Novel fungi-catalyzed reduction of α-alkyl-β-keto esters

Article abstract of DOI:10.1016/j.tetasy.2009.05.031

A screening of 15 fungi and yeast strains was carried out in fermentation processes to perform the diastereo- and enantioselective reduction of ethyl 2-methyl-3-oxobutanoate, to the corresponding (R^*,S^*)-3-hydroxy-2-methyl esters. Overall, biotransformations led to excellent conversions, as well as good to excellent diastereo- and enantioselectivities. A strain of Aureobasidium pullulans (CCM H1) was found to be the most efficient biocatalyst in terms of conversion (100%), syn:anti ratio (3:97), and enantiomeric excess (94% anti-(2S,3S) isomer). This biotransformation was successfully carried out on a preparative level as well. Other microorganisms, such as Fusarium graminearum (CCM HH 224), Aspergillus terreus (BFQU 121), Geotrichum candidum (CCM H38), Trichoderma koningii (ATCC 76666), and Aspergillus niger (CCM H21) also showed excellent diastereo- and enantioselectivities, combined with high conversions (>95% conversion, ≥95% ee, and excellent syn:anti ratios). Many of the strains used in this work had scarcely been described as oxido-reducing agents, or had never been used with the substrates reported herein.

Full text of DOI:10.1016/j.tetasy.2009.05.031

Tetrahedron: Asymmetry 20 (2009) 1393–1397
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Novel fungi-catalyzed reduction of a-alkyl-b-keto esters
*
Silvana P. Ravía, Ignacio Carrera, Gustavo A. Seoane, Silvana Vero, Daniela Gamenara
Organic Chemistry Department, Biosciences Department, Facultad de Química, Universidad de la Republica, Gral. Flores 2124, 11800 Montevideo, Uruguay
a r t i c l e i n f o
a b s t r a c t
Article history:
A screening of 15 fungi and yeast strains was carried out in fermentation processes to perform the diastereo-
and enantioselective reduction of ethyl 2-methyl-3-oxobutanoate, to the corresponding (R*,S*)-3-hydroxy-
2-methyl esters. Overall, biotransformations led to excellent conversions, as well as good to excellent dia-
stereo- and enantioselectivities. A strain of Aureobasidium pullulans (CCM H1) was found to be the most effi-
cient biocatalyst in terms of conversion (100%), syn:anti ratio (3:97), and enantiomeric excess (94% anti-
(2S,3S) isomer). This biotransformation was successfully carried out on a preparative level as well. Other
microorganisms, such as Fusarium graminearum (CCM HH 224), Aspergillus terreus (BFQU 121), Geotrichum
candidum (CCM H38), Trichoderma koningii (ATCC 76666), and Aspergillus niger (CCM H21) also showed
excellent diastereo- and enantioselectivities, combined with high conversions (>95% conversion, P95%
ee, and excellent syn:anti ratios). Many of the strains used in this work had scarcely been described as
oxido-reducing agents, or had never been used with the substrates reported herein.
Received 23 March 2009
Accepted 29 May 2009
Available online 1 July 2009
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
of other competitive enzymes, or the low efficiency associated with
the low permeability of the cell membrane toward the substrate.50
The use of enzymatic transformations with either whole micro-
organisms or isolated enzymes to carry out stereospecific and ster-
eoselective reactions has grown steadily over the last few
decades.1 In particular, fungal or yeast-catalyzed reactions have
been used as stereoselective alternatives for hydroxylations, sul-
foxidations, epoxidations, Baeyer–Villiger oxidations, deracemiza-
tions, and stereo- and enantioselective reductions of ketones.2
The enzymatic reduction of prochiral ketones is a widely inves-
tigated method to produce chiral building blocks.3–8 A broad range
of structurally different chiral alcohols can be obtained depending
on the substrate structure, as a consequence of the huge variety of
enzymes available,9–20 together with the catalytic promiscuity
shown by many of them.21–23 Likewise, recombinant microorgan-
isms overexpressing reductases are also powerful biocatalysts
that can provide excellent yields and enantiomeric excesses due
to their high specificity.24–30 Moreover, different non-conventional
reaction systems can be used, such as ionic liquids as (co-)sol-
vents,31–35 which clearly enhance the practical applicability of
these synthetic approaches.
As a consequence, the importance of finding new microorganisms
that could catalyze highly stereoselective processes in an efficient
manner remains open.
Herein we report our findings on the screening of different
microorganisms, fungi and yeasts, as catalysts for the stereoselec-
tive reduction of ethyl 2-methyl-3-oxobutanoate. We were partic-
ularly interested in identifying new microorganisms that are able
to perform diastereospecific reactions, thus generating two stereo-
genic centers within one biocatalytic turnover. This task is part of
our research program for designing inexpensive as well as environ-
mentally responsible methodologies to prepare chiral building
blocks.
2. Results and discussion
Our model reaction was a diastereoselective synthesis starting
from substrate 1, which contains both a stereogenic and a prochiral
center. In this reduction, up to four stereoisomers can be produced,
due to the presence of two stereogenic centers in the product
(Scheme 1).
The characterization of the four possible isomers was carried out.
A diastereomeric mixture of ethyl a-methyl-b-hydroxy esters was
used as standard for GC analysis. Such a diastereomeric mixture
was obtained by NaBH4 reduction of the corresponding racemic
ethyl a-methyl-b-keto ester according to the procedure described
Filamentous fungi or yeast-catalyzed reductions of carbonyl
compounds have been reported in the past few years as an attrac-
tive methodology to prepare chiral alcohols, since it is not only an
environmentally friendly procedure, but also an economic alterna-
tive compared to expensive commercial reductases.13,36–49 The low
cost of this whole cell-catalyzed process, often contrasts with the
fact that side products are frequently obtained due to the presence
in the Experimental (Section 4.4.1). By GC analysis, two pairs of
peaks of different areas were observed, which corresponded to
the two pairs of enantiomers, and which is consistent with a
* Corresponding author. Tel.: +598 29247881; fax: +598 29241906.
E-mail address: dgamenar@fq.edu.uy (D. Gamenara).
0957-4166/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2009.05.031

1394
S. P. Ravía et al. / Tetrahedron: Asymmetry 20 (2009) 1393–1397
OH
O
OH
O
+
O
O
O
O
2b
2a
Fungal
O
biotransformation
28 °C, 150 rpm
OH
O
OH
O
1
+
O
O
2d
2c
Scheme 1. Fungi-catalyzed reduction of racemic ethyl 2-methyl-3-oxobutanoate.
diastereo- but non-enantioselective chemical reduction mediated
by NaBH4. To assist in the assignment of the absolute configuration,
the (2R,3S)- and (2S,3S)-isomers, 2a and 2d, respectively, were pre-
pared as standards. syn-Isomer 2a was obtained (97.2% de; >99%
ee) using a recombinant Escherichia coli JM 105 strain expressing
Gcy1p aldo keto reductase, according to the procedure described
by Rodríguez et al.30 This compound was then assigned in the GC
analysis (2a, tR = 29.5 min). Considering that the NaBH4-mediated
reduction of a racemic compound is not enantioselective, the enan-
tiomer of 2a was identified as the only other peak with the same area
in the chromatogram. Hence, ethyl (2S,3R)-2-methyl-3-hydroxy-
butanoate, 2b, was assigned as the peak with tR = 31.9 min.. The
anti-isomer 2d was prepared (81% conversion; anti/syn ratio 89/
11; 64% ee) according to the literature,28 using the recombinant
2b [tR = 31.9 min as the (2S,3R)-isomer and 2c (tR = 33.2 min) as
ethyl (2R,3R)-2-methyl-3-hydroxybutanoate] was confirmed.
Once the analytic characterization was finished, 15 different
strains of fungi and yeasts were screened as biocatalysts for the
stereoselective reduction of racemic ethyl 2-methyl-3-oxobutano-
ate (Scheme 1). The reactions were performed using culture media
described in Section 4.1., in an orbital shaker at 28 °C and 150 rpm,
according to the general procedures described in the Experimental
(Section 4.4.1). Aliquots were taken at 24, 48, 72, and 96 h. The
results are summarized in Table 1.
In most cases, biotransformations led to good to excellent
conversions, except for Botrytis cinerea (entry 9, no conversion
observed), and for Rhizopus sp. and Phanerochaete chrysosporum
strains, in which only moderate conversions were obtained
(entries 13 and 15). All microorganisms preferentially afforded
the anti-isomers, except for P. chrysosporum, which gave a syn:anti
ratio 70:30 (entry 15), together with excellent enantiomeric
excesses. The highest diastereoselectivity was obtained with
Aureobasidium pullulans (entry 2, syn:anti 3:97; 100% conversion),
displaying excellent enantioselectivities (for both syn and anti iso-
mers) as well. The biotransformation was successfully conducted
at preparative scale (0.5 g of substrate, 70% isolated yield), showing
the same diastereo- and enantioselectivities. In addition, other
strains such as Fusarium graminearum, Aspergillus terreus,
Geotrichum candidum, Trichoderma koningii, and Aspergillus niger
E. coli strain BL21 (DE3)
(2S,3S)-2-methyl-3-hydroxybutanoate
D
yqhE (pPP4), allowing us to assign
as the peak with
tR = 36.0 min. Finally, the remaining isomer 2c (tR = 33.2 min) was
assigned as ethyl (2R,3R)-2-methyl-3-hydroxybutanoate (Fig. 1).
This assignment of chromatographic peaks was further confirmed
by transesterification of the diastereomeric mixture with vinyl ace-
tate using Candida antarctica B lipase (CaL B) as a biocatalyst. Only
isomers 2b and 2c were acetylated, and no consumption of isomers
2a and 2d was observed. The acetylated compounds had retention
times of 35.5 and 35.7 min. In agreement with Kazlauskas rule,51
only the (3R)-alcohols were transesterified, and the assignment of
OH
2d
O
OH
O
OEt
OEt
2c
OH
2b
O
OH
O
OEt
OEt
2a
Anti-isomers (Enantiomers)
Syn-isomers (Enantiomers)
O
O
O
O
OH
O
E. coli JM 105
OH
O
E. coli BL21 (pPPP4)
Anti/syn 89/11; 64% ee
OEt
97.2%, >99% ee
(Ref. 30)
OEt
OEt
OEt
(Ref. 28)
2a
2d
Figure 1. Characterization of diastereomers.

S. P. Ravía et al. / Tetrahedron: Asymmetry 20 (2009) 1393–1397
1395
Table 1
Reduction of Ethyl 2-methyl-3-oxobutanoate by fungi and yeast strains
Entry
Strain
Reaction time (h)
Conversiona,b (%)
Ethyl 3-hydroxy-2-methylbutanoate
syn:anti
eeb (%)
syn-(2R,3S)
anti-(2S,3S)
1
2
3
4
5
6
7
8
Penicilium expansum (CCM P17)
Aureobasidium pullulans (CCM H1)
Penicilium italicum (CCM L7)
Aspergillus ochraceus (CCM H22)
Fusarium graminearum (CCM H224)
Aspergillus terreus (BFQU 121)
Geotrichum candidum (CCM H38)
Trichoderma koningii (ATCC 76666)
Botrytis cinerea (CCM P18)
96
24
72
96
24
48
96
96
91
100
82
93
95
100
96
100
12:88
3:97
30:70
13:87
6:94
6:94
10:90
7:93
90
>99
64
>99
86
86
>99
60
94
94
84
96
96
96
97
96
9
No conversion
10
11
12
13
14
15
Alternaria sp. (CCM A3)
48
24
24
72
96
96
93
27:73
19:81
25:75
11:89
8:92
>99
>99
98
93
>99
96
97
90
96
97
96
Kluyveromyces marxianus (NRRL Y-8281)
Trichoderma atroviride (CCM M2)
Rhizopus sp. (CCM H43)
Aspergillus niger (CCM H21)
Phanerochaete chysosporum (CCM B4)
100
100
46
100
53
70:30
>99
a
Conversion is expressed as % substrate transformed into alcohol product.
Determined by chiral GC analysis. Culture collections: http://www.atcc.org/, http://nrrl.ncaur.usda.gov/, http://mail.fq.edu.uy/~microbio/.
b
(entries 5–8, 14) performed the bioreduction with excellent con-
versions (higher than 95%), syn:anti ratios (10:90–6:94) and enan-
tiomeric excesses (higher than 95% for the anti isomer). To the best
of our knowledge, microbial reductions using P. chrysosporum and
F. graminearum have been scarcely described for the reduction of
xenobiotics.41,52–55
of prochiral building blocks. Preliminary studies on the scale-up
of the process were successfully carried out, and further experi-
ments on this topic are currently in progress.
4. Experimental
Considering our screening results, in terms of diastereo- and
enantioselectivities, herein we add a new promising group of en-
zymes that can be worth trying for further cloning, over-express-
ing, and deeper biochemical and biocatalytic characterization.
Moreover, despite reductions with A. ochraceus were briefly de-
scribed sometime ago,56,57 no recent progress in this topic has been
reported in the literature. Reductions of aromatic ketones and
keto-acid derivatives using Aureobasidium spp. were described as
well, showing moderate to good performances;58–60 4-chloro-3-
oxoesters were successfully reduced using different reaction sys-
4.1. Fungal biotransformations
Fifteen strains of fungi and yeasts were screened for their
reductase activity. All microorganisms belong to the collection of
the Microbiology Laboratory of the Biosciences Department (Facul-
tad de Química, UdelaR; http://mail.fq.edu.uy/~microbio/) and are
freely available upon request.
The culture medium contained (g/l): sucrose (50), (NH4)2SO4
(5), KH2PO4 (2), CaCl2ꢀ2H2O (0.25), and MgSO4ꢀ7H2O (0.25), except
for the culture of Trichoderma, where Czapec medium (pH 7.3) was
used, according to the literature.41
tems,61 but no reports are known of
a-alkyl-b-ketoesters reduction
with Aureobasidium spp.
Moreover, our reductions using the T. koningii (ATCC 76666)
strain led preferentially to the anti-(2S,3S) enantiomer (entry 8,
with syn:anti ratio 7:93; 94% ee), instead of the syn-(2S,3R) enan-
tiomer previously reported by Iwamoto et al. (anti:syn 28:72).41
4.2. Chemicals and analysis
Solvents were purified and dried by conventional methods.
Commercial reagents were purchased from Sigma–Aldrich Inc.
The purity of the reactants, as well as the development of the
reactions, were controlled using analytical TLC on silica gel (Kie-
selgel HF254 from Macherey-Nagel) and visualized with UV light
(254 nm) and/or p-anisaldehyde in acidic ethanolic solution. Fur-
ther analyses were performed by chiral gas chromatography (GC)
in Shimadzu 2010 equipment, with an FID detector and a Mega-
3. Conclusion
We conducted the screening of fifteen microbial strains of fungi
and yeasts. Most of the microorganisms showed a strong prefer-
ence for the anti-isomer, except for P. chrysosporum where the
syn-isomer was predominant, displaying a syn:anti ratio of 70:30.
Promising results were obtained with A. pullulans, which gave com-
plete conversion (100%) in short reaction times (24 h) via a highly
diastereo- and enantioselective process (syn:anti 3:97; 94% ee of
anti-(2S,3S) isomer). These results were confirmed on a preparative
scale. Moreover, five other strains (F. graminearum, A. terreus, G.
candidum, T. koningii, and A. niger) performed this biocatalytic
reduction with excellent yields (>95%), showing a remarkable pref-
erence for the anti-isomer and excellent enantioselectivities (>95%
ee for the anti-isomer). When using T. koningii (ATCC 76666) we
found opposite results compared to those reported by Iwamoto
et al.,41 preferentially obtaining the anti-(2S,3S) enantiomer with
high enantioselectivity, instead of the syn-(2S,3R).
dex DET-TBS (25 m ꢁ 0.25 mm ꢁ 0.25
lm) column. Temperature
program: 60 °C/1 °C/min/70 °C (10 min)/2 °C/min/150 °C (5 min).
TSPLIT: 220 °C, TFID: 250 °C. The % ee and absolute configuration
of the reduction products were determined by chiral gas chroma-
tography, comparing with the standards, as described in the text.
Column chromatography was performed using silica gel flash
(Kieselgel 60, EM reagent, 230–240 mesh.) from Macherey-Nagel.
NMR spectra (1H and 13C) were carried out in a Bruker Avance
DPX 400 MHz equipment. All experiments were carried out at
30 °C, CDCl3 was used as solvent and TMS was used as an
internal standard. Optical rotations were measured at 25 °C on
a Zuzi 412 polarimeter using a 0.5 dm cell. [a] values are given
D
in units of deg cm2 gꢂ1 and concentration values are expressed in
g/100 mL.
Considering the excellent selectivities uncovered, the findings
reported herein are a valuable contribution to the challenge of dis-
covering new biocatalysts for the highly stereoselective reduction
Biotransformations were conducted in orbital shakers from
Thermoforma (model 420) and Sanyo (model IOX400.XX2.C).

1396
S. P. Ravía et al. / Tetrahedron: Asymmetry 20 (2009) 1393–1397
4.3. Racemic ethyl 2-methyl-3-oxobutanoate 1
MgSO4, filtered, and distilled under vacuum at 30 °C. The residue
was purified by column chromatography (silica gel, Hex/AcOEt,
Ethyl 2-methyl-3-oxobutanoate was prepared from commer-
cially available ethyl 3-oxobutanoate by alkylation with methyl io-
dide, according to a procedure described in the literature.62 Ethyl
3-oxobutanoate (0.2 g, 1.54 mmol) was dissolved in anhydrous
acetone (2 mL) under a nitrogen atmosphere. Dried potassium car-
bonate (0.199 g, 1.44 mmol) was added with magnetic stirring. The
solution was maintained at room temperature for 10 min, and
methyl iodide (0.12 mL, 1.89 mmol) was added with a syringe.
The reaction was refluxed for 5 h. After the reaction was complete,
diethyl ether was added (3 mL), and the mixture was filtered. The
solvent was evaporated under vacuum, and the residue was
purified by flash column chromatography (silica gel, Hex/AcOEt,
v/v, 8:2), affording 2d. Isolated yield: 70%. ½a D20
¼ þ22:5 (c 1.20,
ꢃ
CHCl3).
4.5. CaL B-catalyzed transesterification of a diastereomeric
mixture of ethyl 3-hydroxy-2-methylbutanoate
The experiments were carried out in orbital shaker at 30 °C and
200 rpm. Vinyl acetate was used as the acyl donor, C. antarctica B
lipase (CaL B, Novozym 435) was the catalyst of choice and the dia-
stereomeric mixture of ethyl-3-hydroxy-2-methylbutanoate acted
as the nucleophile. The reactions were performed using 50 mg of
the diastereomeric substrate, the lipase/hydroxy ester ratio (w/
w) was 0.3, and the acyl donor/alcohol ratio (mol/mol) was 0.6.
Hexane was used as solvent. The reaction was complete in 3 h,
the enzyme was filtered-off through a short silica-gel column,
and the solvent was distilled under vacuum. Chiral CG analysis of
the crude was performed, showing complete consumption of the
(3R)-isomers.
v/v, 95:5) giving compound
1 d
(0.211 g, 95%). 1H NMR:
(ppm) = 4.24–4.19 (m, 2H, CH2), 3.49 (q, J = 7.2 Hz, 1H, CH), 2.26
(s, 3H, CH3), 1.35 (d, J = 7.2 Hz, 3H, CH3), 1.29 (t, J = 7.1 Hz, 3H,
CH3). 13C NMR: d (ppm) = 204.0, 170.9, 61.7, 54.0, 28.7, 14.4,
13.1. GC: tR = 27.9, 28.0 min.
4.4. Ethyl 3-hydroxy-2-methylbutanoates 2a–d
Acknowledgments
4.4.1. Chemical reduction
Sodium borohydride (0.244 g, 0.646 mmol) was added in dry
ethanol (15 mL) under a nitrogen atmosphere, and the mixture
was cooled to 0 °C and stirred for 10 min. A solution containing
ethyl 2-methyl-3-oxobutanoate (0.2 g, 1.4 mmol) in dry ethanol
(5 mL) was then added dropwise. After stirring for 3 h at 0 °C, the
reaction was quenched with saturated ammonium chloride, after
which ethanol was distilled under reduced pressure. Next 15 mL
of water was added, and then extracted thrice with ethyl acetate
(3 ꢁ 15 mL). The organic layer was dried over MgSO4, and the sol-
vent was distilled under vacuum. The residue was purified by flash
column chromatography (silica gel, Hex/AcOEt, v/v, 8:2) giving
compound 2a–d (diastereomeric mixture) (0.192 g, 95%). 1H
NMR: d (ppm) = 4.18–4.13 (q, J = 7.1, 4H, CH2), 4.05–4.02 (m, 1H,
CH), 3.89–3.84 (m, 1H, CH), 2.50–2.39 (m, 2H, CH), 1.26 (t, J = 7.1,
6H, CH3), 1.21–1.13 (m, 12H, CH3). 13C NMR: d (ppm) = 176.1,
69.7, 68.0, 61.0, 60.6, 47.4, 45.5, 21.0, 19.7, 14.5, 14.2, 11.5, 11.0.
GC: tR = 36.0 min (27.3%), tR = 33.1 min (26.5%), tR = 31.9 min
(23.2%) and tR = 29.5 min (22.9%).
The authors wish to thank Microbiology Laboratory (Facultad
de Química, Universidad de la República, Uruguay) for the gener-
ous gift of fungal strains, as well as Dr. Pilar Menéndez for kindly
giving us the strain of Aspergillus terreus (BFQU 121), Dr. C. Kurtz-
man for the strain of Kluyveromyces marxianus (NRRL Y-8281), Dr.
Jon D. Stewart and Dr. Sonia Rodríguez for the gift of recombinant
E. coli strains, and specially Dr. S. Rodríguez for helpful discussions.
We also thank OPCW (Organization for the Prohibition of Chemical
Weapons), DINACYT (PDT 77/22, Uruguay), and PEDECIBA (Progra-
ma de Desarrollo de las Ciencias Básicas, Uruguay) for financial
support. The reviewers’ advice on the assignment of configurations
is also appreciated.
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