The dynamic kinetic resolution of 3-oxo-4-phenyl-β-lactam by recombinant E. coli overexpressing yeast reductase Ara1p

Article abstract of DOI:10.1016/j.tetasy.2005.07.029

Using a recombinant E. coli strain overexpressing yeast reductase Ara1p, we reduced racemic 3-oxo-4-phenyl-β-lactam to cis-(3S,4R)-3-hydroxy-4-phenyl- β-lactam as a single enantiopure product. The dynamic kinetic resolution occurred over the course of fermentation at pH 7. Under the same conditions, 3-oxo-4-(2-thiophenyl)-β-lactam 4 and 3-oxo-4-(2-furyl)-β-lactam 5 were not resolved.

Full text of DOI:10.1016/j.tetasy.2005.07.029

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 16 (2005) 2748–2753
The dynamic kinetic resolution of 3-oxo-4-phenyl-b-lactam
by recombinant E. coli overexpressing yeast reductase Ara1p
Yan Yang, Melissa Drolet and Margaret M. Kayser*
Department of Physical Sciences, University of New Brunswick, PO Box 5050, Saint John, New Brunswick, Canada E2L 4L5
Received 9 June 2005; accepted 20 July 2005
Abstract—Using a recombinant E. coli strain overexpressing yeast reductase Ara1p, we reduced racemic 3-oxo-4-phenyl-b-lactam to
cis-(3S,4R)-3-hydroxy-4-phenyl-b-lactam as a single enantiopure product. The dynamic kinetic resolution occurred over the course
of fermentation at pH 7. Under the same conditions, 3-oxo-4-(2-thiophenyl)-b-lactam 4 and 3-oxo-4-(2-furyl)-b-lactam 5 were not
resolved.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
the desired enantiomer is theoretically limited to 50%.
Efforts to overcome this limitation and to obtain com-
pounds with high enantiomeric purity, as well as high
yields, have led to the growing interest in dynamic
kinetic resolution (DKR), a method that combines enzy-
matic kinetic resolution with the in situ racemization of
the substrate.16
3-Hydroxy-b-lactams substituted at the 4-position are
convenient precursors of b-amino-a-hydroxy esters, the
key fragments of biologically important compounds
such as a-hydroxy-b-amino acids,1,2 peptidic enzyme
inhibitors,3 and pharmaceuticals, in particular the anti-
cancer drug Taxol.4 Enantiomerically pure 3-hydroxy-
b-lactams have been prepared via asymmetric synthesis
employing either a combination of chiral imine and
achiral ketenes,5,6 achiral imines and chiral ketenes,7,8
or the direct cyclization of ketene–imine in the presence
of a chiral catalyst.9 Biocatalytic approaches include
lipase-catalyzed resolution10–12 and bakerÕs yeast
reduction.13
The use of biocatalysis in the preparation of enantiopure
compounds is an attractive alternative to other chemical
methods. Over the years, the enzyme-promoted kinetic
resolution of racemates has been extensively explored
while the number of reported chemoenzymatic DKRs
has been increased.14–16 Recently, this approach has
been successfully applied in microbial reductions17,18
and oxidations.19
The biological activity of a compound often depends on
the correct absolute configuration of all the stereogenic
centres in the molecule. Constructing a molecule with
only two stereogenic centres of the required absolute
configuration can be an arduous undertaking. There
are two main access routes to enantiomerically pure or
enriched compounds: the resolution of racemates and
the asymmetrization of prochiral or meso-compounds.
Biocatalysts have been successfully used in both strate-
gies.14,15 The kinetic resolution of a racemic mixture is
a well-travelled route to optically active compounds;
its major disadvantage is that the maximum yield of
BakerÕs yeast (Saccharomyces cerevisiae) is a rich source
of reducing enzymes. In addition to metabolic enzymes
whose function and physiological substrates are already
known, there is a large contingent of enzymes that re-
duce exogenous ketones. Although several 3-oxo-ketone
reductases had been purified and characterized,20 it was
the availability of the yeast genome sequence that
allowed the identification of 49 putative reductases.21
A number of these proteins were found to correspond
to the already known reductases; many others, however,
open the door to further studies.21 Recently, 19 of the
alleged reductases have been overexpressed in Esche-
richia coli (E. coli) as gluthatione (S)-transferase
fusion proteins, and their ability as enantioselective
reducing agents for a series of b-ketoesters has been
*
Corresponding author. Tel.: +1 506 648 5576; fax: +1 506 648
5948; e-mail: kayser@unbsj.ca
0957-4166/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2005.07.029

Y. Yang et al. / Tetrahedron: Asymmetry 16 (2005) 2748–2753
2749
investigated.22,23 The results of this study have shown
that several of these enzymes are highly enantioselective
Ph
Ph
HO
O
O
O
E. coli / Ara1p
R
S R
fast
N
N
`
and diastereoselective vis-a-vis 2-substituted, non-cyclic
PMP
PMP
b-ketoesters.22,23
2
racemization
Ph
Ph
HO
O
O
O
E. coli / Ara1p
slow
S
S S
2. Results and discussion
N
N
PMP
PMP
We screened several yeast reductases for their ability to
reduce 3-keto-b-lactams to the corresponding alcohols
and identified several that are suitable for development
as reducing agents for these substrates.24 Among the
yeast enzymes which, under screening conditions,
reduced 3-oxo-4-phenyl-b-lactam 1 to the corresponding
alcohol, reductase Ara1p was found to be efficient
and was selected for further studies. The construction
and evaluation of the recombinant E. coli strain
BL21(DE3)/Ara1p (E. coli/Ara1p) have already been re-
ported.25–27 The screening of substrate 1 with E. coli/
Ara1p gave a mixture of (3S,4R)- and (3S,4S)-diastereo-
mers (Scheme 1). Interestingly, these are isomers that are
either only minor products or that are not observed in
bakerÕs yeast-catalyzed reductions of 1, thus suggesting
that Ara1p is not efficiently expressed under these
conditions.
3
Scheme 2.
is stopped at 50% conversion. When kinetic resolution
is accompanied by in situ racemization of the substrate,
the slow-reacting enantiomer is continually converted
into the fast-reacting enantiomer, which is transformed
into a product. Thus, theoretically, DKR is capable of
giving the desired enantiomer in 100% yield from the
racemic substrate. It has been stated that in order to
achieve efficient DKR, the ratio of rate constants for
fast and slow reaction (kF/kS) should be more than 30
while the rate of racemization of the less reactive enan-
tiomer has to be equal or greater than the rate of conver-
sion of the more reactive enantiomer.28 Our experiments
show that in the Ara1p-catalyzed reduction of 1 under
fermentation conditions at constant pH 7, DKR takes
place. On the other hand, no significant DKR is ob-
served when this transformation is performed in a shake
flask where the levels of oxygen and glucose deviate
from the optimal values and pH drops from 7 to
approximately 5 as the reaction progresses. Thus,
Ara1p-catalyzed reduction of substrate 1 meets the crite-
ria only under fermentation conditions, while the fine-
tuning of reaction conditions is necessary to achieve
DKR in the reduction of substrate 1.
Ph
Ph
Ph
HO
O
HO
O
O
O
Biocatalyst
3
4
3
4
3
4
+
N
N
N
Conversion
72 h
PMP
PMP
PMP
1
2
3
Baker's yeast
>98%
81%
(3R,4S) 55% ee
1.6 : 1 (3R,4R) > 99% ee
1.4 : 1
E. coli / Ara1p
(3S,4R) >99% ee
(3S,4S) > 99% ee
(shake-flask)
trace
E. coli / Ara1p
100%
(3S,4R) >98% ee
(fermentation)
To corroborate the racemization of substrate, we moni-
tored, by chiral phase HPLC, the fate of enantiopure
(S)-1 stirred in the reaction medium alone and in the
reaction medium containing host E. coli strain
BL21(DE3). The latter experiment was carried out to
ascertain that the racemization is not caused by any race-
mase present in E. coli. In both experiments, some race-
mization was detected (2% after 1 h and 12% after 3 h).
However, it was accompanied by depletion of the start-
ing material, probably through hydrolysis and decom-
position. After 12 h, all of ketone 1 disappeared. These
results explain why despite apparent Ô100% conversionÕ
low isolated yields have been observed in shake-flask
experiments with several reductases. When a reaction
is carried out in a shake flask, the quantity of the avail-
able enzyme is lower than that under the optimized fer-
menter conditions. At the same time, the enzyme activity
may also be diminished, and racemization of 1 is slower
because the pH of the mixture is decreasing from 7 to
approximately 5. Under these conditions, DKR is not
effective and the yields of alcohols are low because of
the loss of starting material through decomposition.
PMP = p-methoxyphenyl
Scheme 1.
Since the screening protocol performed in a shake flask
is not optimized for maximum conversion, we followed
up with a biotransformation in a small fermenter with a
constant supply of oxygen and glucose with the pH
maintained at 7 throughout the reaction. To our sur-
prise, under these conditions, substrate 1 was completely
converted to a single product identified as alcohol
(3S,4R)-2. Only traces of the diastereomer (3S,4S)-2
could be detected on chiral phase HPLC. The alcohol
(3S,4R)-2 was isolated in 65% yield and was confirmed
by HPLC to be >98% enantiopure. These results indi-
cate that, under fermentation conditions at pH 7, enzy-
matic reduction is accompanied by in situ racemization
of the substrate leading to efficient DKR of substrate 1
(Scheme 2).
Because the active site of an enzyme is chiral, one enan-
tiomer of the substrate is converted to the product at a
higher rate than the other. This leads to kinetic resolu-
tion. In an ideal case, when conversion of one enantio-
mer is significantly faster than that of the other, we
can obtain an enantiopure product when the reaction
Several reductases studied gave the best conversion at pH 8.

2750
Y. Yang et al. / Tetrahedron: Asymmetry 16 (2005) 2748–2753
The stringent requirements for a successful DKR are
well illustrated when E. coli/Ara1p-catalyzed reduction
of 1 is compared with the corresponding reductions of
the two closely related compounds: 4-(2-thiophenyl)-b-
lactam 4, and 4-(2-furyl)-b-lactam 7. Here, the results
from screening are very similar to those obtained during
fermentation (Scheme 3). Under fermentation condi-
tions, both reductions proceeded rapidly (100% conver-
sion in 3 h); however, as the rates of conversion of the
(R)- and (S)-enantiomers were almost identical, no
DKR was achieved.
zation at neutral conditions, enzymatic reaction can be
combined with base-catalyzed racemization. This
approach has been applied in microbial reductions of
a-cyano cyclopentanones and cyclohexanones.33 Furstoss
et al. prepared enantiopure lactones by combining
microbial Baeyer–Villiger oxidation with a base-cata-
lyzed (pH 9) racemization of the corresponding a-substi-
tuted cyclopentanone.19 A highly efficient preparation of
D-phenyl glycines by enzymatic hydrolysis and derace-
mization of DL-hydantoins, was achieved at pH >8.34
Racemization of 3-oxo-b-lactams is not obvious; thus
the case of DKR of 3-oxo-4-phenyl-b-lactam 1, reported
here, is clearly an example of scientific serendipity.
R
R
HO
O
HO
O
R
O
O
E. coli / Ara1p
S
S
S
R
+
N
N
N
PMP
PMP
PMP
3. Conclusion
5
8
6
4
7
R =2-thiophenyl
R = 2-furyl
9
In conclusion, we have prepared cis-(3S,4R)-3-hydroxy-
4-phenyl-b-lactam 2 as a single enantiopure product
from racemic ketone 1. The recombinant E. coli strain
overproducing yeast reductase Ara1p was the Ôreducing
agentÕ. The enzymatic DKR occurred during fermenta-
tion at pH 7. This short synthesis of the title compound
can be readily carried out on a scale of several grams.
Parallel experiments with the related substrates, 4-(2-thio-
phenyl) and 4-(2-furyl) substituted 3-oxo-b-lactams, did
not lead to DKR. These results underscore the fact that
the successful DKR depends on the ratio of the fast and
slow reactions and its relationship to the rate of
racemization.
1 : 1
1 : 1
93% (3S,4S) >99% ee
98% (3S,4S) >99% ee
(3S,4R) > 99% ee
(3S,4R) > 99% ee
4
7
72 h
72 h
shake-flask
fermentation
1 : 1
1.2 : 1
4
7
3 h
3 h
100% (3S,4S) >98% ee
100% (3S,4S) >98% ee
(3S,4R) > 99% ee
(3S,4R) > 99% ee
Scheme 3.
While the rates of reduction of (S)-4 and (R)-4 are the
same, the rate of conversion of (S)-7 is slightly faster
than (R)-7. This is reflected in a slightly higher propor-
tion of the cis-alcohol (3S,4S)-8 obtained in fermenta-
tion. Overall, however, the lack of an efficient DKR in
the reduction of both 4 and 7 is a cumulative effect of
the low E ratio and a rapid reduction compared to race-
mization. These points are illustrated in Figures 1–3.
4. Experimental
4.1. General procedure for the biotransformations with
recombinant E. coli strains in a shake flask26
Biocatalytic DKR has been reported for bakerÕs-yeast-
catalyzed reductions of b-keto esters.29–32 a-Monosub-
stituted b-ketoesters are excellent substrates for yeast
reductases and, when the a-substituent is alkyl or aryl
group, DKR may be achieved, since racemization is
quite rapid under the biotransformation conditions. In
the case of substrates that do not undergo rapid racemi-
Fresh plates of engineered E. coli strains were streaked
from the appropriate frozen stock and incubated at
30 °C until the colony was 1–2 mm in size (normally
24 h). A single colony was used to inoculate 10 mL
Ph
Ph
O
Ph
HO
O
HO
O
Ara1p/BL21(DE3)
S
S
R
S
+
N
N
N
M9 minimal salts medium
O
PMP
PMP
PMP
pH=7
ee >98%
120
keto β-lactam
100
80
60
40
20
0
(3S,4R)-hydroxy β-lactam
(3S,4S)-hydroxy β-lactam
0
1
2
3
4
time (h)
Figure 1. Fermentation of 3-oxo-4-phenyl-b-lactam 4 with E. coli/Ara1p.

Y. Yang et al. / Tetrahedron: Asymmetry 16 (2005) 2748–2753
2751
O
O
HO
HO
O
Ara1p / BL21(DE3)
S
S
S
S
S
S
R
+
N
N
N
M9 minimal salts medium
pH=7
PMP
O
PMP
ee >98%
PMP
ee >99%
100
80
60
40
20
0
0
1
2
3
4
5
time(h)
Figure 2. Fermentation of 3-oxo-4-(2-thiophenyl)-b-lactam 4 with E. coli/Ara1p.
O
O
HO
O
HO
O
O
Ara1p / BL21(DE3)
O
O
S
S
S
R
+
N
N
N
M9 salt medium
pH=7
PMP
PMP
PMP
ee >99%
ee 98%
keto β-lactam
(3S,4R)-hydroxy β-lactam
(3S,4S)-hydroxy β-lactam
100
80
60
40
20
0
0
1
2
3
time (h)
Figure 3. Fermentation of 3-oxo-4-(2-furyl)-b-lactam 7 with E. coli/Ara1p.
LB-ampicillin medium in a 50 mL Erlenmeyer flask. The
culture was incubated at 200 rpm at 30 °C on a rotary
shaker overnight, and 0.25 mL of this culture was used
to inoculate a 25 mL fresh LB-ampicillin medium in a
150 mL baffled flask under the same conditions until it
reached an OD600 of 0.5 (ꢀ3 h). Then, IPTG was added
to a final concentration of 1 mM. The substrate (25 mg)
was added along with b-cyclodextrin (1 equiv if needed).
The culture was shaken at 30 °C and sampled periodi-
cally for GC and chiral phase HPLC analysis. The ana-
lytical samples were prepared by mixing 300 lL of the
reaction mixture with 300 lL of ethyl acetate. After vor-
tex mixing for 1 min, the sample was spun in a microcen-
trifuge for 1 min, then 200 lL of the organic layer was
collected and dried under nitrogen. The residue was dis-
solved in 200 lL of iso-propanol and the resulting solu-
tion passed through a Cameo 3 N syringe filter (0.22
micro, 3 mm diameter). One microlitre of sample was
used for GC analysis (where applicable) and 20 lL used
for HPLC analysis.
4.2. Biotransformation with resting cells35
Fresh plates were streaked from a frozen stock on solid
LB medium supplemented with 50 lg/mL ampicillin. A
single colony was used to inoculate a 10 mL LB medium
with 50 lg/mL ampicillin. The culture was incubated
overnight with shaking (200 rpm) at 30 °C. One litre of

2752
Y. Yang et al. / Tetrahedron: Asymmetry 16 (2005) 2748–2753
1
LB medium containing 4 g/L glucose and 50 lg/mL
ampicillin in two 500 mL baffled culture flasks were
inoculated with 5 mL precultures, separately. The cul-
ture was grown at 30 °C with shaking at 200 rpm. When
the OD600 value reached 0.6, IPTG was added to a final
concentration of 0.10 mM. Growth was continued until
the D600 value reached 4.0. The cells were harvested by
centrifugation, and the cell pellet suspended in 250 mL
of M9 minimal salt medium lacking nitrogen. To this
was added 200 mg of b-lactams (ground with 200 mg
b-cyclodextrin when necessary). The biotransformation
was carried out at 32 °C with aeration and vigorously
stirring. Ten millilitres of a 20% glucose solution was
added every 5 h to the reaction mixture. The pH was
controlled at 7.0 with a 3 M NaOH solution. The reac-
tion mixture was sampled periodically and analyzed by
both GC and HPLC as described above. When the reac-
tion reached complete conversion, the mixture was
saturated with NaCl and the cells removed by centrifu-
gation. Then, the reaction mixture was extracted with
ethyl acetate and the organic layers combined, washed
with brine, and dried over magnesium sulfate. The sol-
vent was removed under vacuum. The diastereoisomeric
mixture could be readily separated by column chroma-
tography on silica gel eluting with hexane and ethyl
acetate (4:1 to 3:1 to 2:1) to yield the pure product.
1513, 1252, 1113, 1027; H NMR (250 MHz, CDCl3) d
2.88 (1H, d, J = 9.1, OH), 3.76 (3H, s, OCH3), 5.18
(1H, dd, J = 5.1, J = 9.0, NCH), 5.51 (1H, d, J = 5.1,
OHCH), 6.81 (2H, d, J = 9.1, ArH), 7.07 (1H, dd,
J = 3.5, J = 5.0, ArH), 7.13 (1H, d, J = 2.9, ArH),
7.32 (2H, d, J = 9.0, ArH), 7.36 (1H, dd, J = 1.2,
J = 5.0, ArH); 13C NMR (62.9 MHz, CDCl3) d 55.45
(OCH3), 58.67 (NCH), 77.53 (OHC), 114.41, 118.88,
126.69, 127.29, 127.74, 130.26, 136.55, 156.58; HRMS:
C14H13NO3S (M+), calc.: 275.0616, found: 275.0614.
4.7. (3S,4S)-(À)-3-Hydroxy-1-(4-methoxyphenyl)-4-
(thiophen-2-yl)-2-azetidinone 5
White crystals (X-ray crystal structure);24 29% yield; mp:
178–179 °C; [a]20 = À171.0 (Lit. [a]20 = À115.012); IR
and NMR spectra as above.
4.8. (3R,4S)-(À)-3-Hydroxy-1-(4-methoxyphenyl)-4-
(thiophen-2-yl)-2-azetidinone
White crystals (X-ray crystal structure);24 mp: 98–99 °C;
[a]20 = À43.5; IR (CHCl3) cmax/cmÀ1 3352, 2921, 2836,
1
1728, 1512, 1249, 1138; H NMR (500 MHz, CDCl3) d
3.72 (3H, s, OCH3), 4.67 (1H, s, OH), 4.84 (1H, s,
NCH), 5.13 (1H, s, HOCH), 6.71 (2H, d, J = 8.5,
ArH), 6.99 (1H, t, J = 3.5, ArH), 7.12 (1H, s, ArH),
7.17 (2H, d, J = 8.5, ArH), 7.28 (1H, d, J = 5.0, ArH);
4.3. Compounds isolated from E. coli/Ara1p fermentation
reactions
13C NMR (125 MHz, CDCl3)
d 55.35 (OCH3),
61.82 (NCH), 84.51 (OHCH), 114.27, 119.16, 126.06,
126.29, 127.34, 129.96, 139.62, 156.62, 166.44 (CO);
HRMS: C14H13NO3S (M+), calc.: 275.0616, found:
275.0616.
The syntheses and characterizations of ketones and
alcohols discussed here have been reported earlier.24,36
Full characterizations, including HRMS analyses were
performed for (3R,4S)-2, (3R,4R)-5, (3R,4S)-6,
(3R,4R)-8, and (3R, 4S)-9.37 Their enantiomers dis-
cussed here showed identical IR and NMR spectra (Sup-
plementary Material). Optical rotations for all
compounds were measured in CH2Cl2 (c 1.0).
4.9. (3S,4R)-(+)-3-Hydroxy-1-(4-methoxyphenyl)-4-
(thiophen-2-yl)-2-azetidinone 6
Isolated yield 18%; ee >99%; [a]20 = +47.4; IR and
NMR spectra as above.
4.4. (3R,4S)-(+)-3-Hydroxy-1-(4-methoxyphenyl)-4-phen-
yl-2-azetidinone38
4.10. (3R,4R)-(+)-4-(Furan-2-yl)-3-hydroxy-1-(4-meth-
oxyphenyl)-2-azetidinone
White crystals; mp: 222–229 °C; [a]20 = +192.0; 1H
NMR (400 MHz, CDCl3) d 2.42 (1H, d, J = 9.0, OH),
3.75 (3H, s, OCH3), 5.18 (1H, dd, J = 5.2, J = 9.0,
HOCH), 5.26 (1H, d, J = 5.1, NCH), 6.80 (2H, d,
J = 9.0, ArH), 7.29 (2H, d, J = 9.1, ArH), 7.32–7.42
(5H, m, ArH); 13C NMR (100 MHz, CDCl3) d 55.47
(OCH3), 62.28 (NCH), 76.69 (OCH), 114.49, 118.85,
127.45, 128.97, 129.19, 156.54, 166.25 (CO); HRMS:
C16H15NO3 (M+), calc.: 269.1052, found: 269.1052.
White crystals; mp: 158–159 °C; [a]20 = +216.6; IR
(CHCl3) cmax/cmÀ1 3370, 3120, 2951, 2916, 2847, 2836,
1747, 1510, 1397, 1240, 1119, 1029; 1H NMR
(250 MHz, CDCl3) d 3.20 (1H, d, J = 9.8, OH), 3.75
(3H, s, OCH3), 5.21 (1H, dd, J = 5.0, J = 9.8, OHCH),
5.27 (1H, d, J = 5.1, NCH), 6.43 (1H, dd, J = 1.9,
J = 3.3), 6.53 (1H, d, J = 3.3), 6.81 (2H, d, J = 9.0),
7.29 (2H, d, J = 9.0), 7.48 (1H, d, J = 1.7); 13C NMR
(62.9 MHz, CDCl3) d 55.47 (OCH3), 56.49 (NCH),
77.81 (OHCH), 110.97, 111.18, 114.38, 118.67, 130.45,
143.93, 147.77, 156.57, 165.44 (CO); HRMS:
C14H13NO4(M+), calc.: 259.0845, found: 259.0839.
4.5. (3S,4R)-(À)-3-Hydroxy-1-(4-methoxyphenyl)-4-phen-
yl-2-azetidinone 2
Isolated yield 65%; ee >98%; [a]20 = À170.0 (Lit.
[a]20 = À184.612); IR and NMR spectra as above.
4.11. (3S,4S)-(À)-4-(Furan-2-yl)-3-hydroxy-1-(4-meth-
oxyphenyl)-2-azetidinone 839
4.6. (3R,4R)-(+)-3-Hydroxy-1-(4-methoxyphenyl)-4-
(thiophen-2-yl)-2-azetidinone
Isolated yield 34%; white crystals (X-ray crystal struc-
ture);24 ee >98%; [a]20 = À215.0; IR and NMR spectra
as above.
White crystals; mp: 178–179 °C; [a]20 = +172.6; IR
(CHCl3) cmax/cmÀ1 3299, 3012, 2961, 2907, 2831, 1714,

Y. Yang et al. / Tetrahedron: Asymmetry 16 (2005) 2748–2753
2753
11. Patel, R. N.; Howell, J.; Chidambaram, R.; Benoit, S.;
Kant, J. Tetrahedron: Asymmetry 2003, 14, 3673–3677.
12. Carr, J. A.; Al-Azemi, T. F.; Long, T. E.; Shim, J.-Y.;
Coates, C. M.; Turos, E.; Bisht, K. S. Tetrahedron 2003,
59, 9147–9160.
13. Kayser, M. M.; Yang, Y.; Mihovilovic, M. D.; Feicht, A.;
Rochon, F. D. Can. J. Chem. 2002, 80, 796–800.
14. Faber, K. Biotransformations in Organic Chemistry: A text
book, 4th ed.; Springer: Berlin, 2000.
15. Enzyme Catalysis in Organic Synthesis; Drauz, K., Wald-
mann, H., Eds.; WILEY-VCH: Weinheim, 2002.
16. Turner, N. J. Curr. Opin. Chem. Biol. 2004, 8, 114–119.
17. Dehli, J. R.; Gotor, V. J. Org. Chem. 2002, 67, 6816–6819.
18. Fogal, E.; Forzato, C.; Nitti, P.; Pitacco, G.; Valentin, E.
Tetrahedron: Asymmetry 2000, 11, 2599–2614.
4.12. (3R,4S)-(À)-4-(Furan-2-yl)-3-hydroxy-1-(4-meth-
oxyphenyl)-2-azetidinone
White powder; mp: 96–97 °C; [a]20 = À40.7; IR (CHCl3)
c
max/cmÀ1 3362, 3124, 2935, 2916, 2837, 1729, 1513,
1
1250, 1144; H NMR (250 MHz, CDCl3) d 3.73 (3H,
s, OCH3), 3.74 (1H, d, J = 1.9, NCH), 4.92 (1H, dd,
J = 1.9, J = 4.8, OHCH), 1.09 (1H, d, J = 5.0, OH),
6.37 (1H, dd, J = 1.9, J = 3.2, ArH), 6.46 (1H, d,
J = 3.2, ArH), 6.73 (2H, d, J = 9.1, ArH), 7.20 (2H, d,
J = 9.1, ArH), 7.39 (1H, d, J = 1.0, J = 1.2, ArH); 13C
NMR (62.9 MHz, CDCl3) d 55.34 (OCH3), 58.98
(NCH), 80.62 (OHCH), 110.10, 110.68, 114.22, 118.86,
130.27, 143.40, 1478.71, 156.57, 166.69 (CO); HRMS:
C14H13NO4 (M+), calc.: 259.0845, found: 259.0836.
19. Berezina, N.; Alphand, V.; Furstoss, R. Tetrahedron:
Asymmetry 2002, 13, 1953–1955.
20. Sybesma, W. F. H.; Straathof, A. J. J.; Jongejan, J. A.;
Pronk, J. T.; Heignen, J. J. Biocatal. Biotrans. 1998, 16,
95–134.
4.13. (3S,4R)-(+)-4-(Furan-2-yl)-3-hydroxy-1-(4-meth-
oxyphenyl)-2-azetidinone 9
´
21. Stewart, J. D.; Rodrıguez, S.; Kayser, M. M. In Enzyme
Isolated yield 40%; ee >99%; [a]20 = +44.8; IR and
NMR spectra as above.
Technology for Pharmaceutical and Biotechnological Appli-
cations; Kirst, H. A., Yeh, W.-K., Zmijewski, M., Eds.;
Marcel Dekker: New York, 2001; pp 175–207.
22. Kaluzna, I. A.; Matsuda, T.; Sewell, A. K.; Stewart, J. D.
J. Am. Chem. Soc. 2004, 126, 12826–12827.
Acknowledgements
23. Kaluzna, I. A.; Feske, B. D.; Wittayanan, W.; Ghiviriga,
I.; Stewart, J. D. J. Org. Chem. 2005, 70, 342–345.
Financial support by the Natural Sciences and Engi-
neering Research Council of Canada (MMK), and the
Medical Research Fund of New Brunswick (MMK)
are gratefully acknowledged.
´
24. Yang, Y.; Kayser, M. M.; Rochon, F. D.; Rodrıguez, S.;
Stewart, J. D. J. Mol. Cat. B: Enzym. 2005, 32, 167–174.
´
25. Rodrıguez, S.; Kayser, M. M.; Stewart, J. D. Org. Lett.
1999, 1, 1153–1155.
´
26. Rodrıguez, S.; Kayser, M. M.; Stewart, J. D. J. Am. Chem.
Soc. 2001, 123, 1547–1555.
References
´
27. Rodrıguez, S.; Schroeder, K. T.; Kayser, M. M.; Stewart,
J. D. J. Org. Chem. 2000, 65, 2586–2587.
28. Huerta, F. F.; Minidis, A. B. E.; Ba¨ckvall, J.-E. Chem.
Soc. Rev. 2001, 30, 321–331.
29. Nakamura, K.; Miyai, T.; Nozaki, K.; Ushio, K.; Oka, S.;
Ohno, A. Tetrahedron Lett. 1986, 27, 3155–3156.
30. Nakamura, K.; Kawai, Y.; Ohno, A. Tetrahedron Lett.
1991, 32, 2927–2928.
31. Seebach, D.; Roggo, S.; Maetzke, T.; Braunschweiger, H.;
Sircus, J.; Krieger, M. Helv. Chim. Acta 1987, 70, 1605–
1615.
1. Palomo, C.; Aizpurua, J. M.; Ganboa, I. In Enantio-
selective Synthesis of b-Amino-acids; Juaristi, E., Ed.; The
Synthesis of b-Amino-Acids and Their Derivatives from b-
Lactams; Wiley-VCH: New York, 1997; pp 463–464.
2. Herranz, R.; Castro-Pichel, J.; Vinuesa, S.; Garcia-Lopez,
M. T. J. Org. Chem. 1990, 55, 2232–2234, and references
cited therein.
3. Cutrona, K. J.; Sanderson, P. E. J. Tetrahedron Lett. 1996,
37, 5045–5048.
4. Kingston, D. G. I. J. Nat. Prod. 2000, 63, 727–734.
5. Jayaraman, M.; Deshmukh, A. R.; Bhawal, B. M.
Tetrahedron 1996, 52, 8989–8992.
6. Arun, M.; Joshi, S. N.; Puranik, V. G.; Bhawal, B. M.;
Deshmukh, A. R. A. S. Tetrahedron 2003, 59, 2309–
2318.
32. Abalain, C.; Buisson, D.; Azerad, R. Tetrahedron: Asym-
metry 1996, 7, 2983–2996.
33. Brown, S. A.; Parker, M. C.; Turner, M. J. Tetrahedron:
Asymmetry 2000, 11, 1687–1690.
34. Garcia, M. J.; Azerad, R. Tetrahedron: Asymmetry 1997,
8, 85–92.
7. Joshi, S. N.; Deshmukh, A. R. A. S.; Bhawal, B. M.
Tetrahedron: Asymmetry 2000, 11, 1477–1485.
8. Shinkre, B. A.; Puranik, V. G.; Bhawal, B. M.; Desh-
mukh, A. R. A. S. Tetrahedron: Asymmetry 2003, 14, 453–
459.
9. France, S.; Weatherwax, A.; Taggi, A. E.; Lectka, T. Acc.
Chem. Res. 2004, 37, 592–600, and references cited therein.
10. Brieva, R.; Crich, J. Z.; Sih, C. J. J. Org. Chem. 1993, 58,
1068–1075.
35. Walton, A. Z.; Stewart, J. D. Biotechnol. Prog. 2002, 18,
262–268.
36. Mihovilovic, M. D.; Feicht, A.; Kayser, M. M. J. Prakt.
Chem. 2000, 342, 585–590.
37. Yang, Y. PhD Thesis 2005, University of New Brunswick,
NB, Canada.
38. Holton, R. A.; Liu, J. H. Bioorg. Med. Chem. Lett. 1993,
3, 2475–2478.
39. Patel, R. N. European Patent, EP 0 634492A1, 1993.