.57 (d, J = 13.8 Hz, 1H), 4.47 (d, J = 13.8 Hz, 1H), 4.11
(dd, J = 1.8, 4.8 Hz, 1H), 3.43 (d, J = 15.0 Hz, 1H), 3.11 (dd, J =
4.8, 14.7 Hz, 1H); 13C NMR (75 MHz, CDCl3) d 170.4, 166.4,
136.6, 130.4, 129.6, 126.6, 126.4, 123.3, 50.6, 44.9, 39.1; MS (ESI)
m/z (%) 203 [M+H]+ (100), 225 [M+Na]+ (99). Anal. Calcd. for
C11H10N2O2:C, 65.34; H, 4.98; N, 13.85; Found: C, 65.00; H, 4.98;
N, 13.57.
Following the same procedure for the reduction of ester R-4g, R-8
was converted to intermediate 14 (121 mg, 95%) as colourless oil;
IR(KBr) n 3402, 1734 cm-1; [a]D25 -44◦ (c 0.5, CHCl3); ee 94.0%
(chiral HPLC analysis); 1H NMR (300 MHz, CDCl3) d 5.76–5.89
(m, 1H), 5.20–5.29 (m, 2H), 3.96 (dd, J = 6.0, 15.9 Hz, 1H), 3.69–
3.86 (m, 3H), 2.95 (dd, J = 4.8, 9.6 Hz, 1H), 2.77–2.82 (m, 2H);
13C NMR (75 MHz, CDCl3) d 167.5, 132.4, 118.6, 62.2, 52.5, 43.9,
38.6; MS (ESI) m/z (%) 142 [M+H]+ (78), 164 [M+Na]+ (100).
Anal. Calcd. for C7H11NO2: 141.0790 [M]+. Found: 141.0792 [M]+.
A solution of 14 (70.5 mg, 0.5 mmol) in acetonitrile (1 mL) at 25
◦C was treated with iodine (0.25 g, 1 mmol) and stirred overnight.
The reaction mixture was diluted with ethyl acetate (5 ml), and
then washed twice with a saturated solution of sodium bisulfite.
The organic layer was dried with anhydrous Na2SO4, filtered and
concentrated under vacuum. Chromatography on a silica gel (200–
300 mesh) column eluting with a mixture of petroleum ether and
ethyl acetate (◦3 : 1) gave product 15 (95 mg, 71%)◦as a while solid;
mp 46.0–47.0 C; IR(KBr) n 1738 cm-1; [a]D25 -140 (c 1.0, CHCl3);
ee 94.0% (chiral HPLC analysis); 1H NMR (300 MHz, CDCl3) d
4.17–4.30 (m, 3H), 4.10 (d, J = 13.5 Hz, 1H), 3.84 (s, 1H), 3.73 (dd,
J = 3.6, 13.5 Hz, 1H), 3.42–3.50 (m, 1H), 2.99–3.14 (m, 2H), 2.75
(dd, J = 2.1, 14.8 Hz, 1H); 13C NMR (75 MHz, CDCl3) d 168.2,
80.7, 72.9, 52.9, 49.8, 40.9, 23.4; MS (ESI) m/z (%) 268 [M+H]+
(6), 290 [M+Na]+ (100). Anal. Calcd. for C7H10INO2: C, 31.48; H,
3.77; N, 5.24. Found: C, 31.85; H, 3.86; N, 5.23.
Synthesis of 13
To a solution of ester R-4g (104 mg, 0.35 mmol) in tetrahydrofuran
(0.5 mL) was added anhydrous lithium chloride (30 mg, 0.7 mmol)
and sodium borohydride (27 mg, 0.7 mmol). After addition of
ethanol (1 mL), the mixture was stirred at room temperature for
1 h. The mixture was cooled with an ice-water bath, and water (1
mL) was then added. The mixture was extracted with ethyl acetate
(3 ¥ 5 mL). After drying (Na2SO4) and removing the solvent under
vacuum, the residue of the organic phase was chromatographed on
a silica gel (100–200 mesh) column using a mixture of petroleum
ether and ethyl acetate (1 : 1) as the mobile phase to give pure
product 12 (92 mg, 98%) as a white solid; mp 78.0–79.0 ◦C.
Acknowledgements
We thank the National Science Foundation of China
(20772132), Ministry of Science and Technology (2009CB724704,
2009ZX09501), and Chinese Academy of Sciences for financial
support.
◦
1
IR(KBr) n 3429, 1733 cm-1; [a]D25 +40 (c 0.5, CHCl3); H NMR
(300 MHz, CDCl3) d 7.57 (d, J = 7.9 Hz, 1H), 7.40 (dd, J = 1.4,
7.6 Hz, 1H), 7.30–7.35 (m, 1H), 7.15–7.18 (m, 1H), 4.66 (d, J =
15.4 Hz, 1H), 4.45 (d, J = 15.4 Hz, 1H), 3.76 (d, J = 11.6 Hz, 1H),
3.62–3.69 (m, 2H), 2.98 (dd, J = 4.8, 14.5 Hz, 1H), 2.87 (d, J =
13.7 Hz, 1H) 2.03 (s, 1H); 13C NMR (75 MHz, CDCl3) d 167.6,
135.3, 133.1, 130.7, 129.6, 128.0, 123.5, 61.9, 52.7, 45.4, 38.9; MS
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