Exploring the potential of some yeast strains in the stereoselective synthesis of aldol reaction products and its reduced 1,3-dialcohol derivatives

Article abstract of DOI:10.1016/j.molcatb.2013.03.017

The behavior of two yeast strains has been studied under different conditions. Both microorganims catalyzed the aldol reaction between activated aldehydes and acetone when a large amount of the latter was present in the reaction medium producing, with mod

Full text of DOI:10.1016/j.molcatb.2013.03.017

Journal of Molecular Catalysis B: Enzymatic 92 (2013) 57–61
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Journal of Molecular Catalysis B: Enzymatic
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Exploring the potential of some yeast strains in the stereoselective synthesis of
aldol reaction products and its reduced 1,3-dialcohol derivatives
Cecilia Andreua,∗, Marcel·lí del Olmob,∗∗
a Departament de Química Orgànica, Universitat de València-Estudi General, Spain
b Departament de Bioquímica i Biologia Molecular, Universitat de València-Estudi General, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The behavior of two yeast strains has been studied under different conditions. Both microorganims cat-
alyzed the aldol reaction between activated aldehydes and acetone when a large amount of the latter
was present in the reaction medium producing, with moderate stereoselectivity, the aldol product with
the R configuration. No reduction of any of the products present in the medium was detected. On the
other hand, the carbonyl group of the racemic aldol was reduced to produce chiral 1,3-dialcohol deriva-
tives when water was employed as the only solvent. In this case, the resolution of the racemic starting
material was also possible with one of the biocatalysts, and the aldol was recovered with the S config-
uration. A complementary enantioselectivity was shown by both microorganisms in the generation of
the new stereogenic center, which allowed access to 3 of the 4 possible diastereomeric diols with high
enantiomerical purity.
Received 31 January 2013
Received in revised form 25 March 2013
Accepted 25 March 2013
Available online 3 April 2013
Keywords:
Aldol reaction
Asymmetric catalysis
Biocatalysis
Carbonyl reduction
Yeast
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
strategies for efficient cofactor recycling or multistep conversions
than the utilization of isolated enzymes as catalysts [5].
Biocatalysis refers to the use of natural enzymes to perform
chemical transformations of organic compounds. Enzymes used as
catalysts in synthesis offer considerable advantages, not the least
of which include their high efficiency and their capacity to produce
regio- and stereoselectively building blocks of great value and util-
ity in the pharmaceutical and food industries [1]. If compared with
conventional chemical catalysis, biocatalysis is the most conve-
nient from the experimental and ecological points of view: its high
specificity results in few side products, the reaction conditions are
very mild, and the low production level of waste pollutants involves
a negligible environmental impact [2]. In the biocatalysis field, not
only have isolated enzymes been used, but also whole microorgan-
isms [3]. Whole cells of different yeast species have been widely
used for a number of asymmetric transformations, especially in
bioreduction reactions for the preparation of chiral alcohols [4].
The use of whole cells entails simpler catalyst preparation and easy
Catalytic promiscuity has been recognized as enzymes’ ability
to catalyze different chemical transformations depending on the
reaction conditions [6]. Whole cell catalysts containing multiple
enzymes are also capable of catalyzing different reactions depend-
ing on the medium conditions and the substrates supplied. This
potential has been exploited for synthetic purposes [4a–c].
The stereoselective aldol reaction is considered one of the most
important carbon-carbon bond-forming reactions in organic syn-
thesis, and is a way to obtain chiral ␤-hydroxy carbonyl compounds
[7]. Recently, we described the use of different yeast strains to
study the aldol reaction between acetone and p-nitrobenzaldehyde.
We carried out the reaction using lyophilized cells in an organic
medium (acetone), and studied the influence of different condi-
tions to achieve the best results from the stereoselective viewpoint.
In all cases the excess enantiomer shows R configuration, although
moderate conversion and stereoselectivity were achieved for all the
strains checked. In this study we were unable to identify the respec-
tive enzyme responsible for this catalytic activity, not discarding
the possibility that some amino acid residue acts as organocata-
lyst in the reaction. Under the reported conditions, the carbonyl
reduction of the starting materials did not occur, and only the aldol
product and the aldehyde substrate were recovered in all cases [8].
Chiral 1,3-diols, with two stereogenic centers, are impor-
tant building blocks in the synthesis of pharmaceutically active
compounds [9]. All the possible stereoisomers of these inter-
esting compounds have been obtained by different groups by
∗
Corresponding author at: Departament de Química Orgànica, Facultat de Far-
màcia, Vicent Andrés Estellés s/n, E-46100 Burjassot, València, Spain.
Tel.: +34 96 3543048; fax: +34 96 3544328.
∗∗
Corresponding author at: Departament de Bioquímica i Biologia Molecular, Fac-
ultat de Biologia, Dr. Moliner 50, E-46100 Burjassot, València, Spain.
Tel.: +34 96 3543355; fax: +34 96 3544635.
E-mail addresses: cecilia.andreu@uv.es (C. Andreu), m.del.olmo@uv.es
(M. del Olmo).
1381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.03.017

58
C. Andreu, M. del Olmo / Journal of Molecular Catalysis B: Enzymatic 92 (2013) 57–61
combining organocatalysis, for aldol asymmetric reaction between
an aldehyde and acetone, and biocatalysis, using isolated alcohol
dehydrogenases, for the asymmetric carbonyl reduction of the aldol
product [10].
In this work we describe results obtained with whole cells from
yeasts Pichia jardinii (CECT 1060) [11] and Kluyveromyces marxianus
(CECT 1018) [11] as biocatalysts under different reaction condi-
tions. Selection of the medium, substrate and biocatalyst allowed
us to prepare chiral aldols in both configurations and/or their chiral
1,3-diols derivatives.
R; tr = 49.8 min S); Chiralpak IC column, Hex/iprOH 92/8, 1 mL/min,
254 nm (tr = 24.4 min R; tr = 41.9 min S).
2.2.3. 4-(3-Nitrophenyl)-4-hydroxybutan-2-one (2c) [12]
1H NMR (CDCl3): 2.23 (s, 3H), 2.90 (m, 2H), 3.59 (s, 1H), 5.30
(m, 1H), 7.53 (m, 1H), 7.70 (m, 1H), 8.09 (m, 1H), 8.24 (m, 1H).
Enantiomeric excess: HPLC, Chiralpak IA column, Hex/iprOH 97/3,
0.8 mL/min (tr = 70 min R; tr = 80 min S).
2.2.4. 4-(4-Chlorophenyl)-4-hydroxybutan-2-one (2d) [12]
1H NMR (CDCl3): 2.19 (s, 3H), 2.80 (m, 2H), 3.38 (s, 1H), 5.11
(dd, 8, 4 Hz, 1H), 7.30 (m, 4H). Enantiomeric excess: HPLC, Chiral-
pak IA column, Hex/iprOH 97/3, 1 mL/min, 214 nm (tr = 25.5 min R;
tr = 27.3 min S).
2. Experimental
2.1. General
All the commercially available reagents were purchased
from Sigma–Aldrich. Reactions were monitored by thin layer
chromatography (TLC) on Merck silica plates 60 F254. Flash chro-
matography was performed on Merck silica gel (60 particle size:
0.040–0.063 mm). The NMR spectra were recorded with Bruker
DRX 300 spectrometers using deuterated chloroform as solvent.
Chemical shifts are reported in ppm in relation to the residual
solvent peak. Absolute configurations were determined by compar-
ison with the optical rotations reported in the literature and these
were performed on a Perkin Elmer 241 Polarimeter at ꢀ = 589 nm.
Determination of enantiomeric excess was carried out by high
performance liquid chromatography (HPLC), with a Merck Hitachi
Lachrom system. The specific conditions are described for each case.
Racemic mixtures of diastereomeric dialcohols were synthe-
sized by reduction of the respective racemic aldol with NaBH4 in
methanol following standard methods. They were used as HPLC
patterns.
2.2.5. 4-(2-Chlorophenyl)-4-hydroxybutan-2-one (2e) [12]
1H NMR (CDCl3): 2.21 (s, 3H), 2.68 (dd,18, 9 Hz, 1H), 2.98 (dd,18,
2.2 Hz, 1H), 3.65 (s, 1H), 5.50 (dd, 9, 2.1 Hz, 1H), 7.20 (m, 1H), 7.34
(m, 2H), 7.62 (m, 1H). Enantiomeric excess: HPLC, Chiralpak IA col-
umn, Hex/iprOH 97/3, 1 mL/min, 214 nm (tr = 16 min R; tr = 17 min
S).
2.3. General procedure for the reduction of racemic aldols 2
To ensure that in independent experiments the same amount of
biomass was used, the OD600 (which measures the turbidity of the
cellular suspension) was determined in overnight cultures of the
microorganisms in YPD medium (1% (w/v) yeast extract, 2% (w/v)
bactopeptone, 2% (w/v) glucose), and the volume corresponding to
500 OD600 was taken for each experiment (for example, 50 mL of a
culture with OD600 of 10). After centrifugation, the pellet was resus-
pended in 25 mL of sterile water containing 2.5% glucose (to ensure
the metabolic activity of the cells, and guarantee the regeneration
of the cofactor NADH). The mixture was incubated in a flask at 30 ◦C
for 30 min with orbital shaking. Then racemic aldol 2 (10 mg) was
added, and the reaction was maintained under the above condi-
tions for the time indicated in each case (previously determined
by a chiral chromatography analysis of the various aliquots taken
at different times). Next 1 mL of 40% glucose was added peri-
odically every 24 h during the incubation time. Afterwards, the
reaction mixture was centrifuged and the aqueous supernatant was
extracted with ethyl acetate (3× 20 mL). Organic phases were com-
bined and dried with sodium sulfate. After solvent evaporation, the
crude material was analyzed by 1H NMR, to determine the percent-
age of transformation, and also by chiral HPLC. Afterward, it was
purified by column chromatography (hexanes:ethyl acetate 5:1) to
afford quantitatively the products described latter.
2.2. Typical procedure for biocatalytic aldol condensation using
dried whole cells [8]
Reactions were carried out in capped vials (15 mL), where
lyophilized cells (40 mg), prepared as we described previously [8],
were resuspended in 2.5 mL of the solvent mixture (acetone:water,
97.5:2.5 or 1:1). The corresponding aldehyde (4 mg) was added
and the mix was shaken on an orbital shaker at 25 ◦C. The reac-
tion was monitored by tlc and, finally, the mixture was centrifuged
(3500 rpm, 3 min), treated with an ammonium chloride solution
and acetone was evaporated in vacuum. The aqueous solution was
extracted with methylene chloride and the organic phase was dried
over anhydrous sodium sulfate. Crude material was employed to
determine the yield by NMR and the ee by HPLC using the chiral
stationary phase.
The reaction products were purified by column chromatography
(hexane:ethyl acetate 4:1) and characterized by NMR and chiral
HPLC. Data were consistent with those described in the literature
[12].
2.3.1. 1-(4-Nitrophenyl)-1,3-butanediol (3a)[10]
syn- 1H NMR (CDCl3): 1.27 (d, 6.2 Hz, 3H), 1.76–1.82 (m, 2H),
2.34 (s, 1H), 4.05 (s, 1H), 4.2–4.26 (m, 1H), 5.05–5.09 (m, 1H), 7.54
(d, 8.3 Hz, 2H), 8.20 (d, 8.3 Hz, 2H) ppm.
anti- 1H NMR (CDCl3): 1.28 (d, 6.3 Hz, 3H), 1.89–1.92 (m, 2H),
2.00 (s, 1H), 3.55 (s, 1H), 4.04–4.10 (m, 1H), 5.16–5.20 (m, 1H), 7.55
(d, 8.4 Hz, 2H), 8.21 (d, 8.4 Hz, 2H) ppm.
Enantiomeric excess: HPLC, Chiralpak IC column, Hex/iprOH
96/4, 1 mL/min, 254 nm, ((+)1R,3R 49.2 min; (−)1S,3S 42.0 min;
(+)1R,3S 33.4 min).
2.2.1. 4-(4-Nitrophenyl)-4-hydroxybutan-2-one (2a)[12]
1H NMR (CDCl3): 2.21 (s, 3H), 2.84 (m, 2H), 3.60 (s, 1H), 5.26 (m,
1H), 7.53 (d, 8.8 Hz, 2H), 8.20 (d, 8.8 Hz, 2H) ppm. Enantiomeric
excess: HPLC, Chiralpak IC column, Hex/iprOH 94/6, 1 mL/min,
254 nm (tr = 31.5 min S; tr = 33.6 min R).
2.2.2. 4-(2-Nitrophenyl)-4-hidroxybutan-2-one (2b) [12]
2.3.2. 1-(2-Nitrophenyl)-1,3-butanediol (3b)
1H NMR (CDCl3): 2.16 (s, 3H), 2.63 (dd, 17.8 Hz, 9.4 Hz, 1H), 3.09
(dd, 17.8 Hz, 2.0 Hz, 1H), 3.90 (s, 1H), 5.60 (m, 1H), 7.46 (m, 1H), 7.67
(m, 1H), 7.88 (m, 1H), 7.96 (m, 1H). Enantiomeric excess: HPLC, Chi-
ralpak IA column, Hex/iprOH 97/3, 0.8 mL/min, 254 nm (tr = 47 min
syn- 1H NMR (CDCl3): 1.27 (d, 6.2 Hz, 3H), 1.5 (s, 1H). 1.69–1.81
(m, 1H),1.97–2.09 (m, 1H), 4.0 (s, 1H), 4.20–4.34 (m, 1H), 5.49 (dd,
9.8, 1.8 Hz, 1H), 7.41 (dd, 8.3, 1.6 Hz, 1H), 7.66 (dd, 8, 1 Hz, 1H),
7.91–7.94 (m, 2H) ppm.

C. Andreu, M. del Olmo / Journal of Molecular Catalysis B: Enzymatic 92 (2013) 57–61
59
anti- 1H NMR (CDCl3): 1.27 (d, 6.3 Hz, 3H), 1.5 (s, 1H), 1.90–1.93
(m, 2H), 3.6 (s, 1H), 4.05–4.15 (m, 1H), 5.58–5.62 (m, 1H), 7.35 (dd,
8.0, 1.4 Hz, 1H), 7.57 (dd, 7, 1.2 Hz, 1H), 7.85–7.89 (m, 2H) ppm.
Enantiomeric excess: HPLC, Chiralpak IC column, Hex/iprOH
92/8, 1 mL/min, 254 nm, ((+)1S,3S 25.81; (−)1R,3S 17.38 min).
O
OH
O
O
Biocat.
+
H
X
X
2a (X= 4-NO2)
2b (X= 2-NO2)
2c (X= 3-NO2)
2d (X= 4-Cl)
1a (X= 4-NO2)
1b (X= 2-NO2)
1c (X= 3-NO2)
1d (X= 4-Cl)
2.3.3. 1-(3-Nitrophenyl)-1,3-butanediol (3c)
syn- 1H NMR (CDCl3): 1.27 (d, 6.3 Hz, 3H), 1.6 (s, 2H), 1.79–1.84
(m, 2H), 4.17–4.24 (m, 1H), 5.04–5.09 (m, 1H), 7.54–7.64 (m, 1H),
7.71–7.74 (m,1H), 8.11–8.14 (m, 1H), 8.24–8.26 (m, 1H) ppm.
anti- 1H NMR (CDCl3): 1.29 (d, 6.3 Hz, 3H), 1.58 (s, 1H), 1.90–1.94
(m, 2H), 3.57 (s, 1H), 4.06–4.12 (m, 1H), 5.16–5.20 (m, 1H),
7.50–7.53 (m, 1H), 7.71–7.73 (m,1H), 8.10–8.14 (m, 1H), 8.25–8.27
(m, 1H) ppm.
2e (X= 2-Cl)
1e (X= 2-Cl)
Scheme 1. Biocatalytic aldol reaction.
(2.5% water in acetone used as a solvent) [8]. In all cases, the R
enantiomer was obtained in moderate excess, the stereoselectivity
being slightly better when K. marxianus was used as a catalyst.
In reactions carried out in a 1:1 mixture of acetone and water as
a solvent at 30 ◦C, the reaction was quantitative after 72 h (as shown
in Scheme 1), but no stereoselectivity was achieved with any of the
catalysts under these conditions (less than 5% ee). Unfortunately,
the reaction was effective only when strongly activated aldehydes
were used as substrates.
It is worth mentioning that under both conditions the carbonyl
reduction of the starting materials does not occur, and only the
aldol product and the aldehyde substrate are recovered in all cases.
Based on these results, we decided to explore the behaviour of
these microorganisms under different conditions. So, first of all, we
checked their ability to reduce the racemic aldol product derived
from 4-nitrobenzaldehyde and acetone ( 2a) using fresh cells in
aqueous medium (2% glucose w/v). Scheme 2 shows the comple-
mentary results obtained for both biocatalysts.
K. marxianus showed excellent stereoselectivity in the resolu-
tion of the racemic starting material and allowed the recovery of the
S enantiopure aldol (S-2a,95% ee). Moreover excellent enantioselec-
tivity (95%) was achieved in the 1,3-diol product of the reduction
reaction. Indeed, the syn diol with the 1R,3R configuration was
almost the only product. On the other hand, P. jardinii was unable
to resolve the racemic mixture, and both diastereomers (rela-
tion syn:anti 1.17) with excellent enantioselectivity were obtained
(1S,3S–3a, 95% ee and 1R,3S–3a, 99% ee).
Enantiomeric excess: HPLC, Chiralpak IC column, Hex/iprOH
95/5, 1 mL/min, 254 nm, ((−)1S,3S 60.1 min; (+)1R,3S 31.4 min).
2.3.4. 1-(4-Chlorophenyl)-1,3-butanediol (3d)[10]
syn- 1H NMR (CDCl3): 1.24 (d, 6.0 Hz, 3H), 1.69–1.87 (m, 2H),
2.64 (s, 1H), 3.38 (s, 1H), 4.10–4.19 (m, 1H), 4.93 (dd,9.6, 3.3 Hz,1H),
7.31 (s, 4H) ppm.
anti- 1H NMR (CDCl3): 1.25 (d, 6.3 Hz, 3H), 1.84–1.89 (m, 2H),
2.1 (s, 1H), 3.04 (s, 1H), 4.04–4.10 (m, 1H), 5.03–5.07 (m,1H), 7.31
(s, 4H) ppm.
Enantiomeric excess: HPLC, Chiralpak IA column, Hex/iprOH
97/3, 1 mL/min, 214 nm, ((+)1R,3R 29.1 min; (−)1S,3S 28.04 min;
(+)1R,3S 36.8 min).
2.3.5. 1-(2-Chlorophenyl)-1,3-butanediol (3e) [10]
syn- 1H NMR (CDCl3): 1.19 (d, 6.0 Hz, 3H), 1.56–1.67 (m, 1H),
1.81–1.87 (m, 1H), 2.7 (s, 1H), 3.46 (s, 1H), 4.13–4.22 (m, 1H), 5.28
(dd, 9.9, 1.8 Hz; 1H), 7.13 (dd, 6.6, 1.8 Hz,1H) 7.21–7.26 (m, 2H),
7.55–7.58 (m, 1H) ppm.
anti- 1H NMR (CDCl3): 1.22 (d, 6.0 Hz, 3H), 1.84–1.87 (m, 2H), 2.1
(s, 1H), 3.2(s, 1H), 3.98–4.04 (m, 1H), 5.36–5.42 (m,1H), 7.13 (dd, 6.9,
1.8 Hz,1H) 7.24 (dd, 7.8, 0.9 Hz, 2H), 7.55–7.59 (m, 1H) ppm.
Enantiomeric excess: HPLC, Chiralcel ODH column, Hex/iprOH
98/2, 1 mL/min, 214 nm, ((+)1R,3R 24.13 min; (−)1S,3S 45 min;
(+)1R,3S 32.7 min).
Both microorganisms showed a different enantioselectivity in
the generation of the new stereogenic center (R in the case of
K. marxianus and S when P. jardinii was used), and three of the
four possible diol stereoisomers were available. In order to obtain
the 1,3-diol with 1S,3R configuration, we unsuccessfully screened
seven other yeast strains [11]. Saccharomyces cerevisiae Lalvin T-
73, Torulospora delbrueckii, Saccharomyces cerevisiae FY86, Pichia
2.3.6. 1-Phenyl-1,3-butanediol (3f)[13]
syn- 1H NMR (CDCl3): 1.23 (d, 6.0 Hz, 3H), 1.73–1.93 (m, 2H),
2.89 (s, 1H), 3.09 (s, 1H), 4.13–4.18 (m, 1H), 4.95 (dd, 9.9, 3 Hz, 1H),
7.35–7.37 (m, 5H) ppm.
anti- 1H NMR (CDCl3): 1.25 (d, 6.0 Hz, 3H), 1.86–1.93 (m, 2H), 2.2
(s, 1H), 2.84 (s, 1H), 4.05–4.11 (m, 1H), 5.04–5.1 (m, 1H),7.35–7.40
(m, 5H) ppm.
Enantiomeric excess: HPLC, Chiralpak IC column, Hex/iprOH
96/4, 1 mL/min, 214 nm, ((+)1R,3R 35.2 min; (−)1S,3S 24.7 min;
(+)1R,3S 28.46 min).
Table 1
Scope of aldehydes for the aldol reaction with acetone under optimized conditions.
Aldehyde
Biocatalyst
Conversion (%)a
Product/ee (%)b
3. Results and discussion
1a[8]
1a[8]
1b
1b
1c
1c
1d
1d
1e
P. jardinii
K. marxianus
P. jardinii
K. marxianus
P. jardinii
K. marxianus
P. jardinii
K. marxianus
P. jardinii
K. marxianus
35
42
25
25
30
30
nd
nd
80
60
(R)–2a/30
(R)–2a/38
(R)–2b/35
(R)–2b/47
(R)–2c/27
(R)–2c/44
(R)–2d/33
(R)–2d/50
(R)–2e/39
(R)–2e/50
Microorganisms used as whole cell catalysts can display dif-
ferent enzymatic activities depending on the medium conditions
and the substrates supplied [3,4]. In a previous work, we stud-
ied the ability of several yeast strains to carry out asymmetric
aldol reaction between 4-nitrobenzaldehyde and acetone to yield
4-(4-nitrophenyl)-4-hydroxybutan-2-one (2a). For this purpose,
lyophilized cells from stationary phase cultures were employed.
In the present work, we wanted to extend the reaction described
above to other biocatalysts and substrates. Scheme 1 and Table 1
show the results obtained with the microorganisms K. marxianus
and P. jardinii for the reaction of different activated benzaldehy-
des and acetone under conditions that were previously optimized
1e
Conditions [8]: lyophilized biocatalyst (40 mg), substrate (4 mg), water in acetone
(2.5-vol.%, 2.5 mL), 25 ◦C; 96 h.
a
Conversion: determined by 1H NMR. No reaction was observed in the absence
of biocatalyst.
b
Determined by chiral HPLC. Absolute configuration according to literature data
[12].

60
C. Andreu, M. del Olmo / Journal of Molecular Catalysis B: Enzymatic 92 (2013) 57–61
O
OH
O
O
K. marxianus
H2O 2.5%
+
H
R-2a (38% ee)8
O2N
O2N
1a
H2O 50%
K. marxianus
OH
OH
O
OH
+
K. marxianus
(1R, 3R)-3a (95% ee)
S-2a (95% ee)
H2O/Glucose
O
OH
O2N
O2N
OH
OH
OH
OH
+
(+/-) 2a
O2N
P. Jardinii
H2O/Glucose
O2N
(1S, 3S)-3a (95% ee)
O2N
(1R, 3S)-3a (99% ee)
H2O 50%
P. Jardinii
O
OH
O
O
P. Jardinii
H
+
R-2a (30% ee) 8
H2O 2.5%
O2N
O2N
1a
Scheme 2. Use of both yeast strains allows access to a structural and stereochemical diversity of compounds, depending on the starting reagents and conditions.
Table 2
Reduction of several racemic aldols with yeast K. marxianus.
Substrate/time (h)/conversion (%)b
Product 2/ee (%)d
Product 3- syn/antic
Product 3-conf/ee (%)d
(
(
(
(
(
(
)2a/96/60
)2b/96/10
)2c/96/10
)2d/96/50
)2e/72/52
)2fa/72/62
S–2a/95
–
–
S–2d/57
S–2e 78
S–2f/92
83/17
–
–
83/17
90/10
60/40
1R,3R-3a/95
–
–
1R,3R-3d/99
1R,3R-3e/85
1R,3R-3f/99
Conditions: biocatalyst (500 OD600 unit) in 25 mL of sterile water containing 2.5% glucose, substrate (10 mg), 30 ◦C.
a
2f (X = H).
b
Calculated by 1H NMR.
c
Relation between diastereomeric dialcohols was determined by both 1H NMR and chiral HPLC. Assignation of the 1H NMR signals for each one was done in accordance
with the literature data [10,13] and was confirmed by NOE experiments (Supplementary Data).
d
Absolute configuration was established in accordance with the literature data [10,12,13]. Enantioselectivity was less than 5% for the minoritary anti diastereomer.
glucozyma, and Pichia fermentants were inactive in the reduction
of the starting material. Ogatea minuta showed the same stere-
oselectivity than P. jardinii, but was considerably less active in
the reduction reaction. On the other hand, Debaromyces etchellsii
produced the four possible diasteroemers with some diastereose-
lectivity (syn:anti 2) but without enantioselectivity.
Then, we decided to explore the general scope of this method-
ology. Racemic aldols obtained by condensation of acetone
and several aromatic aldehydes were employed as substrate
(Tables 2 and 3). The stereoselectivity and the reactivity of the reac-
tion were affected by the nature and position of the substituents
in the aromatic ring when K. marxianus was the biocatalyst. Thus,
2- and 3-nitro derivatives (2b, 2c) were not transformed by this
microorganism, but surprisingly 2-chloro (2e) was. In those cases
in which reduction was possible, the transformation of the starting
material fell between 50 and 60% after 72–96 h and the enan-
tioselectivity in the resolution of the racemic aldol was between
good and excellent. The stereoselectivity in syn diol production was
Table 3
Reduction of several racemic aldols with yeast P. jardinii.
Substrate/time (h)/conversion (%)b
Product 3 [syn/anti]c
Product 3 syn/conf/ee(%)d
Product 3 anti/conf/ee (%)d
(
(
(
(
(
(
)2a/48/96
)2b/96/72
)2c/48/86
)2d/96/90
)2e/72/95
)2fa/72/89
3a [54/46]
3b [63/37]
3c [53/47]
3d [55/45]
3e [51/49]
3f [50/50]
(1S,3S)/95
(1S,3S)/99
(1S,3S)/99
(1S,3S)/99
(1S,3S)/99
(1S,3S)/99
(1R,3S)/99
(1R,3S)/99
(1R,3S)/99
(1R,3S)/99
(1R,3S)/99
(1R,3S)/99
Conditions: biocatalyst (500 OD600 unit) in 25 mL of sterile water containing 2.5% glucose, substrate (10 mg), 30 ◦C.
a
2f (X = H).
b
Calculated by 1H NMR.
c
Relation between diastereomeric dialcohols was determined by both 1H NMR and chiral HPLC. Assignation of the 1H NMR signals for each one was done in accordance
with the literature data [10,13] and was confirmed by NOE experiments (Supplementary Data).
d
Absolute configuration was established according to the literature data [10,12,13].

C. Andreu, M. del Olmo / Journal of Molecular Catalysis B: Enzymatic 92 (2013) 57–61
61
always excellent. In the case of P. jardinii, all the racemic aldols
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Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.molcatb.2013.03.017.