Arthrobacter sp.: a lipase of choice for the kinetic resolution of racemic arylazetidinone precursors of taxanoid side chains

Article abstract of DOI:10.1016/j.tetasy.2007.04.031

The native strain of Arthrobacter sp. (MTCC 5125) bearing a lipase has been found to be the most effective in the kinetic resolution of racemic arylazetidinones for producing cis-(3R,4S)-3-acetoxy-1-(4-methoxyphenyl)-4-phenyl-2-azetidinone, cis-(3R,4S)-3-

Full text of DOI:10.1016/j.tetasy.2007.04.031

Tetrahedron: Asymmetry 18 (2007) 1059–1069
Arthrobacter sp.: a lipase of choice for the kinetic resolution
of racemic arylazetidinone precursors of taxanoid side chains
Naveen Anand,a Munish Kapoor,a Khursheed Ahmad,a Surrinder Koul,a Rajinder Parshad,a
Kuldip S. Manhas,a Rattan L. Sharma,b Ghulam N. Qazia and Subhash C. Tanejaa,*
aIndian Institute of Integrative Medicine (CSIR), Jammu Tawi 180 001, India
bDepartment of Chemistry, University of Jammu, Jammu Tawi 180 005, India
Received 23 February 2007; accepted 26 April 2007
Abstract—The native strain of Arthrobacter sp. (MTCC 5125) bearing a lipase has been found to be the most effective in the kinetic res-
olution of racemic arylazetidinones for producing cis-(3R,4S)-3-acetoxy-1-(4-methoxyphenyl)-4-phenyl-2-azetidinone, cis-(3R,4R)-3-
acetoxy-1-(4-methoxyphenyl)-4-(2-furanyl)-2-azetidinone, cis-(3R,4R)-3-acetoxy-1-(4-methoxyphenyl)-4-(2-thienyl)-2-azetidinone, and
cis-3-acetoxy-4-(t-butyl)-2-azetidinone products. The resolved compounds, which were obtained in high enantiopurity are important
intermediates of amino acid side chains of paclitaxel as well as a new generation of taxanoids. The use of co-solvents dramatically
improved the resolution efficacy of the lipase.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
O
H
H
N
O
Paclitaxel, a complex polyoxygenated diterpenoid isolated
from the bark of Taxus brevifolia,1 has been extensively
used in anti-cancer therapy.2 Continuing efforts are being
made to meet its high world-wide demand by semi-syn-
thetic methods. The discovery of 10-deacetyl baccatin III
(10-DAB) in 1981 opened the gateway to semi-synthetic
paclitaxel, wherein 10-DAB was obtained from Taxus bac-
cata and the C-13 side chain was prepared synthetically.3,4
M/s Bristol-Myers Squibb, regarded as the major producer
of paclitaxel, reportedly exploited the semi-synthetic
route.5 The basic requirement in this method is to prepare
the N-benzoyl-b-phenylisoserine side chain 2 with a pro-
tected 20-OH group and 10-DAB protected at the 7-OH.
Ever since the initial work of Potier,6 a number of attempts
have been made to convert 10-DAB to paclitaxel, all of
these methods generally relied on the coupling of a 7-pro-
tected baccatin derivative to a protected phenylisoserinate,
which in turn can be prepared from either glycidates3 or
azetidinones, for example, 1 (Scheme 1).4
Ph
NH
O
O
O
OH
OH
O
1
2
Scheme 1.
biotics.7 Furthermore, asymmetric syntheses,8 and the
preparation of their chiral intermediates either by chemi-
cal9 or chemoenzymatic10 methods have also been
described. However, biocatalytic methods are now
increasingly being employed due to the efficacy of enzy-
matic resolution and simple reaction conditions. Thus,
the use of the lipase from Pseudomonas sp. has been more
frequent,4,11 whereas PPL and PLE have also been used for
the stereoselective hydrolysis of various 2-azetidinone
derivatives.12
Studies on the structure modifications of paclitaxel have
been carried out to design newer analogs, which display
improved bioactivity and low toxicity. The phenyl group
at the 30-position on the C-13 side chain has been an impor-
tant and easy target for the synthesis of new compounds.
A number of such new generation taxanoids have been
2-Azetidinones are well known due to their potent bioactiv-
ity and importance as a part of the family of b-lactam anti-
*
Corresponding author. Tel.: +91 191 2569000–06; fax: +91 191 2569111/
2569016; e-mail: sc_taneja@yahoo.co.in
0957-4166/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2007.04.031

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N. Anand et al. / Tetrahedron: Asymmetry 18 (2007) 1059–1069
synthesized and are currently under various stages of
clinical trials, for example, MAC-321 (TL-00139)13 3;
MST-997 (TL-909)14 4; BMS-275183 5.15
phenyl)-4-t-butyl-2-azetidinone 8, cis-3-propyloxy-1-(4-meth-
oxyphenyl)-4-phenyl-2-azetidinone 10, cis-3-butyloxy-1-(4-
methoxyphenyl)-4-phenyl-2-azetidinone 11, and cis-3-hex-
O
O
O
O
O
O
HO
O
O
H
O
O
NH
O
H
O
O
NH
O
OH
O
O
NH
O
O
O
OH
O
O
O
O
O
OH
S
OH
OH
H
HO
HO
HO
O
O
O
O
O
O
O
O
O
O
O
O
H3CO
3
4
5
The azetidinone precursors of side chain of new generation
of semi-synthetic paclitaxel analogues 3–5 are represented
by structures 6–8, respectively. A process of resolution of
b-lactam ( )-6 via enantioselective hydrolysis using
homogenized beef liver to give a product in >95% enantio-
purity in 40% yield has been disclosed in a patent by Hol-
tan.11a The enzymatic resolution of a side chain precursor
of 4 has also been described by Bisht et al.4a using PS-30
to obtain both enantiomers in >97% enantiopurity. The
side chain precursor of Taxotere, that is, ( )-cis-3-acet-
oxy-4-(1,1-dimethylethyl)-2-azetidinone 8 with lipase PS-
30 or crude BMS lipase from Pseudomonas, has also been
reported by Patel et al.11b,c However, an improved and
highly efficient kinetic resolution process of the above race-
mic precursors with high enantiopurity of the products
(ꢀ99%) will remain a prerequisite for industry due to their
biological importance as well as future demand. In our
ongoing program of the development of efficient kinetic
resolutions methods, we have reported the preparation of
various chiral drugs/drug intermediates and chiral auxilia-
ries using indigenous and commercial sources of biocata-
lysts, whole cell preparations as well as immobilized
enzymes.16 Herein, we report a highly efficient kinetic reso-
lution protocol for the preparation of 1 and 6–8 wherein
the whole cells preparation of ABL was successfully used
for the kinetic resolution of all the racemates.
yloxy-1-(4-methoxyphenyl)-4-phenyl-2-azetidinone 12, were
first carried out via a Staudinger reaction. The Schiff’s base
from the benzaldehyde and p-anisidine was prepared using
microwaves and the rest by acid catalyzed (p-toluenesulf-
onic acid) reaction between the corresponding aldehydes
and p-anisidine using a Dean–Stark apparatus.
The Schiff’s bases thus prepared were reacted with acetoxy-
acetyl chloride (AAC) in the presence of triethylamine
(TEA) in dry dichloromethane to yield b-lactams (72–
90%) via a [2+2] cycloaddition reaction (Scheme 2). In all
of the reactions, only one diastereomer with a cis-configu-
1
ration was obtained as confirmed by H NMR.
After the formation of racemic 2-azetidinones, a prelimin-
ary screen was undertaken to select the most suitable lipase
for enantioselective hydrolysis. A set of 14 lipases and
whole cell preparations, both from commercial sources,
as well as the institutes microbial repository were used to
affect the hydrolysis. The results of the preliminary screen-
ing are summarized in Table 1.
It is apparent from Table 1 that four lipases, that is, PS,
PS-C, AS, and Arthrobacter sp. (MTCC 5125, ABL) were
able to hydrolyze the substrates and ABL was the only
one which hydrolyzed all four substrates (shown with a
H
H
N
H H
N
H
H
N
AcO
O
AcO
O
S
O
AcO
O
O
O
O
(+)-6
(+)-7
(+)-8
2. Results and discussion
positive sign). Detailed studies of the kinetic resolution of
racemic b-lactams were undertaken via stereoselective
hydrolysis of the corresponding racemic esters, which were
incubated with selected lipases, that is, Aspergillus niger
(Amano AS), Pseudomonas sp. now known as Burkholderia
cepacia (Amano PS), B. cepacia on ceramics (Amano
PS-C), and whole cells of Arthrobacter sp. (ABL).
For the proposed kinetic resolution studies, the syntheses
of racemic cis-3-acetoxy-1-(4-methoxyphenyl)-4-phenyl-2-
azetidinone 1, cis-3-acetoxy-1-(4-methoxyphenyl)-4-(2-fur-
yl)-2-azetidinone 6, cis-3-acetoxy-1-(4-methoxyphenyl)-4-
(2-thienyl)-2-azetidinone 7, cis-3-acetoxy-1-(4-methoxy-

N. Anand et al. / Tetrahedron: Asymmetry 18 (2007) 1059–1069
1061
Table 2. Enzymatic hydrolysis of ( )-cis-3-acetoxy-1-(4-methoxyphenyl)-
4-phenyl-2-azetidinone ( )-1 at 26 ꢁC at 20 g/L
H
H
N
AcO
R
R
AcO
O
CH2Cl2
TEA
+
Enzyme
Co-solvent Conversion T (h) ees
eep
E
N
PMP
O
PMP
Cl
Amano AS
CH3CN
DMF
DMSO
46
49
49
78
78
78
84
82
83
96
85
87
125
31
38
1, R = phenyl
7, R = 2-thienyl
6, R = 2-furyl
8, R = t-butyl
Amano PS
CH3CN
DMF
DMSO
49
48
49
24
24
24
96
93
97
99
98.5
98
752
425
357
Scheme 2.
Amano PSC CH3CN
DMF
50
50
49
10
6
9
99
99
99
99
99
99
>1000
>1000
752
Table 1. Screening of lipases on substrates 1, 6, 7, and 8
Enzyme
Substrates
DMSO
1
6
7
8
ABL
CH3CN
DMF
DMSO
50
50
50
6
99
99
>1000
>1000
>1000
4.5
5
>99 >99
>99 >99
PS
PS-C
PF
AS
ABL
CRL
PLAP
MJ
RRL–1789
MM
Lipase G
RRLBB-1
PPL
+
+
ꢁ
+
+
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
+
+
ꢁ
+
+
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
+
+
ꢁ
+
+
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
nd
ꢁ
+
ꢁ
Table 3. ABL catalyzed hydrolysis of ( )-1 after 5 h at 20 g/L concen-
tration at 26 ꢁCa
ꢁ
nd
ꢁ
Co-solvent
10%
20%
30%
40%
50%
nd
nd
ꢁ
DMF
50
45
41
37
20
17
47
10
<5
45
Nd
Nd
<1
<1
Nil
50
Nd
Nd
Nil
Nil
Nil
42
Nd
Nd
Nil
Nil
Nil
<1
Nd
Nd
Acetone
CH3CN
DMSO
Toluene
DCM
nd
nd
CCL
(+) = Hydrolysis; (ꢁ) = no reaction; (nd) = not determined; PS (Burk-
holderia cepacia, Amano PS), PS-C (Burkholderia cepacia on ceramics,
Amano PS-C), PF (Pseudomonas florescence), MJ (Mucor javanicus), ABL
(Arthrobacter sp.), CRL (Candida rugosa lipase), PPL (Porcine pancreatic
lipase), RRLBB1 (Bacillus subtilis), RRL-1789 (Bacillus sp.), Acylase
(Amano Acylase), MM (Mucor miehei), AS (Aspergillus niger, Amano
AS), Lipase G, CCL (Candida cylindracea), PLAP (Pig Liver Acetone
Powder).
Nd—not determined.
ABL concentration 2% (wet weight/v).
a 8.2% conversion in 5 h in buffer only (in the absence of any co-solvent).
observed that when compared to commercial lipases,
ABL was more effective in stereoselective hydrolysis, fur-
nishing only one enantiomer (ee >99%) with high space–
time yields. ABL also displayed high activity in almost all
the co-solvents used and remained active even in the pres-
ence of 30–40% DMSO, whereas other enzymes became
inactive at those concentrations (Table 3). Only PSC which
is a ceramics immobilized lipase could match the efficacy of
ABL for the resolution of ( )-1.
2.1. Resolution of ( )-cis-3-acetoxy-1-(4-methoxyphenyl)-4-
phenyl-2-azetidinone 1
The stereoselective hydrolysis was carried out in an aque-
ous 0.1 M-phosphate buffer at pH 7.0 in the temperature
range 25–26 ꢁC. Due to the slow rate of hydrolysis (15%
conversion in 5 h, ee = 99%) the use of co-solvents was
envisaged (Scheme 3). The addition of organic co-solvents
during enzymatic hydrolysis is known to influence the rates
of hydrolysis as well as enatioselectivity.17
In order to further optimize the reaction conditions, stereo-
selective hydrolysis at varied concentrations ranging from
20 to 100 g/L was attempted. It was observed that ABL
was quite active at 40–50 g/L (Fig. 1). At a concentration
of 20 g/L, 50% conversion was obtained in 5 h, whereas
at 40 g/L, 50% conversion was achieved in 10 h and 24 h
at 60 g/L. Space–time yield calculations show that a con-
centration of 40 g/L is the optimum (6.43 mmol/h). On
The addition of organic co-solvents in ratios ranging from
10% to 50% in aqueous pH 7.0 phosphate buffer medium
was studied and their effect on the rates of hydrolysis and
enantioselectivity are shown in Tables 2 and 3. It was
H
H
N
H
H
N
H
H
N
HO
Ph
AcO
Ph
AcO
Ph
Lipase
pH 7
+
O
O
O
OCH3
OCH3
OCH3
(+)-1
(+)-1
(-)-9
Scheme 3.

1062
N. Anand et al. / Tetrahedron: Asymmetry 18 (2007) 1059–1069
60
55
50
45
40
35
30
25
20
15
10
5
20g/l
40g/l
60g/l
80g/l
100g/l
0
1
2
3
4
5
10
prolonged
Time (hours)
Figure 1. Concentration v/s conversion in DMF using ABL at 26 ꢁC.
increasing the concentrations to 80–100 g/L, no conversion
beyond 35% was observed.
During the hydrolysis of esters 10–12, ABL behaved more or
less in a similar manner as in 1, whereas both PS and PS-C
showed a marked improvement of enantioselectivity as well
as rates of hydrolysis of 11 and 12 in the presence of 10%
DMF. The commercial lipases PS and PS-C hydrolyzed
the racemic propionate ester 10 in only 8 h (ꢀ50% conver-
sion, ee 99%) whereas it took more than 24 h to hydrolyze
the racemic acetoxy derivative 1. A significant improvement
in the rates of hydrolysis with higher esters for both PS and
PS-C was probably missed by other workers.
In addition to the above studies, the effect of the size of acyl
groups at C-3 were also investigated for the resolution of
racemic
cis-3-acetoxy-1-(4-methoxyphenyl)-4-phenyl-2-
azetidinone 1 with respect to different lipases. Therefore,
the alkyl acylates, that is, cis-3-propyloxy-1-(4-methoxy-
phenyl)-4-phenyl-2-azetidinone 10, cis-3-butyloxy-1-(4-
methoxyphenyl)-4-phenyl-2-azetidinone 11, and cis-3-hex-
yloxy-1-(4-methoxyphenyl)-4-phenyl-2-azetidinone 12 were
prepared and subjected to kinetic resolution using all four
lipases (Scheme 4, Table 4).
In order to fully explore the ability of the native strain bear-
ing lipase from Arthrobacter sp., a unit wise comparative
H
H
N
R
O
O
O
(3R,4S)
OCH3
H
H
H
H
N
Lipase
pH 7
R
O
HO
1, (RCO)2O, DMAP
2, R'COOH, DCC
+
O
N
O
O
H
H
HO
OCH3
10-12
9
OCH3
N
O
10, R = C2H5
11, R = C3H7
12, R = C5H11
OCH3
(3S,4R)
Scheme 4.
Table 4. Effect of alky acyloxy groups with 10% DMF at 26 ꢁC
Enzyme
Propyloxy (10)
Butyloxy (11)
Hexyloxy (12)
T (h)
C (%)
ees
eep
T (h)
C (%)
ees
eep
T (h)
C (%)
ees
eep
AS
PS
PSC
ABL
8
8
8
8
34
44
48
49
24
89
96
99
78
99
99
99
48
48
48
48
0
47
49
21
—
92
99
27
—
99
99
92
24
24
24
24
0
49
46
48
—
99
87
<5
—
99
99
<5

N. Anand et al. / Tetrahedron: Asymmetry 18 (2007) 1059–1069
1063
Table 5. Unit wise comparative study of ABL on ( )-1 at 26 ꢁC
Table 6. Kinetic resolution of ( )-cis-3-acetoxy-1-(4-methoxyphenyl)-4-
(2-furyl)-2-azetidinone ( )-6 (30 ꢁC)
Enzyme
Conversion percentage
Enzyme
AS
Co-solvent
T (h)
C (%)
ees
eep
E
1 h 2 h 3 h 4 h 5 h 6 h
Nil
DMF
DMSO
24
24
24
23
44
38
5
45
24
27
58
41
<5
6
<5
ABL (wet pellet)
9
18
42
21
13
33
49
31
20
41
50
34
26
49
50
39
34
50
50
42
38
ABL (lyophilized whole cells) 26
ABL (cell free)
ABL (cell free, lyophilized)
19
8
PS
Nil
DMF
DMSO
24
24
24
27
50
43
41
99
85
99
99
99
285
>1000
449
Reactions were carried out using ꢀ40,000 units in each experiment.
PS-C
ABL
Nil
DMF
DMSO
15
15
15
41
50
50
69
99
99
99
99
99
412
>1000
>1000
study was carried out by using (a) whole cells wet pellet; (b)
whole cells lyophilized; (c) cell free extract; and (d) cell free
lyophilized powder.
Nil
DMF
DMSO
24
24
20
30
50
50
45
98.5
99
99
98.5
99
302
650
>1000
The results of these studies clearly show that ABL prepared
in the form of whole cells lyophilized powder was the most
suitable to affect the enantioselective hydrolysis as shown
in Table 5.
acid side chain of paclitaxel analogue 3 [MAC-321(TL-
00139)]. Racemate 6 was subjected to kinetic resolution
studies using the panel of lipases in the presence of organic
co-solvents (Scheme 5) and the results are summarized in
Table 6. It is apparent from these results that ABL as well
as PS-C were the most suitable lipases for producing the
desired ester with high enantiopurity (E = 1057).
As already discussed, a new generation of taxanoids such as
3–5 are undergoing clinical trials, therefore the synthesis
and resolution to obtain the optically active amino acid side
chain of these analogues is a challenging task. Moreover, it
was also our endeavor to explore the stereoselective hydro-
lytic capability of ABL as well as other commercial lipases
with respect to other important azetidinones. The racemic
azetidinones, that is, cis-3-acetoxy-1-(4-methoxyphenyl)-4-
(2-furyl)-2-azetidinone 6, cis-3-acetoxy-1-(4-methoxyphen-
yl)-4-(2-thienyl)-2-azetidinone 7, and cis-3-acetoxy-1-(4-
methoxyphenyl)-4-t-butyl-2-azetidinone 8 were synthesized
for kinetic resolution studies and development of amino
acid side chains having 2-furyl, 2-thienyl and t-butyl groups,
respectively, at C-4.
On the basis of the reported specific rotation values, the
absolute configuration at C-3 was also established as (R)
in case of unhydrolyzed esters and (S) in case of the alcohols.
2.3. Resolution of ( )-cis-3-acetoxy-1-(4-methoxyphenyl)-4-
(2-thienyl)-2-azetidinone 7
Racemic cis-3-acetoxy-1-(4-methoxyphenyl)-4-(2-thienyl)-
2-azetidinone 7 is another important precursor of paclit-
axel analogue 4 [MST-997 (TL-909)]. Its racemate was also
successfully resolved using ABL and other lipases in the
presence of organic co-solvents (Scheme 6) to produce
(+)-7 and (ꢁ)-14, respectively. The results of these experi-
ments are summarized in Table 7. Here again ABL clearly
2.2. Resolution of ( )-cis-3-acetoxy-1-(4-methoxyphenyl)-4-
(2-furyl)-2-azetidinone 6
Enantiomerically pure cis-3-acetoxy-1-(4-methoxyphenyl)-
4-(2-furyl)-2-azetidinone 6 is the precursor of the amino
AcO
O
AcO
O
HO
O
O
O
Lipases
+
N
O
N
N
O
O
O
(-)-13
(+)-6
6
Scheme 5.
AcO
S
AcO
O
HO
O
S
S
Lipases
+
N
O
N
N
O
O
O
(-)-14
(+)-7
7
Scheme 6.

1064
N. Anand et al. / Tetrahedron: Asymmetry 18 (2007) 1059–1069
Table 7. Kinetic resolution of ( )-cis-3-acetoxy-1-(4-methoxyphenyl)-4-
(2-thienyl)-2-azetidinone 7 at 30 ꢁC
Table 8. Kinetic resolution of ( )-cis-3-acetoxy-4-t-butyl-2-azetidinone 15
(22 ꢁC)
Enzyme
AS
Co-solvent
T (h)
C (%)
ees
eep
E
Enzyme
AS
Co-solvent
T (h)
C (%)
ees
eep
E
Nil
8
8
8
8
30
48
48
49
42
83
76
84
80
86
83
87
13
Nil
CH3CN
DMF
4
4
4
41
22
26
68
12
4
99
99
99
8
<5
<5
CH3CN
DMF
DMSO
32
25
38
PS
Nil
CH3CN
DMF
4
4
4
48
50
50
98
99
99
98
99
99
311
PS
Nil
8
8
8
8
36
44
47
50
58
79
89
92
98
98
99
93
173
232
581
94
>1000
>1000
CH3CN
DMF
DMSO
PS-C
ABL
Nil
CH3CN
DMF
4
4
4
50
50
50
99
99
99
99
99
99
>1000
>1000
>1000
PS-C
ABL
Nil
8
8
8
8
36
49
48
47
55
95
87
86
98
98
96
97
173
357
146
183
CH3CN
DMF
DMSO
Nil
CH3CN
DMF
4
4
4
50
50
50
99
99
99
99
99
99
>1000
>1000
>1000
Nil
8
8
8
8
32
46
48
47
47
85
92
89
99
99
99
99
316
536
645
581
CH3CN
DMF
DMSO
The problem of hydrolyzing azetidinone derivative 8 was,
however, resolved by removing the p-methoxyphenyl group
on reacting with ammonium ceric nitrate (CAN) 15. The
resulting cis-3-acetoxy-4-t-butyl-2-azetidinone 15 was now
easily amenable to ABL, PS, and PS-C. Thus racemic 15
was hydrolyzed stereoselectively to produce enantiomers
(+)-15 and (ꢁ)-16 in 99% ee after 4 h (Scheme 8, Table 8).
scored over other lipases, thus affecting 48% conversion in
only 8 h (E = 645).
2.4. Resolution of ( )-cis-3-acetoxy-4-t-butyl-2-azetidinone
15
3. Conclusions
Racemic
cis-3-acetoxy-1-(4-methoxyphenyl)-4-t-butyl-2-
azetidinone 8, which is a side chain precursor of an oral
taxane known as Taxotere,18 was also subjected to lipase
catalyzed kinetic resolution. It was observed that all the lip-
ases used were unable to hydrolyze it, including ABL which
could hydrolyze up to 10% in 16 h with very low ee (<5%).
The low acceptance of 8 by lipases is probably due to the
steric effect of the t-butyl group (Scheme 7).
In conclusion the hydrolytic activity of a series of lipases
toward 2-azetidinone derivatives which are important pre-
cursors of amino acid side chain of paclitaxel. 2-Azetidi-
none derivatives with varying substitutions at C-3 were
synthesized and kinetically resolved using lipases. The
kinetic resolutions of ( )-cis-3-acetoxy-1-(4-methoxy-
phenyl)-4-phenyl-2-azetidinone,
( )-cis-3-acetoxy-1-(4-
methoxyphenyl)-4-(2-thienyl)-2-azetidinone, and ( )-cis-3-
acetoxy-1-(4-methoxyphenyl)-4-(2-furyl)-2-azetidinone were
achieved using ABL with ee ꢀ99%. The use of co-sol-
vents, such as CH3CN, DMF, and DMSO, dramatically
improved the enantioselectivity and reduced reaction tim-
ings. Taxotere side chain intermediate ( )-cis-3-acetoxy-
1-(4-methoxyphenyl)-4-t-butyl-2-azetidinone, which could
not be directly hydrolyzed by any one of the lipases used,
was effectively resolved (ee 99%) after its conversion to
( )-cis-3-acetoxy-4-t-butyl-2-azetidinone. In conclusion
Arthrobacter sp. (ABL), the native strain bearing lipase
H
H
N
O
O
Products
(with low EE)
Arthrobacter sp.
O
8
OCH3
pH 7
Lipase
No hydrolysis
Scheme 7.
H
H
O
O
NH
O
H
H
N
H
H
O
O
O
O
(+)-15
Lipase
pH 7
(3R,4S)
CAN
O
O
NH
+
8
15
H
H
HO
O
OCH3
NH
(-)-16
(3S,4R)
Scheme 8.

N. Anand et al. / Tetrahedron: Asymmetry 18 (2007) 1059–1069
1065
was found to be the most effective that could kinetically
4.4. General method of preparation of the imines
resolve all the substrates used in the present study with high
ee.
An imine from the benzaldehyde and p-anisidine was pre-
pared by irradiating for 3 min an equimolar solution
(50 mmol) in ethanol (50 mL) in a commercial microwave
oven at 150 W, and the rest were prepared by refluxing in
toluene (100 mL). The water formed during the reaction
was removed azeotropically using Dean–Stark apparatus.
Excess of the solvent was removed and the imines were
stored under moisture free conditions and used as such
without further purification.
4. Experimental
4.1. General
NMR spectra were recorded on Bruker 200 MHz and
500 MHz spectrometers in the indicated solvents with
tetramethylsilane as the internal standard. IR was recorded
on FT-IR Bruker 270-30 spectrophotometer. Optical rota-
tions were measured with a Perkin–Elmer Model 241
polarimeter in the indicated solvents. MS were recorded
on Bruker esquire 3000 and Thermo-Finnigan TSQ 5000.
GC–MS was carried out on Shimadzu GC–MS QP2000.
Elemental analysis was performed on an Elementar CHNS
analyzer. Melting points were determined by open capillary
method on Buchi B-542 apparatus and are uncorrected.
TLC was performed on Silica gel 60 F254 (Merck), using
ethyl acetate–hexane (20:80, 30:70, or 40:60) as mobile
phase. Chiral HPLC was performed on Shimadzu and
Thermo-Finnigan equipments using (R,R) Whelk-O1,
Chiralcel OJ-H and OD-H chiral columns, and hexane–
isopropyl alcohol–acetic acid (97:3:0.1), hexane–ethyl
alcohol (98.1:1.9) and hexane–isopropyl alcohol–acetic
acid (95:5:0.1) as mobile phases. The UV detection was
done at 210, 254, and 260 nm.
4.5. ( )-cis-3-Acetoxy-1-(4-methoxyphenyl)-4-phenyl-2-aze-
tidinone 1
Triethylamine (1.51 g, 15.0 mmol) was slowly added to a
stirred mixture of imine (from benzaldehyde and p-anisi-
dine) (2.11 g, 10.0 mmol) in dry dichloromethane (75 mL)
at 0–5 ꢁC. Acetoxyacetyl chloride (1.71 g, 10.0 mmol) dis-
solved in 50 mL dichloromethane was added over a period
of 30 min while maintaining the temperature between 0 and
5 ꢁC and stirring continued for 8 h at room temp. After the
reaction completed (TLC), the mixture was washed with
5% aq NaHCO3 (50 mL), 5% HCl (50 mL), and water
(3 · 50 mL). The organic layer was dried and evaporated
under reduced pressure. The product was purified using
column chromatography (230–400 mesh silica gel and
dichloromethane and hexane). Yield: 2.7 g (87%), mp
160–162 ꢁC. IR (KBr): 3476, 2953, 1744, 1515, 1399,
1371, 1223, 1107, 1025 cmꢁ1. 1H NMR (500 MHz, CDCl3):
d 1.69 (s, 3H, –OCOCH3), 3.75 (s, 3H, –OCH3), 5.35 (d,
1H, J = 4.82 Hz, C4–H), 5.94 (d, 1H, J = 4.83 Hz, C3–H),
6.81 (d, 2H, J = 8.91 Hz, Ar–H), 7.29 (d, 2H,
J = 8.92 Hz, Ar–H), 7.34 (s, 5H, Ar–H). 13C NMR
(125 MHz): d 19.8, 55.5, 61.5, 76.4, 114.4, 118.8, 127.9,
128.5, 128.8, 130.1, 132.3, 156.0, 161.3 172.0. MS (m/z):
311, 212, 167, 162, 149, 120. Anal. Calcd for C18H17NO4:
C, 69.44; H, 5.50; N, 4.50. Found: C, 69.30; H, 5.47; N,
4.51.
4.2. Preparation of the cell biomass of ABL
The microorganism Arthrobacter sp. (ABL) was isolated
from the fresh water samples collected from Jammu hills
and has been deposited in MTCC culture collection at
IMT, Chandigarh (MTCC No. 5125) under Budapest
Treaty (2004).
ABL cell biomass was prepared in 10 L fermentor contain-
ing medium (1% peptone, and 0.5% NaCl and 0.5% beef
extract, pH 7.0). The medium was inoculated with an over-
night preculture prepared in the same broth. The culture
was grown at 30 ꢁC for 16–18 h at 200 rpm. The cell pellet
was separated from the broth by centrifugation at 10,000g
for 15 min at 4 ꢁC. The cell pellet was preserved at ꢁ20 ꢁC
till further use. The moisture contents in the wet cell pallet
was estimated as 75–80%.
4.6. ( )-cis-3-Acetoxy-4-(2-furyl)-1-(4-methoxyphenyl)-2-
azetidinone 6
Compound 6 was prepared from triethylamine (3.0 g,
30.0 mmol), imine (from furfural and p-anisidine) (2.01 g,
10.0 mmol), and acetoxyacetyl chloride (1.71 g, 10.0 mmol)
following the above mentioned procedure for 1 and main-
taining the temperature at ꢁ5 to 0 ꢁC. Yield: 2.7 g (90%),
mp 159–161 ꢁC. IR (KBr): 3476, 2959, 1743, 1516,
4.3. ABL enzyme isolation
1
1378 cmꢁ1. H NMR (500 MHz, CDCl3): d 1.93 (s, 3H,
Lipase from Arthrobacter sp. was obtained by ultrasonica-
tion of cells (1 U/mg wet biomass) in phosphate buffer, pH
7.0 using MSE Manor Roya Crawley RH 10 2QQ cell dis-
rupter at 16 kHz. Cell free extract (CFE) as crude enzyme
preparation was used directly. The CFE obtained from the
above method was partially purified by 60% ammonium
sulphate precipitation. The precipitates were then dissolved
in phosphate buffer (0.1 M, pH 7.0) and dialyzed against
the phosphate buffer (10 mM, pH 7.0). The partially puri-
fied enzyme was lyophilized (specific activity 40 units per
mg protein) and stored at ꢁ20 ꢁC till use.
–OCOCH3), 3.76 (s, 3H, –OCH3), 5.39 (d, 1H,
J = 4.72 Hz, C4–H), 5.95 (d, 1H, J = 4.73 Hz, C3–H),
6.39 (dd, 1H, J = 3.19 and 1.36 Hz, furyl C4–H), 6.43 (d,
1H, J = 3.26 Hz, furyl C3–H), 6.83 (d, 2H, J = 9.00 Hz,
Ar–H), 7.29 (d, 2H, J = 9.00 Hz, Ar–H), 7.45 (s, 1H,
J = 0.97 Hz, furyl C5–H). 13C NMR (125 MHz): d 20.0,
55.3, 55.4, 76.1, 110.8, 110.8, 114.4, 118.6, 130.3, 143.7,
146.8, 156.7, 161.0, 169.6. MS (m/z): 301, 260, 242, 232,
230, 214, 202, 186, 153, 111. Anal. Calcd for C16H15NO5:
C, 63.78; H, 5.02; N, 4.65. Found: C, 63.72; H, 5.00; N,
4.61.

1066
N. Anand et al. / Tetrahedron: Asymmetry 18 (2007) 1059–1069
4.7. ( )-cis-3-Acetoxy-1-(4-methoxyphenyl)-4-(2-thienyl)-2-
azetidinone 7
Calcd for C16H15NO3: C, 71.36; H, 5.61; N, 5.20. Found:
C, 71.29; H, 5.60; N, 5.18.
Compound 7 was prepared from triethylamine (1.51 g,
15.0 mmol), imine (from thienaldehyde and p-anisidine)
(2.17 g, 10.0 mmol), and acetoxyacetyl chloride (1.71 g,
10.0 mmol), following the method of the preparation of 1
while maintaining the temperature from ꢁ5 to 0 ꢁC. Yield:
2.65 g (84%), mp 148–149 ꢁC. IR (KBr): 3479, 2985, 1748,
4.10. ( )-cis-3-Propyloxy-1-(4-methoxyphenyl)-4-phenyl-2-
azetidinone 10
A solution of ( )-cis-3-hydroxy-1-(4-methoxyphenyl)-4-
phenyl-2-azetidinone (1.0 g, 3.72 mmol), propionic anhy-
dride (520 mg, 4.0 mmol), and DMAP (10 mg) in dichloro-
methane (20 mL) was kept overnight at room temperature.
After the completion of the reaction, the contents were
poured into ice-cold water and extracted with dichloro-
methane (3 · 50 mL). The organic layer was washed, dried,
and evaporated to furnish the crude product which was
further purified by crystallization to give 10. Yield: 1.16 g
(96%), mp 127 ꢁC. IR (KBr): 3473, 1743, 1515, 1401
1514, 1375 cmꢁ1 1H NMR (500 MHz, CDCl3): d 1.85
.
(s, 3H, –OCOCH3), 3.74 (s, 3H, –OCH3), 5.60 (d, 1H,
J = 4.7 Hz, C4–H), 5.94 (d, 1H, J = 4.7 Hz, C3–H), 6.80
(d, 2H, J = 9.0 Hz, Ar–H), 7.0 (dd, 1H, J = 3.59 and
3.60 Hz, thienyl C4–H), 7.09 (d, 1H, J = 2.81 Hz, thienyl
C4–H), 7.30 (d, 2H, J = 9.10 Hz, Ar–H), 7.31 (s, 1H, thie-
nyl C5–H). 13C NMR (125 MHz), 20.0, 55.4, 57.6, 76.5,
114.4, 118.8, 126.8, 127.1, 128.1, 130.1, 135.6, 156.7,
161.0, 169.3. MS (m/z): 317, 218, 217, 202, 173, 168, 149,
126, 97. Anal. Calcd for C16H15NO4S: C, 60.55; H, 4.76;
N, 4.41; S, 10.10. Found: C, 60.56; H, 4.73; N, 4.42; S,
10.08.
1
1243, 1110, 1032, 981 cmꢁ1. H NMR (200 MHz, CDCl3):
d 0.71 (t, 3H, J = 7.56 Hz, –OCOCH2CH3), 1.77–2.10 (m,
2H, –OCOCH2CH3), 3.73 (s, 3H, –OCH3), 5.34 (d, 1H,
J = 4.83 Hz, C4–H), 5.94 (d, 1H, J = 4.83 Hz, C3–H),
6.79 (d, 2H, J = 8.93 Hz, Ar–H), 7.27 (d, 2H,
J = 8.92 Hz, Ar–H), 7.28 (s, 5H, phenyl). 13C NMR
(50 MHz, CDCl3): d 8.6, 26.8, 55.4, 61.5, 76.1, 114.4,
118.8, 127.9, 128.5, 128.7, 130.3, 132.4, 156.6, 161.5,
172.7. MS (m/z): 325, 212, 196, 176, 149, 134, 120, 91.
Anal. Calcd for C19H19NO4: C, 70.14; H, 5.89; N, 4.30.
Found: C, 70.16; H, 5.87; N, 4.26.
4.8. ( )-cis-3-Acetoxy-4-(t-butyl)-1-(4-methoxyphenyl)-2-
azetidinone 8
Compound 8 was prepared from triethylamine (3.02 g,
30.0 mmol), imine (pivaldehyde and p-anisidine) (1.91 g,
10.0 mmol),
and
acetoxyacetyl
chloride
(1.71 g,
10.0 mmol), following the above mentioned procedure for
1 and maintaining temperature at –78 ꢁC. Yield: 2.1 g
(72%), mp 187–189 ꢁC. IR (KBr): 2964, 1758, 1740, 1516,
4.11. ( )-cis-3-Butyloxy-1-(4-methoxyphenyl)-4-phenyl-2-
azetidinone 11
1371, 1228, 1181, 1121, 1031, 824 cmꢁ1
.
1H NMR
It was prepared from ( )-cis-3-hydroxy-1-(4-methoxyphe-
nyl)-4-phenyl-2-azetidinone (1.0 g, 3.72 mmol), butyric
anhydride (630 mg, 4.0 mmol), and DMAP (10 mg) in
dichloromethane (20 mL) following the procedure for 10.
Yield: 1.2 g (95%), mp 129 ꢁC. IR (KBr): 3477, 1747,
(200 MHz, CDCl3): d 0.97 (s, 9H, t-butyl), 2.14 (s, 3H,
–OCOCH3), 3.75 (s, 3H, –OCH3), 4.22 (d, 1H,
J = 5.36 Hz, C4–H), 6.12 (d, 1H, J = 5.36 Hz, C3–H),
6.85 (d, 2H, J = 8.95 Hz, Ar–H), 7.27 (d, 2H,
J = 8.95 Hz, Ar–H). 13C NMR (125 MHz): d 20.9, 27.0,
34.6, 55.5, 66.9, 73.5, 114.3, 122.3, 130.0, 157.2, 164.0,
169.4. MS (m/z): 292, 291, 192, 165, 164, 150, 149, 134.
Anal. Calcd for C16H21NO4: C, 65.96; H, 7.27; N, 4.81.
Found: C, 65.93; H, 7.22; N, 4.78.
1519, 1456, 1402 1245, 1111, 1029, 842, 805 cmꢁ1 1H
.
NMR (200 MHz, CDCl3): d 0.61 (t, 3H, J = 7.34 Hz,
–OCOCH2CH2CH3), 1.11–1.25 (h, 2H, –OCOCH2-
CH2CH3), 1.75–2.05 (m, 2H, –OCOCH2CH2CH3), 3.73
(s, 3H, –OCH3), 5.33 (d, 1H, J = 4.85 Hz, C4–H), 5.94
(d, 1H, J = 4.85 Hz, C3–H), 6.80 (d, 2H, J = 8.98 Hz,
Ar–H), 7.28 (d, 2H, J = 8.96 Hz, Ar–H), 7.30 (s, 5H,
phenyl). 13C NMR (50 MHz): d 13.3, 17.8, 35.2, 55.4,
61.6, 76.2, 114.4, 118.8, 127.9, 128.5, 128.7, 130.3, 132.4,
156.6, 161.5, 171.9. MS (m/z): (M+1) 340, 213, 197, 149,
134, 120, 91, 71. Anal. Calcd for C20H21NO4: C, 70.78;
H, 6.24; N, 4.13. Found: C, 70.71; H, 6.20; N, 4.11.
4.9. ( )-cis-3-Hydroxy-1-(4-methoxyphenyl)-4-phenyl-2-
azetidinone 9
cis-3-Acetoxy-1-(4-methoxyphenyl)-4-phenyl-2-azetidinone
(4 g, 12.8 mmol) dissolved in 250 mL of THF was added
dropwise over a period of 1 h to a stirring mixture of
1 M KOH (240 mL) and THF (160 mL) at 0 ꢁC. The reac-
tion mixture was stirred further for another hour. After
completion of the reaction, it was diluted with 200 mL of
THF and saturated solution of NaHCO3, extracted
with ethyl acetate (3 · 200 mL), washed, dried, and evapo-
rated under reduced pressure to give 9. Yield: 3.36 g
(97%), mp 210–212 ꢁC. IR (KBr): 3309, 1719, 1514, 1405,
4.12. ( )-cis-3-Hexyloxy-1-(4-methoxyphenyl)-4-phenyl-2-
azetidinone 12
A solution of ( )-cis-3-hydroxy-1-(4-methoxyphenyl)-4-
phenyl-2-azetidinone (1.0 g, 3.72 mmol), caproic acid
(500 mg, 4.3 mmol), and DCC (in excess) in dichlorometh-
ane (100 mL) was stirred at room temperature for 24 h.
After completion of the acylation reaction, the contents
were passed through a pad of silica (230–400 mesh). The
solution upon evaporation furnished the crude product
which on purification by crystallization gave 12. Yield:
1.3 g (95%), mp 110 ꢁC. IR (KBr): 3427, 1747, 1517,
1251, 1181, 1116, 1029 cmꢁ1
.
1H NMR (500 MHz,
CDCl3+DMSO-d 6) d 3.74 (s, 3H, –OCH3), 5.16 (d, 1H,
J = 4.7 Hz, C4–H), 5.19 (d, 1H, J = 4.7 Hz, C3–H), 6.78
(d, 2H, J = 9.01 Hz, Ar–H), 7.28 (d, 2H, J = 9.01 Hz,
Ar–H), 7.35 (m, 5H, Ar–H), 13C NMR (125 MHz), 55.3,
62.6, 77.0, 114.2, 118.6, 127.8, 128.1, 128.4, 130.8, 134.1,
156.1, 166.3. MS (m/z): 269, 212, 196, 167, 149, 120. Anal.
1455, 1400, 1244, 1157, 1111, 1065, 986, 835, 698 cmꢁ1
.

N. Anand et al. / Tetrahedron: Asymmetry 18 (2007) 1059–1069
1067
1H NMR (500 MHz, CDCl3): d 0.77 (t, 3H, J = 7.3 Hz,
–OCO(CH2)4CH3), 0.90–0.96 (m, 2H, –OCO(CH2)3-
CH2CH3), 1.07–1.16 (m, 2H, –OCO(CH2)2CH2CH2CH3),
1.81–1.87 (m, 2H, –OCOCH2CH2(CH2)2CH3), 1.97–2.03
(m, 2H, –OCOCH2(CH2)3CH3), 3.72 (s, 3H, –OCH3),
5.31 (d, 1H, J = 4.77 Hz, C4–H), 5.92 (d, 1H,
J = 4.79 Hz, C3–H), 6.78 (d, 2H, J = 8.71 Hz, Ar–H),
7.25 (d, 2H, J = 8.62 Hz, Ar–H), 7.30 (s, 5H, phenyl).
13C NMR (125 MHz): d 13.7, 22.1, 23.9, 30.8, 33.3,
55.4, 61.5, 76.1, 114.4, 118.8, 127.9, 128.3, 128.8, 130.3,
132.4, 156.6, 161.5, 172.1. MS (m/z): 367, 218, 213, 196,
149, 134, 120, 99, 91. Anal. Calcd for C22H25NO4:
C, 71.91; H, 6.86; N, 3.81. Found: C, 71.86; H, 6.82; N,
3.80.
sure to furnish a mixture comprising hydrolyzed alcohol
and unhydrolyzed ester, which was separated by column
chromatography (230–400 mesh silica gel using dichloro-
methane and hexane as eluent) giving (+)-cis-(3R,4S)-3-
acetoxy-1-(4-methoxyphenyl)-4-phenyl-2-azetidinone (+)-
25
1 (180 mg, 90%) having ee >99.5%, ½aꢂD ¼ þ8:1 (c 1.0,
CHCl3) and hydrolyzed alcohol (ꢁ)-cis-(3S,4R)-3-hydr-
oxy-1-(4-methoxyphenyl)-4-phenyl-2-azetidinone
(160 mg, 92%), ee >99.5% (chiral HPLC), ½aꢂD ¼ ꢁ179:0
(c 1.0, CHCl3).
(ꢁ)-9
25
5.2. (+)-cis-(3R,4R)-3-Acetoxy-1-(4-methoxyphenyl)-4-(2-
furyl)-2-azetidinone (+)-6
A mixture of racemic ( )-cis-3-acetoxy-1-(4-methoxyphen-
yl)-4-(2-furyl)-2-azetidinone (200 mg), aqueous phosphate
buffer (9 mL, 0.1 M, pH 7.0), dimethylsulfoxide (1 mL),
and whole cells of ABL (200 mg) was stirred continuously
at 30 1 ꢁC. After a certain degree of conversion, the reac-
tion was stopped following the above general procedure to
give unhydrolyzed ester (+)-cis-(3R,4R)-3-acetoxy-1-
4.13. ( )-cis-3-Acetoxy-4-(1,1-dimethylethyl)-2-azetidinone
15
A solution of ( )-cis-3-acetoxy-1-(4-methoxyphenyl)-4-
(1,1-dimethylethyl)-2-azetidinone (2.91 g, 10.0 mmol) dis-
solved in 200 mL of acetonitrile was added slowly to a solu-
tion of ceric ammonium nitrate (16.0 g, 30 mmol) in
100 mL of water at 0 ꢁC. The mixture was stirred for 2 h,
diluted with 200 mL water, extracted with ethylacetate
(3 · 100 mL), and neutralized with 5% NaHCO3 solution.
The organic layer was combined, washed with 10%
Na2SO3, 5% NaHCO3, and brine successively, dried over
sodium sulfate, and concentrated under reduced pressure.
The crude oil obtained was chromatographed over a silica
gel column (60–120 mesh using ethylacetate and hexane as
eluent) to give 15. Yield: 1.65 g (89%), mp 68–69ꢁC. IR
(4-methoxyphenyl)-4-(2-furyl)-2-azetidinone (+)-6 (89 mg,
25
89%), ee >99.5%, ½aꢂD ¼ þ13:6 (c 1.0, CHCl3) and hydro-
lyzed alcohol (ꢁ)-cis-(3S,4S)-3-hydroxy-1-(4-methoxy-
phenyl)-4-(2-furyl)-2-azetidinone (ꢁ)-13 (81 mg, 92%), ee
25
>99.5% (chiral HPLC), ½aꢂD ¼ ꢁ222:0 (c 1.0, CHCl3).
5.3. (+)-cis-(3R,4R)-3-Acetoxy-1-(4-methoxyphenyl)-4-(2-
thienyl)-2-azetidinone (+)-7
1
(KBr): 3265, 2919, 1792, 1740, 1375, 1228, 1128 cmꢁ1. H
A mixture of racemic ( )-cis-3-acetoxy-1-(4-methoxy-
phenyl)-4-(2-thienyl)-2-azetidinone (200 mg), aqueous phos-
phate buffer (9 mL, 0.1 M, pH 7.0), dimethylformamide
(1 mL), and whole cells of ABL (200 mg) was stirred con-
tinuously at 30 1 ꢁC. After a certain degree of conversion
the reaction was stopped following the above general
procedure to give unhydrolyzed ester (+)-cis-(3R,4R)-3-
NMR (200 MHz, CDCl3): d 0.98 (s, 9H, t-butyl), 2.16 (s,
3H, –OCOCH3), 3.61 (d, 1H, J = 5.09 Hz, C4–H), 5.99
(d, 1H, J = 5.07 Hz, C3–H). 13C NMR (50 MHz): d 20.8,
26.0, 32.8, 63.2, 74.8, 166.6, 169.3. MS (m/z): 186, 142,
127, 100, 85. Anal. Calcd for C9H15NO3: C, 58.36; H,
8.16; N, 7.56. Found: C, 58.33; H, 8.11; N, 7.57.
acetoxy-1-(4-methoxyphenyl)-4-(2-thienyl)-2-azetidinone
25
(+)-7 (91 mg, 90%) having ee >99%, ½aꢂD ¼ þ4:5 (c 0.94,
5. General method for enzyme catalyzed resolution of
2-azetidinones
CHCl3) and hydrolyzed alcohol (ꢁ)-cis-(3S,4S)-3-hydro-
xy-1-(4-methoxyphenyl)-4-(2-thienyl)-2-azetidinone (ꢁ)-14
25
(81 mg, 91%), ee 99% (chiral HPLC), ½aꢂD ¼ ꢁ162:8 (c
5.1. (+)-cis-(3R,4S)-3-Acetoxy-1-(4-methoxyphenyl)-4-
phenyl-2-azetidinone (+)-1
0.42, CHCl3).
Racemic ( )-cis-3-acetoxy-1-(4-methoxyphenyl)-4-phenyl-
2-azetidinone (400 mg) was added to a solution of aqueous
phosphate buffer (18 mL, 0.1 M, pH 7.0) and dimethyl-
formamide (2.0 mL). A wet pallet of whole cell preparation
of ABL (400 mg, 1100 units/mg) was added to the above
solution with continuous stirring while maintaining tem-
perature at 26 1 ꢁC. Thin-layer chromatography (TLC)
and chiral high performance liquid chromatography
(HPLC) was carried out to monitor the progress of the
reaction after every hour. The completed reaction (5–6 h
approx., 50% conversion) was terminated by adding ethyl
acetate and centrifuged to remove cells and the suspended
particles. The decanted clear solution and the centrifuged
mass were extracted separately with ethyl acetate
(3 · 40 mL). The organic layers combined and washed with
water and dried. The solvent removed under reduced pres-
5.4. (+)-cis-(3R,4S)-3-Propyloxy-1-(4-methoxyphenyl)-4-
phenyl-2-azetidinone (+)-10
A mixture of racemic ( )-cis-3-propyloxy-1-(4-methoxy-
phenyl)-4-phenyl-2-azetidinone (400 mg), aqueous phos-
phate buffer (18 mL, 0.1 M, pH 7.0), dimethylformamide
(2 mL), and whole cells of ABL (400 mg) was stirred con-
tinuously at 25 1 ꢁC. After a certain degree of conversion
the reaction was stopped following the above general
procedure to get unhydrolyzed ester (+)-cis-(3R,4S)-3-pro-
pyloxy-1-(4-methoxyphenyl)-4-phenyl-2-azetidinone (+)-
25
10 (175 mg, 87%), ee >99.5%, ½aꢂD ¼ þ3:5 (c 0.6, CHCl3)
and hydrolyzed alcohol (ꢁ)-cis-(3S,4R)-3-hydroxy-1-(4-
methoxyphenyl)-4-phenyl-2-azetidinone (ꢁ)-9 (160 mg,
25
92%), ee >99.5% (chiral HPLC), ½aꢂD ¼ ꢁ190 (c 0.4,
CHCl3).

1068
N. Anand et al. / Tetrahedron: Asymmetry 18 (2007) 1059–1069
5.5. (+)-cis-(3R,4S)-3-Butyloxy-1-(4-methoxyphenyl)-4-
phenyl-2-azetidinone (+)-11
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A mixture of racemic ( )-cis-3-butyloxy-1-(4-methoxyphen-
yl)-4-phenyl-2-azetidinone (400 mg), aqueous phosphate
buffer (18 mL, 0.1 M, pH 7.0), dimethylformamide
(2 mL), and lipase PS-C (400 mg) was stirred continuously
at 25 1 ꢁC. After a certain degree of conversion, the reac-
tion was stripped following the above general procedure to
obtain unhydrolyzed ester (+)-cis-(3R,4S)-3-butyloxy-1-
(4-methoxyphenyl)-4-phenyl-2-azetidinone (+)-11 (180 mg,
25
90%) having ee >99.5%, ½aꢂD ¼ þ7:6 (c 0.54, CHCl3)
and hydrolyzed alcohol (ꢁ)-cis-(3S,4R)-3-hydroxy-1-
(4-methoxyphenyl)-4-phenyl-2-azetidinone (ꢁ)-9 (159 mg,
25
92%), ee >99.5% (chiral HPLC), ½aꢂD ¼ ꢁ174:0 (c 1.0,
CHCl3).
5.6. (+)-cis-(3R,4S)-3-Hexyloxy-1-(4-methoxyphenyl)-4-
phenyl-2-azetidinone (+)-12
A mixture of racemic ( )-cis-3-hexyloxy-1-(4-methoxyphen-
yl)-4-phenyl-2-azetidinone (400 mg), aqueous phosphate
buffer (18 mL, 0.1 M, pH 7.0), dimethylformamide
(2 mL), and lipase PS (400 mg) was stirred continuously
at 25 1 ꢁC. After a certain degree of conversion, the reac-
tion was stopped following the above general procedure to
obtain unhydrolyzed ester (+)-cis-(3R,4S)-3-hexyloxy-1-(4-
4. (a) Carr, J. A.; Al-Azemi, T. F.; Shim, J.-Y.; Coates, C. M.;
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methoxyphenyl)-4-phenyl-2-azetidinone (+)-12 (173 mg,
25
88%), ee >99.5%, ½aꢂD ¼ þ11:6 (c 1.0, CHCl3) and hydro-
6. Gueritte-Voegelein, F.; Senilh, V.; David, B.; Guenad, D.;
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phenyl)-4-phenyl-2-azetidinone (ꢁ)-9 (161 mg, 92%)
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25
having ee >99.5% (chiral HPLC), ½aꢂD ¼ ꢁ174:0 (c 1.0,
CHCl3).
5.7. Kinetic resolution of ( )-cis-3-acetoxy-4-(1,1-dimethyl-
ethyl)-2-azetidinone 15
A mixture of racemic ( )-cis-3-acetoxy-4-(1,1-dimethyl-
ethyl)-2-azetidinone (130 mg, 0.72 mmol) and whole cells
of ABL (150 mg) in aqueous phosphate buffer (4 mL,
0.1 M, pH 7.0) with the continuous stirring at 20 1 ꢁC.
After a certain degree of conversion, the reaction was ter-
minated and contents extracted with ethyl acetate
(3 · 20 mL). The organic phase was washed with water,
dried over sodium sulfate and concentrated in vacuo to
give the crude product, which was heated in a mixture of
toluene–heptane (55:45) for 3 h. The reaction mixture on
cooling gave (+)-cis-3-acetoxy-4-(1,1-dimethylethyl)-2-aze-
25
tidinone (+)-15 (56 mg, 86%), ee 98.5%, ½aꢂD ¼ þ67:4
(c 1.1, CHCl3) recovered from the organic layer and (ꢁ)-
cis-3-hydroxy-4-(1,1-dimethylethyl)-2-azetidinone (ꢁ)-16
25
(42 mg, 82%), ee 99%, ½aꢂD ¼ ꢁ91:2 (c 0.6, CH3OH) as a
white solid.
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Acknowledgments
The authors are thankful to M/s AMANO Enzymes Inc.
Japan, for the gift of AS, PS, and PS-C samples and the
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