Asymmetric reduction of aryl imines using Candida parapsilosis ATCC 7330

Article abstract of DOI:10.1016/j.tetasy.2007.11.039

A highly enantioselective one pot, novel biocatalytic method for the asymmetric reduction of aryl imines is reported. Treatment of aryl imines with Candida parapsilosis ATCC 7330 in aqueous medium produces the enantiomerically pure (R)-secondary amines in

Full text of DOI:10.1016/j.tetasy.2007.11.039

Available online at www.sciencedirect.com
Tetrahedron: Asymmetry 19 (2008) 93–96
Asymmetric reduction of aryl imines using
Candida parapsilosis ATCC 7330
T. Vaijayanthia and Anju Chadhab,*
aDepartment of Chemistry, Indian Institute of Technology Madras, Chennai 600 036, India
bLaboratory of Bioorganic Chemistry, Department of Biotechnology and National Center for Catalysis Research,
Indian Institute of Technology Madras, Chennai 600 036, India
Received 30 October 2007; accepted 14 November 2007
Available online 26 December 2007
Abstract—A highly enantioselective one pot, novel biocatalytic method for the asymmetric reduction of aryl imines is reported. Treat-
ment of aryl imines with Candida parapsilosis ATCC 7330 in aqueous medium produces the enantiomerically pure (R)-secondary amines
in moderate to good yields (55–80%) with excellent enantiomeric excesses (95–>99%).
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
reduction of imines catalyzed by Bronsted acid using a
Hantzsch dihydropyridine as the hydrogen source gives
yields of 46–91% and ee of 68–98%.13 The biocatalytic
asymmetric reduction of C@O bond is a very well-known
reaction,14 but the asymmetric reduction of imines by en-
zymes is very rare in the literature in comparison to the
chemical methods that have been reported. The enzyme,
NAD(P)-dependent oxidoreductase isolated from Pseudo-
monas putida ATCC 12633, catalyzes the reduction of
cyclic imines such as 1-piperideine-2-carboxylate and
1-pyrroline-2-carboxylate to L-pipecolate and L-proline,
respectively, but the yields and ee are not reported.15 Imine
reductase activity was found in the anaerobic bacterium
Acetobacterium woodii by screening a dynamic combinato-
rial library of virtual imine substrates, using a biphasic
water–tetradecane solvent system.16 Clearly, these studies
were not directed towards developing biocatalytic methods
for the asymmetric reduction of imines. As part of our con-
stant effort in ‘green’ chemistry towards developing efficient
biocatalytic methods for organic transformations, we at-
tempted the asymmetric reduction of imines using Candida
parapsilosis ATCC 7330. This biocatalyst is well known for
the asymmetric reduction of a- and b-keto esters and
deracemization of a- and b-hydroxy esters as reported by
us.17–23 The obvious chemical analogy between the reduc-
tion of a carbonyl group (C@O) and an imine (C@N) also
extends the scope of the biocatalyst, C. parapsilosis ATCC
7330. Herein we report a benchmark for the biocatalytic
asymmetric reduction of imines.
Asymmetric reduction of C@N bond to the corresponding
enantiomerically pure amines is a very useful transforma-
tion because enantiomerically pure amines are important
precursors to compounds that are of much interest in the
pharmaceutical and agricultural industries.1 For example,
enantiomerically pure N-(1-phenylethyl)benzenamine is
an important chiral intermediate, which finds widespread
use in the synthesis of anticholinergic drugs such as des-
oxyeseroline, physostigmine and esermethole.2,3 While the
reduction of the C@N bond is well established,4 reports
on the asymmetric reduction of C@N are comparatively
rare. These enantioselective reductions generally employ
catalysts derived from transition metals in low-oxidation
states.5–9 Enantioselective reduction of imines using poly-
methylhydrosiloxane (PMHS) as a hydrogen source in
the presence of a chiral ligand with binaphthol and metal
catalysts such as Sn(OTf)2, Zn(OTf)2, In(OTf)3, Cd(CHB)2
is known to give reasonable yields (50–99%) but poor enan-
tiomeric excesses of the product amine (29–60% ee).10
Asymmetric reduction of ketimines with trichlorosilane
activated with N-methyl L-valine derived Lewis base organ-
ocatalyst (up to 92% ee) and N-formylproline derivatives
(up to 66% ee) has been reported.11,12 The enantioselective
*
Corresponding author. Tel.: +91 44 2257 4106; fax: +91 44 2257 4102;
e-mail: anjuc@iitm.ac.in
0957-4166/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2007.11.039

94
T. Vaijayanthi, A. Chadha / Tetrahedron: Asymmetry 19 (2008) 93–96
2. Results and discussion
the enantiomeric excesses of the reduced products. This is
the first report of the preparation of enantiomerically pure
chiral amines 1b–6b (Scheme 1, Tables 1 and 2) by the
asymmetric reduction of prochiral imines using whole cells.
Thus, whole cells of C. parapsilosis ATCC 7330 catalyzes
the formation of enantiomerically pure secondary aromatic
amines, that is, 1b–6b with excellent enantiomeric excesses
(95–>99%) and high yields (55–80%). Since the reaction is
carried out in an aqueous medium and no cofactor is
added, it is an attractive process for the preparation of
enantiomerically pure secondary aromatic amines. The
absolute configurations of amines 1b, 3b and 4b were as-
signed to be (R) by comparing their specific rotation values
with those reported in the literature.11 Interestingly, as re-
ported by us earlier,18,21 this biocatalyst C. parapsilosis
ATCC 7330 reduces the prochiral keto esters [a and b] to
the (S)-enantiomer and it also has an (R) specific oxidore-
ductase, which is involved in the deracemization.20,21 The
specific rotation values of amines 2b, 5b and 6b are re-
ported here for the first time. Compounds 2b, 5b and 6b
had the same elution profile as compounds 1b, 3b and 4b;
the (R)-enantiomer is the early eluting enantiomer and
the (S)-enantiomer is the late eluting enantiomer (Table 2).
Biocatalytic asymmetric reduction of aromatic imines 1a–
6a (Scheme 1, Tables 1 and 2) by whole cells of C. parapsi-
losis ATCC 7330 gave the corresponding secondary aro-
matic amines 1b–6b (Scheme 1, Tables 1 and 2) in 95–
>99% ee and 55–80% yields. As can be seen in Table 1,
the reduction of unsubstituted (E)-N-(1-phenylethyli-
dene)benzenamine 1a (Scheme 1, Tables 1 and 2) using
C. parapsilosis ATCC 7330 showed high ee (98%) and good
yield (71%) of the reduced product. Compounds 5b and 6b
(Scheme 1, Tables 1 and 2) with the electron-withdrawing
substituents (2-NO2 and 3-NO2) were formed in high ee
(>99% and 98%) in moderate yields (59% and 55%). Com-
pound 2b with the 2-hydroxy substituent (Scheme 1, Tables
1 and 2) was formed in high ee (99%) and marginally better
yield (65%). Compounds 3b and 4b with electron-releasing
substituents (4-MeO and 4-Cl) (Scheme 1, Tables 1 and 2)
were formed in high ee (97% and 95%) and excellent yields
(80% and 74%), respectively. The nature (electron-with-
drawing or electron-donating) and position of the substitu-
ent on the aromatic ring with 4-chloro, 4-methoxy, 2-
hydroxy, 2- and 3-nitro groups does not seem to influence
Candida parapsilosis
3. Conclusions
ATCC 7330
N
N
H
R'
R'
Water, 25 ºC, 3 h
Enantiomerically pure (R) secondary amines were obtained
with excellent ee (up to >99% ee) and good yields (up to
80%) using C. parapsilosis ATCC 7330 as a biocatalyst.
Substrates with electron-withdrawing and electron-donat-
ing groups also gave good ee and yields. Mild reaction con-
ditions (pH 6.8, 25 °C) and no additional requirement of
the addition of cofactors make this water-based biotrans-
formation mediated by C. parapsilosis ATCC 7330 a very
R
R
(R)
1b-6b
1a-6a
R= H, 2-OH, 4-MeO, 4-Cl, 2-NO2
R'= H, 3-NO2
Yield: 55-80%
ee:95->99%
Scheme 1. Asymmetric reduction of aromatic imines 1a–6a using the
whole cells of Candida parapsilosis ATCC 7330.
Table 1. Asymmetric reduction of aryl imines 1a–6a using whole cells of Candida parapsilosis ATCC 7330
25
Product number
R
R0
eea (%)
Yield (%)
½aꢀD CHCl3
Abs. Config.
1b
2b
3b
4b
5b
6b
H
H
H
H
H
H
3-NO2
98
99
97
95
>99
98
71
65
80
74
59
55
ꢁ11.4 (c 1.1)
ꢁ8.1 (c 1.3)
ꢁ3.97 (c 0.8)
ꢁ11.9 (c 1.2)
ꢁ 7.9 (c 1.0)
ꢁ 5.4 (c 0.6)
(R)b
(R)c
(R)b
(R)b
(R)c
(R)c
2-OH
4-MeO
4-Cl
2-NO2
H
a Enantiomeric excess was determined by Chiral HPLC.
25
b The absolute configurations of 1b, 3b and 4b were found to be (R) by comparing the ½aꢀD values with the reported literature data.11
c Compounds 2b, 5b and 6b had the same elution profile as compounds 1b, 3b and 4b, that is, the (S)-enantiomer is the late eluting enantiomer, while the
(R)-enantiomer is the early eluting enantiomer (see Table 3).
Table 2. Retention times of enantiomerically pure aromatic amines 1b–6b
Product number
R
R0
Chiral column
Elution of HPLC peaks
(retention time in minutes)
Early (major)
Later (minor)
1b
2b
3b
4b
5b
6b
H
H
H
H
H
H
3-NO2
ODH
ODH
ODH
OJH
OJH
ODH
14.7
15.3
17.5
16.7
24.7
19.3
17.1
18.7
19.7
19.3
29.0
22.7
4-Cl
4-OMe
2-OH
4-NO2
H

T. Vaijayanthi, A. Chadha / Tetrahedron: Asymmetry 19 (2008) 93–96
95
centrated. Compound (E)-N-(1-phenylethylidene)benzen-
amine 1a (Scheme 2 and Table 3) was obtained as a very
pale yellow solid in 57% yield (0.449 mg, 2.2 mmol) after
deactivated silica gel column purification using hexane/
ethyl acetate (99:1) as solvent. The same procedure was
followed for compounds 2a–6a (Scheme 1 and Table 1).
NH2
O
Molecular sieves 4Å
Microwave 5 mins
N
+
R'
R'
R
R
R= H, 2-OH, 4-MeO, 4-Cl, 2-NO2
R'= H, 3-NO2
4.3. Asymmetric reduction of various aryl imines 1a–6a
Scheme 2. Synthesis of aryl imines 1a–6a.
4.3.1. Growth of C. parapsilosis ATCC 7330. Cells of the
yeast C. parapsilosis ATCC 7330 were grown as reported
earlier25 and used for the asymmetric reduction.
Table 3. Synthesis of aryl imines 1a–6a
Product number
R
R0
Yield (%)
1a
2a
3a
4a
5a
6a
H
H
H
H
H
H
57
70
61
69
72
70
4.3.2. A typical experimental procedure for the asymmetric
reduction of (E)-N-(1-phenylethylidene)benzenamine 1a
using the whole cells of C. parapsilosis ATCC 7330. To
the harvested cells (18 g) suspended in distilled water
2-OH
4-MeO
4-Cl
2-NO2
H
[16.2 ml],
(E)-N-(1-phenylethylidene)benzenamine
1a
3-NO2
(Scheme 2 and Table 2) (36 mg, 0.182 ꢂ 10ꢁ3 moles) dis-
solved in ethanol (900 ll) was added. The biotransforma-
tion was carried out for 3 h at 150 rpm and 25 °C.
Control experiments were carried out wherein only the sol-
vent (ethanol) was added to the cell suspension. After 3 h,
the product N-(1-phenylethyl)benzenamine 1b (Scheme 1
and Table 1) was extracted using ethyl acetate (3 ꢂ 2 ml).
The organic phase was then dried over anhydrous Na2SO4
and concentrated under vacuum. The other imine com-
pounds 2a–6a (Scheme 1 and Table 1) were also used as
substrates in the same manner. The experiments with the
imine substrates 1a–6a (Scheme 1 and Table 1) were done
in duplicates. The ee of amines 1b–6b (Scheme 1 and Tables
1 and 2) was determined by chiral HPLC using Chiralcel
OD-H and OJ-H columns using hexane/isopropanol
(98:2) as the mobile phase.
efficient asymmetric reduction reaction of imines to the cor-
responding amines. Moreover, its operational simplicity
makes this procedure extremely attractive and a practical
alternative to the existing methods, and we believe this will
find general acceptance in organic synthesis.
4. Experimental
4.1. General methods
C. parapsilosis ATCC 7330 was purchased from ATCC,
Manassas, VA 20108, USA. All chemicals used for media
preparation were purchased locally. All substrates were
synthesized using the reported method24 modified by us (re-
1
fer Scheme 2, Table 3 and Experimental Section 4.2). H
4.3.3. Spectral data for compounds 2b, 5b and 6b
4.3.3.1. (R)-N-(1-(2-Hydroxyphenyl)ethyl)benzenamine
and 13C NMR spectra were recorded in CDCl3 on a JEOL
GSX400 and Bruker AV-400 spectrometers operating at
400 MHz and 100 MHz. Chemical shifts are expressed in
ppm values using TMS as an internal standard. Infrared
spectra were recorded on a Shimadzu IR 470 Instrument.
Mass spectra were recorded on a Q TOF micromass spec-
trometer. The enantiomeric excess (ee) was determined by
HPLC analysis using a chiral column on a Jasco PU-
1580 liquid chromatograph equipped with PDA detector.
The chiral columns used were Chiralcel OD-H and Chiral-
cel OJ-H (Daicel, 4.6 ꢂ 250 mm). The solvent used was
hexane/isopropanol (98:2) at a flow rate of 1 ml minꢁ1
and the absorbance monitored using a PDA detector at
254 nm. Optical rotations were determined on an AutopalÒ
digital polarimeter. TLC was carried out using Kieselgel 60
F254 aluminium sheets (Merck 1.05554).
1
2b. Yellow liquid; H NMR (CDCl3, 400 MHz) d ppm:
1.5 (d, 3H), 4.4 (q, 1H), 6.68–7.13 (m, 9H); 13C NMR
(CDCl3, 100 MHz) d ppm: 21.0, 45.5, 113.0, 115.1, 116.1,
121.0, 128.0, 128.4, 130.0, 131.3, 147.2, 154.0; IR mmax
:
3332.5, 2981.5, 1728.2, 1602.1, 1499.0, 1372.6, 1238.4,
1043.2, 751.4 cmꢁ1
;
HRMS(ESI): found 214.1236,
C14H16NO (M+H)+ requires 214.1232.
4.3.3.2. (R)-N-(1-(2-Nitrophenyl)ethyl)benzenamine 5b.
1
Yellow liquid; H NMR (CDCl3, 400 MHz) d ppm: 1.5
(d, 3H), 4.4 (q, 1H), 6.36–8.15 (m, 9H); 13C NMR (CDCl3,
100 MHz) d ppm: 24.9, 53.3, 113.2, 117.9, 123.7, 124.0,
126.1, 126.6, 129.2, 137.8, 147.7, 148.5; IR mmax: 3379.8,
2924.6, 1628.6, 1522.5, 1350.1, 815.5, 794.6, 735.4,
670.7 cmꢁ1; HRMS(ESI): found 243.1130, C14H15N2O2
(M+H)+ requires 243.1134.
4.2. Synthesis of aryl imines
4.3.3.3. (R)-3-Nitro-N-(1-phenylethyl)benzenamine 6b.
1
Condensation of acetophenone (4.0 mmol, 500 mg) and
aniline (6.0 mmol, 0.6 ml) was carried out using a domestic
microwave oven (Power 30) for 5 min. Molecular sieves
Yellow liquid; H NMR (CDCl3, 400 MHz) d ppm: 1.4
(d, 3H), 4.4 (q, 1H), 6.66–7.54 (m, 9H); 13C NMR (CDCl3,
100 MHz) d ppm: 29.6, 53.4, 109.0, 112.3, 113.1, 117.8,
120.5, 125.7, 128.8, 129.3, 129.8, 147.3; IR mmax: 3375.5,
2921.2, 1736.0, 1628.7, 1523.3, 1350.3, 1259.8, 794.7,
734.7 cmꢁ1; HRMS(ESI): found 243.1136, C14H15N2O2
(M+H)+ requires 243.1134.
˚
4 A were used to remove the side product, water, to facili-
tate the completion of the reaction. After completion, the
reaction mixture was neutralized with dil. HCl (1 M),
extracted with ethyl acetate (3 ꢂ 10 ml), dried and con-

96
T. Vaijayanthi, A. Chadha / Tetrahedron: Asymmetry 19 (2008) 93–96
11. Malkov, A. V.; Mariani, A.; MacDougall, K. N.; Koc-ovsky,
Acknowledgement
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We thank the Indian Institute of Technology Madras, for
providing financial assistance and Sophisticated Analytical
Instrumentation Facility (SAIF), Indian Institute of Tech-
nology Madras, Chennai, for NMR data.
14. Kroutil, W.; Mang, H.; Edegger, K.; Faber, K. Curr. Opin.
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