Asymmetric reductions of ethyl 2-(benzamidomethyl)-3-oxobutanoate by yeasts

Article abstract of DOI:10.1016/j.tetasy.2009.02.023

The stereoselective reduction of ethyl 2-(benzamidomethyl)-3-oxobutanoate 1 using yeasts was investigated among a restricted number (12) of yeasts. Kluyveromyces marxianus var. lactis CL69 diastereoselectively produced (2R,3S)-ethyl 2-(benzamidomethyl)-3-hydroxybutanoate 2, whereas Pichia glucozyma CBS 5766 gave (2S,3S)-2 as the major stereoisomer. The biotransformations were independently optimized for minimizing by-product formation and maximizing the diastereoselectivity. Under optimized conditions, K. marxianus var. lactis CL 69 gave the (2R,3S)-ethyl 2-(benzamidomethyl)-3-hydroxybutanoate 2 with ee > 99% and de = 98%, while P. glucozyma CBS 5766 allowed for the production of (2S,3S)-2 with ee > 99% and de = 86%.

Full text of DOI:10.1016/j.tetasy.2009.02.023

Tetrahedron: Asymmetry 20 (2009) 411–414
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Tetrahedron: Asymmetry
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Asymmetric reductions of ethyl 2-(benzamidomethyl)-3-oxobutanoate by yeasts
b
c
c
b
Raffaella Gandolfi a, , Edoardo Cesarotti , Francesco Molinari , Diego Romano , Isabella Rimoldi
*
a Università degli Studi di Milano, Institute of Organic Chemistry ‘A. Marchesini’, Via Venezian 21, 20133 Milan, Italy
b Department of Inorganic, Metallorganic and Analytical Chemistry ‘L. Malatesta’, Via Venezian 21, 20133 Milan, Italy
c Department of Food Science and Microbiology, Via Mangiagalli 25, 20133 Milan, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The stereoselective reduction of ethyl 2-(benzamidomethyl)-3-oxobutanoate 1 using yeasts was investi-
gated among a restricted number (12) of yeasts. Kluyveromyces marxianus var. lactis CL69 diastereoselec-
tively produced (2R,3S)-ethyl 2-(benzamidomethyl)-3-hydroxybutanoate 2, whereas Pichia glucozyma
CBS 5766 gave (2S,3S)-2 as the major stereoisomer. The biotransformations were independently opti-
mized for minimizing by-product formation and maximizing the diastereoselectivity. Under optimized
conditions, K. marxianus var. lactis CL 69 gave the (2R,3S)-ethyl 2-(benzamidomethyl)-3-hydroxybutano-
ate 2 with ee > 99% and de = 98%, while P. glucozyma CBS 5766 allowed for the production of (2S,3S)-2
with ee > 99% and de = 86%.
Received 25 November 2008
Accepted 10 February 2009
Available online 25 March 2009
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
b-ketoacids which are spontaneously decarboxylated to the corre-
sponding ketones; the ketones thus produced can be further re-
The use of microbial cells as catalysts in organic chemistry is
attractive since they provide a number of stereoselective enzymes,
such as oxidoreductases and hydrolases.1 The simultaneous occur-
rence of different enzymes or isoenzymes in whole cells may affect
the overall selectivity; isolation of the desired enzyme or optimiza-
tion of culture and biotransformation conditions can reduce side-
reactions allowing for excellent yields and (stereo)-selectivity.2,3
Yeasts are commonly used for catalyzing different biotransforma-
tions, most often carbonyl reductions, since this microbial group
is known to produce stereoselective membrane-bound dehydro-
genases; decarboxylases and esterases are also produced as cell-
bound enzymes.4–6
duced. Several strategies have been employed to limit the
formation of these by-products, including the use of esters with
different alkyl groups,11,12 medium engineering13,14 or the addition
of specific inhibitors.15,16
Enantiomerically pure b-amino acids are useful building blocks
in the synthesis of many natural products and/or biologically active
compounds and a few reviews deal with their production by chem-
ical17–19 and enzymatic20 means. b-Hydroxy-b-amino acids are
valuable intermediates for drug syntheses such as in the preparation
of b-lactams;21,22 these moleculescan be obtained by hydrogenation
of the corresponding b-keto-b-amido esters with organometallic
catalysts23–25 or with reductases/hydrogenases.26–28
The yeast-mediated reduction of racemic
a-substituted b-keto-
In this work, we have focused our attention on the biotransfor-
esters has been thoroughly studied since in a few cases the car-
bonyl reduction occurs with concomitant dynamic kinetic
resolution of the C-2 stereocentre furnishing high yields of the cor-
mations of ethyl 2-(benzamidomethyl)-3-oxobutanoate 1 by
yeasts. This biotransformation has been previously carried out
using plant cells furnishing (2R,3S)-ethyl 2-(benzamidomethyl)-
3-hydroxybutanoate 2 using cultures of Parthenocissus tricuspidata
and (2S,3S)-2 with Gossypium hirsutum;26 the reactions occurred
with excellent stereoselectivity, but the substrate concentrations
used were quite low (0.12 mg/mL) and reaction times were 3 days.
Biotransformations of ethyl 2-phtalimidomethyl-3-oxobutanoate
catalyzed by whole cells and partially purified enzymes from dif-
ferent Kluyveromyces marxianus strains gave low diastereomeric
and enantiomeric excesses.27,28
responding enantiomerically pure
a-substituted b-hydroxyester
stereoisomers.7–9 This can be due to the action of dehydrogenases
on the enol form or occurrence of an enantioselective keto-reduc-
tase acting only on one enantiomer, leaving the opposite enantio-
mer unreacted and prone to enolization.8 It must be observed that
the processes of enolization might be also enzymatically
catalyzed.10
A drawback of the use of whole cells is the possible occurrence
of esterase activities catalyzing ester hydrolysis and furnishing
Herein, we report the stereoselective reduction of 1 with differ-
ent yeasts. The best results were obtained with K. marxianus var.
lactis CL69 which gave (2R,3S)-2, while Pichia glucozyma CBS
5766 gave (2S,3S)-2 as the predominant stereoisomer.
* Corresponding author. Tel.: +39 02 503 14609; fax: +39 02 503 14615.
E-mail address: raffaella.gandolfi@unimi.it (R. Gandolfi).
0957-4166/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2009.02.023

412
R. Gandolfi et al. / Tetrahedron: Asymmetry 20 (2009) 411–414
OH
O
OH
O
O
O
O
O
O
NH
NH
NH
+
O
O
O
1
(2R,3S)-
2
(2S,3S)-2
OH
O
O
O
OH
NH
NH
NH
O
O
O
CO2
3
4
(R)-5
Scheme 1. Biotransformations of ethyl 2-(benzamidomethyl)-3-oxobutanoate 1.
In the light of these preliminary results, we studied different
parameters (glucose and cell concentration, temperature, pH, sub-
strate concentration) of the biotransformations with K. marxianus
var. lactis CL 69 and P. glucozyma CBS 5766 with the aim of obtain-
ing high yields of 2 with high stereoselectivity. The best results
with K. marxianus var. lactis CL 69 were obtained with 30 g Lꢀ1 of
cells (dry weight), employed at pH 7.0, 30 °C and substrate concen-
tration of 0.5 g Lꢀ1 in the presence of 50 g Lꢀ1 of glucose; P. glu-
cozyma CBS 5766 furnished the best results with 20 g Lꢀ1 of cells
(dry weight), employed at pH 6.0, 27 °C and substrate concentra-
tion of 4.5 g Lꢀ1 in the presence of 30 g Lꢀ1 of glucose. The reduc-
tion of ethyl 2-(benzamidomethyl)-3-oxobutanoate 1 under these
conditions is reported in Table 2.
2. Results and discussion
A preliminary screening using conventional and non-conven-
tional yeasts for the modification of ethyl 2-(benzamidomethyl)-
3-oxobutanoate 1 was performed; the biotransformations of 1 cat-
alyzed by yeasts are summarized in Scheme 1.
Two products were observed in all cases: ethyl 2-(benzamidom-
ethyl)-3- hydroxybutanoate 2 and N-(3-oxobutyl)benzamide 4, the
latter product was further reduced to (R)-5 (ee > 99%) only by Pichi-
a etchellsii (Table 1). Carbonyl reduction of 1 occurred with high
enantioselectivity furnishing the (S)-stereocentre at C3 (Prelog’s
rule) with all the yeasts tested. N-(3-Oxobutyl)benzamide 4 is
formed through the action of cell-bound esterase(s) which pro-
Finally, preparative biotransformations aimed at producing
(2R,3S)-2 and (2S,3S)-2 were independently carried out on a 1 L
scale under optimized conditions. The reduction with K .marxianus
var. lactis CL69 was carried out with recycling of the biocatalyst:
the whole cells were re-used after centrifugation and a decrease
in activity was observed after 6 cycles, when 3 g of substrate was
totally converted and 2.67 g of (2R,3S)-2 was obtained after
duced the carboxylic acid
decarboxylation.
3 and subsequent spontaneous
The (2R,3S)/(2S,3S) ratio of 2 was variable. Among the microor-
ganisms which preferentially produce (2S,3S)-2 (anti-configura-
tion), P. glucozyma CBS 5766 gave the best diastereomeric excess,
although with low yields. K. marxianus var. lactis CL 69 furnished
the highest de of the syn-diastereoisomer (2R,3S).
Table 1
Reduction of ethyl 2-(benzamidomethyl)-3-oxobutanoate 1 by different yeasts
Microorganisms
Time (h)
2 (%)
ee 2 (%)
(2R,3S)/(2S,3S) 2 (%)
4 (%)
5 (%)
C. boidini CBS6056
K. lactis CBS2359
K. marxianus CBS1553
K. marxianus var. lactis CL69
P. anomala CBS110
P. etchellsii MIM
P. glucozyma CBS5766
S. cerevisiae CBS1782
S. cerevisiae CBS3093
S. cerevisiae CBS3081
S. cerevisiae NCYC73
S. cerevisiae Zeus
24
96
96
72
96
120
72
24
24
24
24
96
23
49
44
70
62
45
21
14
34
40
11
15
>99
>99
14
>99
>99
>99
> 99
>99
57
30/70
86/14
32/68
89/11
58/42
71/29
19/81
36/64
41/59
33/67
36/64
38/62
50
26
52
26
20
6
32
15
8
—
—
—
—
—
37
—
—
—
—
—
—
85
>99
>99
11
12
67
Table 2
Reduction of ethyl 2-(benzamidomethyl)-3-oxobutanoate 1 under the optimized conditions
Microorganisms
Time (h)
2 (%)
ee 2 (%)
(2R,3S)/(2S,3S) 2 (%)
4 (%)
P. glucozyma CBS5766
K. marxianus var. lactis CL69
24
24
72
100
>99
>99
7/93
99/1
15
—

R. Gandolfi et al. / Tetrahedron: Asymmetry 20 (2009) 411–414
413
purification. The biotransformation with P. glucozyma CBS 5766
was performed in a batch mode: 4.5 g Lꢀ1 of 1 was converted into
(2S,3S)-2 (2.75 g after purification) having a de = 86% and ee > 99%.
autoclave was pressurized (50 atm). At the end the autoclave was
vented and the mixture was analyzed by NMR spectra and HPLC.
By asymmetric hydrogenation of substrate 4 using (ꢀ)-tetraMe
BITIOP–Ru(II) complex we have obtained the product 5 with an
(R)-configuration.32
3. Conclusion
4.4. Microorganisms: culture conditions
We have shown that biotransformations of ethyl 2-(benzami-
domethyl)-3-oxobutanoate 1 with different yeasts lead to different
products with satisfactory yields and high stereoselectivity. Under
optimized conditions, K. marxianus var. lactis CL 69 gave the
(2R,3S)-ethyl 2-(benzamidomethyl)-3-hydroxybutanoate 2 with
ee > 99% and de = 98%, while P. glucozyma CBS 5766 allowed for
the production of (2S,3S)-2 with ee > 99% and de = 86%. These bio-
transformations are easier to perform and have better yields and
reaction times compared with the ones catalyzed by cell plants26
and have much higher stereoselectivities than the ones catalyzed
by dehydrogenases from K. marxianus CBS 6556 and KCTC 7155
using ethyl 2-phthalimidomethyl-3-oxobutanoate as substrate.27,28
Strains from official collections or from our collection (Microbi-
ologia Industriale Milano) were routinely maintained on malt ex-
, , pH 5.5). To obtain cells for
tract (8 g Lꢀ1 agar 15 g Lꢀ1
biocatalytic activity tests, the microorganisms were cultured in
500 mL Erlenmeyer flasks containing 100 mL of medium and were
incubated for 48 h at 28 °C on a reciprocal shaker (100 spm). The
yeasts were grown on malt extract with 5 g Lꢀ1 Difco yeast extract,
except P. glucozyma CBS 5766 grown on malt extract with 50 g Lꢀ1
Difco yeast extract. Fresh cells from submerged cultures were cen-
trifuged (5000 rpm per 100) and washed with 0.1 M phosphate buf-
fer, pH 7, prior to use.
4. Experimental
4.5. Bioreduction conditions
4.1. Ethyl 2-(benzamidomethyl)-3- hydroxybutanoate 2
General procedure for the screening: reductions were carried
out in 10 mL screw-capped test tubes with a reaction volume of
3 mL with cells (20 g Lꢀ1, dry weight) suspended in 0.1 M phos-
phate buffer, pH 7, containing 5% of glucose and 4 g Lꢀ1 of ethyl
2-(benzamidomethyl)-3-oxobutanoate as substrate 1. The reac-
tions were carried out at 28 °C with magnetic stirring.
Optimization studies were carried out with K. marxianus var.
lactis CL69 and P. glucozyma CBS 5766. The desired amount of cells
was suspended in different 0.1 M phosphate buffers containing
glucose and neat substrate was added to reach the desired concen-
tration; the suspensions obtained were magnetically stirred at dif-
ferent temperatures.
The biotransformation was stopped by centrifugation of the cel-
lular suspension; the liquid fraction was extracted with ethyl ace-
tate. The organic extracts were dried over Na2SO4, and the solvent
was removed under reduced pressure. After flash chromatography
on silica gel eluted with CH2Cl2–di-iso-propyl ether (2:1) the pure
products were obtained.
The standard was obtained by reduction with NaBH4 (1:5) in
ethanol. The absolute configuration was assigned in accordance
with the literature.29,30
4.2. Preparation of N-(3-oxobutyl)benzamide 4
N-(3-Oxobutyl)benzamide 4 was obtained by biotransforma-
tion. Therefore, 2 g Lꢀ1 of ethyl 2-(benzamidomethyl)-3- oxobut-
anoate 1 and 10 g Lꢀ1 of lipase from Candida cylindracea were
added in 0.1 M phosphate buffer, pH 7. The biotransformation
was carried out at 30 °C under magnetic stirring. After 72 h, the
reaction was extracted three times with ethyl acetate. The col-
lected organic phases were dried over Na2SO4 and reduced under
vacuo. The crude extract was purified with preparative TLC on sil-
ica gel (Kiesel 60 with fluorescent indicator) and was characterized
by mass spectroscopy (Thermo Finnigan LCQ Advantage system)
and 1H NMR (Varian 200 MHz). 1H NMR(CDCl3): d = 2.20 (s, CH3);
2.81–2.85 (t, CH2); 3.68–3.74 (q, CH2); 6.89 (br, NH); 7.41–7.77
(m, C6H5). C11H13NO2 calculated: 191.23, found 214.08
(M++Na(23)).
4.6. Analytical methods
Five hundred microliters of the crude reaction mixture were ex-
tracted with ethyl acetate. The organic phases were dried on
Na2SO4 and were analyzed. The course of the reaction was moni-
tored by TLC on silica gel Kiesel 60 with fluorescent indicator
(CH2Cl2:diethyl ether = 2:1) and the molar conversion and diaste-
reoisomeric and enantiomeric excess were determined by chiral
HPLC analysis (Merck-Hitachi L-7100 equipped with Detector
UV6000LP) on CHIRALPACK AD column (hexane:iso-propa-
nol = 90:10, flow:0.6 mL minꢀ1, detector: diode array).
4.3. Preparation of N-(3-hydroxybutyl)benzamide 5
The N-(3-oxobutyl)benzamide was reduced by NaBH4 (1:5) in
ethanol. The reaction was maintained at room temperature under
magnetic stirring. After 12 h, were added water and CH2Cl2 to re-
cover the product 5. The organic extract was dried over Na2SO4
and concentrated in vacuo. The product was purified via prepara-
tive TLC and was analyzed. 1H NMR(CDCl3): d = 1.27 (d, CH3);
1.65–1.77 (m, CH2); 3.27–3.42 (m, CH); 3.91–3.95 (m, CH2); 6.80
(br, NH); 7.41–7.80 (m, C6H5), C11H15NO2 calculated: 193.25, found
216.0 (M++Na(23)).
The absolute configuration was assigned by catalytic asymmet-
ric hydrogenation with Ru(II) complexes prepared with (ꢀ)-tet-
raMe BITIOP31 as a chiral ligand.
In a Schlenk tube sealed under argon the substrate was added to
the precatalyst followed by 20 mL of a solvent, the solution was
stirred for 30 min and then transferred to an autoclave with a
cannula.
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