Rhizopus arrhizus mediated asymmetric reduction of alkyl 3-oxobutanoates

Article abstract of DOI:10.1016/j.tetasy.2004.09.026

t-Butyl 3-oxobutanoate gives the hydroxyester in ～68% yield and 99% ee. Alkyl 3-oxobutanoates (alkyl: methyl, ethyl, allyl, isobutyl, t-butyl) were reduced enantioselectively to the corresponding (S)-alcohols by the fungus Rhizopus arrhizus and other Rhizopus sp. The best result obtained was with t-butyl 3-oxobutanoate, which was reduced by R. arrhizus with 99% enantiomeric excess and ～68% isolated yield.

Full text of DOI:10.1016/j.tetasy.2004.09.026

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 15 (2004) 3397–3400
Rhizopus arrhizus mediated asymmetric reduction of alkyl
-oxobutanoates
3
Neeta A. Salvi and Subrata Chattopadhyay*
Bio-Organic Division BARC, Mumbai 400 085, India
Received 14 July 2004; accepted 24 September 2004
Abstract—Alkyl 3-oxobutanoates (alkyl: methyl, ethyl, allyl, isobutyl, t-butyl) were reduced enantioselectively to the corresponding
(
S)-alcohols by the fungus Rhizopus arrhizus and other Rhizopus sp. The best result obtained was with t-butyl 3-oxobutanoate,
which was reduced by R. arrhizus with 99% enantiomeric excess and ꢀ68% isolated yield.
2004 Published by Elsevier Ltd.
ꢀ
1
. Introduction
yeast-mediated method often proceeds with a poor
chemical yield and varying enantioselectivity (70–
4
b
The synthesis of chiral compounds with the aid of
microorganisms is a useful method in organic synthesis
with the reduction of ketones to chiral alcohols being
a typical example. In this connection, the reduction of
b-keto esters to b-hydroxy esters is extremely useful as
the products are important building blocks for the syn-
thesis of a large number of bioactive compounds, inter-
mediates and chiral auxillaries. Chiral b-hydroxy
esters can be obtained in enantiomeric forms by chemi-
cal asymmetric reducing agents.
enzymatic protocol offers the advantage of better enan-
tioselectivity, but their widespread use for asymmetric
reduction is restricted due to the prohibitive costs of
commercial enzymes and the required co-factors. In this
regard, the use of a whole cell system is emerging as an
economical and eco-friendly alternative. 3-Hydroxybut-
anoic acid and its esters are prominent members in this
category and have been used as synthetic building
blocks and intermediates for the syntheses of several
classes of natural products and many therapeutic agents.
In particular its ethyl ester has been exploited exten-
sively for the synthesis of compounds with diverse struc-
tural features viz. macrolides, pheromones, antibiotics,
97%), better results have been reported using non-fer-
6
menting yeast in an organic solvent. More recently, we
have developed an efficient method for the enantiomeric
synthesis of both the enantiomers of 3-hydroxybutano-
7
ates via a lipase-catalyzed esterification protocol.
8
a–d
During our work on microbial transformations,
1
a–f
we found that the fungus Rhizopus arrhizus can
efficiently carry out the asymmetric reduction of a
wide variety of ketones. Herein, we report the potential
of the fungus for the enantiomeric synthesis of the
3-hydroxybutanoates.
2
a,2b
In comparison, the
2. Results and discussion
One of the prime requirements of a microbial transfor-
mation is the optimization of the reaction parameters
such as substrate structure, reaction conditions etc.
Hence, for the present study, we chose a series of alkyl
3-oxoalkanoates 1a–e differing in the alkyl chain length
(R) of the alcohol moiety and studied their bio-reduc-
tion with R. arrhizus (Scheme 1). The reactions with
all the substrates were carried out for 4 and 8days using
almost the same substrate concentrations. After the
usual work-up, product alcohols 2a–e were isolated
from the respective substrates, purified by preparative
3
a–f
etc.
Amongst the enzymatic methods, bakerÕs yeast
is extensively used for the asymmetric reduction of ethyl
4
3
-oxobutanoate, while other microbial methods have
5
also been found inadequate. Although the bakerÕs
1
TLC and characterized by IR and H NMR spectra.
The enantiomeric excesses (ees) of the products were
1
determined from the H NMR spectra of the corre-
*
Corresponding author. Fax: +91 22 25505151; e-mail: schatt@
apsara.barc.ernet.in
9
sponding MTPA esters, while their configurations were
0
957-4166/$ - see front matter ꢀ 2004 Published by Elsevier Ltd.
doi:10.1016/j.tetasy.2004.09.026

3398
N. A. Salvi, S. Chattopadhyay / Tetrahedron: Asymmetry 15 (2004) 3397–3400
O
O
OH
O
Table 2. Asymmetric reduction of t-butyl acetoacetate 1e with
a
different Rhizopus sp.
Rhizopus arrhizus
OR
OR
b
Yield (%)
c
Entry
Organism
% Ee
1
a-e
2a-e
1
2
3
4
5
6
7
8
Rhizopus arrhizus
68
67
67
69
62
49
75
86
99
91
91
91
91
93
96
93
Rhizopus arrhizus (NCIM 877)
Rhizopus arrhizus (NCIM 878)
Rhizopus arrhizus (NCIM 879)
Rhizopus arrhizus (NCIM 997)
Rhizopus oryzae (NCIM 1009)
Rhizopus nivius (NCIM 958)
Rhizopus nivius (NCIM 959)
a: R = CH ; b: R = Et; c: R = Allyl; d: R = iso-Bu; e: R = t-Bu
3
Scheme 1.
assessed by comparison of their specific rotation values
1
0d,g,6
with those reported.
in Table 1.
The results are summarized
a
The reactions were carried out at 27 ± 2ꢁC by adding the substrates
(
ꢀ0.1g) in EtOH (1.5mL) to different Rhizopus cultures (150mL)
and shaking (90–95rpm) the mixture for 2days.
Isolated yield.
b
c
Table 1. Rhizopus arrhizus mediated reduction of alkyl 3-oxo-
a
butyrates 1a–e
1
From H NMR spectrum of the MTPA ester of 2e obtained from
different experiments.
b
Entry Substrate Incubation time (d) Yield (%) % Ee
c
1
2
3
4
5
6
7
8
9
1
1a
1a
1b
1b
1c
1c
1d
1d
1e
1e
4
8
4
8
4
8
4
8
4
8
15
22
37
39
17
26
51
59
71
65
70
71
74
89
80
89
89
90
94
94
partly due to the their higher hydrophobicity, which
assisted better recovery during isolation. In these cases,
the incubation period did not matter much, both in terms
of yields and % ees. The t-butyl ester 1e was the best sub-
strate furnishing (S)-2e with 94% ee and 65–70% yield.
Although the above results, especially with 1e, were
encouraging, further improvement was warranted for
the application of the protocol as a synthetic route. A re-
duced reaction time with concurrent increases in yields
and ees would be an ideal combination towards this
objective. In principle, this can be accomplished by a
subtle change in the substrate or screening an alternate
microorganism. Very recently, we were able to improve
the enantioselectivity of a Rhizopus mediated hydrolysis
0
a
The reactions were carried out 27 ± 2ꢁC by adding the substrates
(
ꢀ0.1g) in EtOH (1.5mL) to the microbial culture (150mL) and
shaking (90–95rpm) the mixture.
Isolated yield.
b
c
1
From H NMR spectra of the respective MTPA esters.
8
d
Irrespective of the alkyl chain length (R) of the alcohol
moieties, all esters 1a–e, were reduced without any
noticeable decarboxylation furnishing the (S)-hydroxy
esters 2a–e with good ees. Elongation of the alkyl chain
protocol by proper choice of a microbial strain.
Hence, a similar strategy was extended for the present
work. For this, the microbial reduction of 1e was carried
out using various Rhizopus species for 2days. All the
microorganisms transformed 1e to (S)-2e, albeit in dif-
ferent yields and enantiomeric excesses (Table 2). With
the exception of Rhizopus oryzae NCIM 1009 (Table
2, entry 6), the reactions with other microorganisms
proceeded with better enantioselectivities (90–99% ee)
furnishing the product in 62–85% isolated yields.
The two-day incubation protocol with R. arrhizus was
found to be the best furnishing (S)-2e in appreciable
yield (ꢀ68%) and excellent enantiomeric purity (99%).
The fungi Rhizopus nivius NCIM 958 and 959 were also
very efficient providing (S)-2e in excellent yields (75%
and 86%, respectively), and enantiomeric excesses
(96% and 93%, respectively).
(
methyl to t-butyl) in the alkoxy group led to a gradual
increase in the yields and enantioselectivities of the re-
duced products. For the lower homologous 1a–c con-
taining the linear chain alcohols, the yields and % ees
of the products were poor. However, increasing the
chain length of R-group improved the yields and even
the % ees of the products. The apparent lesser yield of
2
c was because of the recovery of significant amounts
of the unreacted substrates, which were not accounted
in the data shown in Table 1. With 1a and 1b also, the
amounts of the recovered substrates could partly explain
the poor yields. In these cases, the substrates and the
resultant products could not be completely extracted
from the reaction mixture, in view of their lower hydro-
phobicity. The reactivities of these lower homologues
1
a–c were poor and even after 8days of incubation,
3. Conclusion
the products were only obtained in modest yields. This,
however, could be improved significantly by using the
substrates 1d and 1e, which possess sterically bulky
branched R-groups.
In view of its simplicity, the bakerÕs yeast-mediated
reduction of alkyl 3-oxobutanoates is the most preferred
method for the synthesis of the corresponding (S)-3-
hydroxybutanoates. However, the ees of the products
are known to be strongly dependent on the reduction
conditions. In view of this, several modifications in the
experimental conditions have been suggested to improve
the results. Different protocols, such as stirring condi-
Better enantioselectivity with these substrates was antic-
ipated, as the enzyme could easily differentiate between
the small and large groups flanking the carbonyl func-
tion. The higher yields of 2d and 2e, however, may be

N. A. Salvi, S. Chattopadhyay / Tetrahedron: Asymmetry 15 (2004) 3397–3400
3399
1
0a
10b
tions, and pretreatment of yeast with 5% ethanol,
have been employed, especially with ethyl 3-oxobutano-
ate. For a large scale preparation, a continuous feed of
the substrate and nutrient was prescribed, as the % ee
of the product was found to be dependent on the sub-
TLC (silica gel, 10% EtOAc/hexane) to furnish the prod-
uct alcohols.
4.3. Typical procedure for the synthesis of (S)-t-butyl-
3-hydroxybutanoate 2e via microbial reduction
5
a
strate concentration. A related paper reported better
results (94–96% ee) by carrying out the reaction in tap
water and terminating it after 4h.
In five cotton plugged Erlenmeyer flasks each containing
the 72h grown R. arrhizus culture (150mL) was added
1
0c,d
1
e (0.570 g, 3.6mmol) in EtOH (7.5mL) in equal
The problem of enantioselectivity in the bakerÕs yeast
reduction is ascribed to the presence of multiple reduc-
tases that show complementary enantiopreferences.
Innovative approaches involving selective suppression
of some of these enzymes with allyl alcohol and by
immobilization on polyurethane (for anticipated change
in configuration) proved counter productive, as both
amounts. The mixtures were incubated on a rotary
shaker (90–95rpm) at room temperature for 2days.
The mycelial mass was removed, washed thoroughly
with water and squeezed. The aqueous washings were
mixed with the aqueous filtrate and extracted with
CHCl3 (3 · 50mL). The organic extract was washed
with H O, dried and concentrated in vacuo to obtain a
2
1
0e,f
these led to the destruction of the pro-(S) reductases.
residue, which was subjected to preparative TLC (silica
gel, 10% EtOAc/hexane, visualization by staining with I2
vapour) to furnish 2e (0.390g, 68%).
In contrast, the fungus Rhizopus arrhizus has been found
to be an excellent bioreduction system for the enantio-
meric synthesis of 3-hydroxybutanoates. By judicious
choice of substrate, microorganism and incubation per-
iod, it is possible to obtain enantiomerically pure 3-
hydroxybutanoates in very high yields and ees with high
reproducibility. It is worth noting that the bakerÕs yeast
reduction in aqueous medium often furnishes low yields
of the products due to the difficulty in their extraction.
The results obtained herein compare very well, with
4
.4. Methyl 3-hydroxybutanoate 2a
22
10g
½
aꢁ ¼ þ24:2 (c 0.82, CHCl3) (71% ee), lit.
D
½
aꢁ ¼ þ33:3 (c 1.2, CHCl ); IR: 3423, 2977, 2933,
D
3
ꢂ1
1
1
2
728cm ; H NMR: d 1.18 (d, J = 6.2Hz, 3H), 2.32–
.41 (m, 2H), 3.1 (br s, 1H), 3.67 (s, 3H), 4.07–4.20
(m, 1H).
6
one recent report, even surpassing the reported results
in some cases.
4.5. Ethyl 3-hydroxybutanoate 2b
2
D
2
10d
23:5
D
½
aꢁ ¼ þ30:0 (c 0.77, CHCl ) (89% ee), lit.
½aꢁ
¼
3
4. Experimental
þ43:9 (c 1.35, CHCl ); IR: 3432, 2977, 2933,
728cm ; H NMR: d 1.17 (d, J = 7.0Hz, 3H), 1.23
(t, J = 6.2Hz, 3H), 2.22–2.48 (m, 2H), 3.2 (br s, 1H),
3
ꢂ1
1
1
4
.1. General
4.06–4.22 (m, 3H).
Substrates 1a–e (Aldrich, Fluka and Lancaster) were
used as received. (R)-(+)-a-Methoxy-a-trifluoromethyl-
phenylacetic acid (MTPA) was supplied by Aldrich.
The Rhizopus species were received from the National
Collection of Industrial Microorganisms, National
Chemical Laboratory, Pune, India. Fungus, from
0
4
.6. 2 -Propenyl 3-hydroxybutanoate 2c
22
½
aꢁ ¼ þ28:2 (c 0.97, CHCl ) (89% ee); IR: 3433, 2986,
D
3
ꢂ1
1
2
2
4
5
8
933, 1728cm ; H NMR: d 1.16 (d, J = 7.0Hz, 3H),
.32–2.49 (m, 2H), 3.2 (br s, 1H), 4.08–4.16 (m, 1H),
.54 (dd, J = 4.5, 6.0Hz, 2H), 5.18–5.26 (m, 2H), 5.78–
.92 (m, 1H). Anal. Calcd for C H O : C, 58.31; H,
8
d
potato/dextrose/agar slants were grown on sterilized
modified Czepek Dox medium (150mL) in 500mL
Erlenmeyer flasks at 27 ± 2ꢁC with shaking (150rpm).
The IR spectra were recorded as films on a Nicolet
7
12
3
.39. Found: C, 58.16; H, 8.58.
1
FT-IR model Impact 410 spectrometer. The H NMR
spectra were recorded in CDCl with a Bruker AC-200
4
.7. Isobutyl 3-hydroxybutanoate 2d
3
(200MHz) spectrometer. The optical rotations were
recorded with a Jasco 360 DIP digital polarimeter.
2
D
2
½
aꢁ ¼ þ27:0 (c 1.16, CHCl ) (90% ee); IR: 3432, 2968,
3
ꢂ1
1
2
881, 1745cm ; H NMR: d 0.88 (d, J = 6.7Hz, 6H),
.18 (d, J = 6.3Hz, 3H), 1.82–1.98 (m, 1H), 2.31–2.52
m, 2H), 3.2 (br s, 1H), 3.84 (d, J = 6.7Hz, 2H), 4.06–
1
(
4
.2. General procedure for microbial reductions
4
1
.19 (m, 1H). Anal. Calcd for C H O : C, 59.97; H,
8 16 3
0.07. Found: C, 59.77; H, 10.25.
Substrates (0.1g) in EtOH (1.5mL) were added to the
2h grown culture in cotton plugged flasks and incu-
7
bated on a rotary shaker (90–95rpm) at room tempera-
ture for the period specified in Tables 1 and 2. At the end
of the reaction, the mycelial mass was removed, washed
thoroughly with water and squeezed. The aqueous
washings were mixed with the aqueous filtrate and ex-
tracted with CHCl3 (3 · 50mL). The organic extract
4.8. t-Butyl 3-hydroxybutanoate 2e
22
6
½aꢁ ¼ þ32:3 (c 1.03, CHCl3) (99% ee), lit.
D
½aꢁ ¼ þ34:05 (c 1.0, CHCl ); IR: 3423, 2977, 2933,
D
3
ꢂ1
1
1727cm ; H NMR: d 1.09 (d, J = 7.0Hz, 3H), 1.35
(s, 9H), 2.24–2.27 (m, 2H), 3.25 (br s, 1H), 4.02–4.06
(m, 1H).
was washed with H O, dried and concentrated in vacuo
2
to obtain a residue, which was subjected to preparative

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N. A. Salvi, S. Chattopadhyay / Tetrahedron: Asymmetry 15 (2004) 3397–3400
5. (a) Wipf, B.; Kupfer, E.; Bertari, R.; Leuenberger, H. G.
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