Bioreduction of prochiral ketones by growing cells of Lasiodiplodia theobromae: Discovery of a versatile biocatalyst for asymmetric synthesis

Article abstract of DOI:10.1016/j.molcatb.2010.01.023

Growing cells of the phytopathogen fungus Lasiodiplodia theobromae in potato dextrose broth have shown their potential for the stereoselective bioreduction of different prochiral aromatic and aliphatic ketones. Optically active alcohols were obtained under mild reaction conditions in high conversions (up to 90%) and moderate to excellent enantioselectivity (35-≥99% ee) depending on the ketone structure. Prelog alcohols were isolated, except in the bioreduction of cyclohexylmethylketone and octan-2-one where anti-Prelog alcohols were obtained.

Full text of DOI:10.1016/j.molcatb.2010.01.023

Journal of Molecular Catalysis B: Enzymatic 65 (2010) 37–40
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Bioreduction of prochiral ketones by growing cells of Lasiodiplodia theobromae:
Discovery of a versatile biocatalyst for asymmetric synthesis
Bartholomeu A. Barros-Filhoa, Fátima M. Nunesb, Maria da Conceic¸ ão F. de Oliveirac,∗
Telma L.G. Lemosc, Marcos C. de Mattosc, Gonzalo de Gonzalod,
Vicente Gotor-Fernándezd, Vicente Gotord,∗∗
,
a Instituto Federal do Piauí, Rua Francisco Urquiza Machado, 462, 64800-000 Floriano, PI, Brazil
b Departamento de Biologia, Universidade Federal do Pará, Caixa Postal 479, Belém, PA, Brazil
c Departamento de Química Orgânica e Inorgânica, Laboratório de Biotecnologia e Síntese Orgânica (LABS), Caixa Postal 6044, Campus do Pici, 60455-970 Fortaleza, CE, Brazil
d Departamento de Química Orgánica e Inorgánica, Instituto Universitario de Biotecnología de Asturias, Universidad de Oviedo, c/Julián Clavería 8, 33006 Oviedo, Asturias, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 1 February 2010
Growing cells of the phytopathogen fungus Lasiodiplodia theobromae in potato dextrose broth have shown
their potential for the stereoselective bioreduction of different prochiral aromatic and aliphatic ketones.
Optically active alcohols were obtained under mild reaction conditions in high conversions (up to 90%)
and moderate to excellent enantioselectivity (35–≥99% ee) depending on the ketone structure. Prelog
alcohols were isolated, except in the bioreduction of cyclohexylmethylketone and octan-2-one where
anti-Prelog alcohols were obtained.
Keywords:
Bioreduction
Chiral alcohols
Ketones
Lasiodiplodia theobromae
Stereoselective transformations
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
the corresponding regeneration system are generally required for
the correct working of the enzyme. In contrast, the use of whole
In the last years, biocatalysis has been established as a very effi-
cient and environmentally friendly methodology for the production
of a varied set of enantiopure compounds with application to indus-
trial sectors [1–7]. Among the vast number of enzymes, lipases,
lyases, oxidoreductases and transferases have demonstrated their
outstanding potential in the production of optically active alco-
hols under generally benign reaction conditions, catalyzing clean
bioprocesses with remarkable levels of chemo-, regio- and stere-
ospecificity [8].
Chiral alcohols and their derivatives are of special interest in
organic chemistry due to their presence in a variety of biologically
active compounds such as agrochemicals and pharmaceuticals.
Besides, enantiomerically pure alcohols are essential and versatile
chiral building-blocks for the preparation of more complex struc-
tures. Probably, the bioreduction of prochiral ketones is the most
common biotransformation for the production of a wide range of
optically active alcohols in high enantiomeric excesses and under
very mild reaction conditions [9]. For that aim, whole cells systems
or isolated enzymes are used as ideal catalysts. Unfortunately, for
isolated dehydrogenases, the addition of an external coenzyme and
cells seems adequate for carrying out this type of processes because
the presence of cofactors is not required.
Microorganisms and vegetables have been established as effi-
cient biocatalyst for the introduction of chirality in prochiral
compounds. The use of whole cells systems has led to the prepa-
ration of the corresponding chiral alcohols in high yields and
selectivities, being achieved in most cases the compounds of (S)-
configuration [10–15], the so-called Prelog product.
In our ongoing project focused on the identification, analysis and
application of novel biocatalysts from the Brazilian biodiversity,
vegetables and microorganisms are being investigated in reactions
such as the bioreduction of carbonyl compounds and the kinetic
resolution of racemic alcohols and esters through acetylation or
hydrolysis procedures, respectively [16–19]. Recently, we have
focused our attention in Lasiodiplodia theobromae (Deuteromycete),
a phytopathogen fungus responsible for the infection of several cul-
tures in tropical areas and associated to more than 500 species of
plants. In Brazil, this phytopathogen is considered a serious prob-
lem in agriculture sector [20]. The literature reports the use of
growing cells of L. theobromae in the biotransformation of natu-
ral products, such as steroids [21], terpenes [22,23], ionones [22],
and a sesquiterpene lactone [24]. The kinetic resolution of a racemic
epoxide by this microorganism has also been reported [25]. In all
the reactions tested, this microorganism has shown remarkable
activities in the catalysis of redox processes. Herein, we describe the
∗
Corresponding author. Tel.: +55 8533669441; fax: +55 8533669782.
Corresponding author. Tel.: +34 985103448; fax: +34 985103448.
E-mail addresses: mcfo@ufc.br (M.C.F. de Oliveira), vgs@fq.uniovi.es (V. Gotor).
∗∗
1381-1177/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2010.01.023

38
B.A. Barros-Filho et al. / Journal of Molecular Catalysis B: Enzymatic 65 (2010) 37–40
use of L. theobromae as a novel source of a stereoselective enzyme
for the reduction of prochiral aromatic and aliphatic ketones to the
corresponding enantiomerically enriched alcohols.
(S)-1-Phenylethanol (1b). Determination of the conversion by
HPLC: Spherisorb, 0.8 mL/min; 5% 2-propanol/hexane; 20 ◦C; tR (1a)
5.8 min, tR (1b) 8.3 min; determination of the ee by HPLC: Chiralcel
OB-H, 0.8 mL/min; 5% 2-propanol/hexane; 20 ◦C; tR (S) 10.2 min, tR
(R) 15.4 min. [˛]D25 = −22.5 (c = 1.0, CH2Cl2) for the (S)-enantiomer
in >99% ee. Absolute configuration was assigned after comparing
their retention times with the ones from commercially available
enantiopure compounds.
2. Experimental
2.1. Materials and methods
(S)-1-(2-Methoxyphenyl)ethanol (2b). 1H NMR (CDCl3,
300.13 MHz): 1.52 (3H, d, J = 6.6 Hz, H2), 3.88 (3H, s, OCH3),
L. theobromae (strain #009) was obtained from EMBRAPA
Agroindústria Tropical (Fortaleza, CE, Brazil) where the microor-
ganism was isolated in the Laboratory of Phytopathology from
infected guava. Flash chromatography columns were performed
using silica gel 60 (230–240 mesh). High performance liquid chro-
matography (HPLC) analyses were carried out in a Hewlett Packard
1100 chromatograph, UV detector using a Tracer Spherisorb
(25 cm × 4.6 mm), Daicel Chiralcel OB-H (25 cm × 4.6 mm) or Chi-
ralpak AS (25 cm × 4.6 mm) columns, varying the conditions
according to the specific substrate. Gas chromatography (GC/FID)
analyses were performed on a Hewlett Packard 6890 Series II chro-
matograph equipped with a Hewlett Packard HP-1 (crosslinked
methyl siloxane, 30 m × 0.25 mm × 0.25 ␮m), a Varian CP-Chiralsil
DEX CB or a Restek RT-␤DEXse (30 m × 0.25 mm × 0.25 ␮m). In
all the experiments, the injector temperature was 225 ◦C and the
detector temperature was heated at 250 ◦C. 1H, 13C NMR and DEPT
experiments were obtained using a DPX-300 (1H, 300.13 MHz and
13C, 75.5 MHz). The chemical shifts are given in delta (ı) values and
the coupling constants (J) in Hertz (Hz). Measurements of the opti-
cal rotation values were done in a Perkin-Elmer 241 polarimeter.
Starting ketones 1–9a and alcohols ( )-1b and ( )-7–9b were
purchased from Sigma–Aldrich or Fluka. Dry solvents were distilled
over an appropriate desiccant under nitrogen. All other reagents
and solvents were of the highest quality grade available, and pur-
chased from Sigma–Aldrich, Fluka or Acros Organics.
ꢀ
5.11 (1H, q, J = 6.6 Hz, H1), 6.90 (1H, dd, J = 7.5 and 1.5 Hz, H3 ), 6.98
ꢀ
ꢀ
(1H, dt, J = 8.5 and 1.5 Hz, H5 ), 7.29 (1H, dt, J = 8.5 and 1.5 Hz, H6 ),
7.36 (1H, dd, J = 7.5 and 1.5 Hz, H4 ); 13C NMR (CDCl3, 75.5 MHz):
ꢀ
ꢀ
ꢀ
ꢀ
22.8 (C2), 55.2 (OCH3), 66.3 (C1), 110.3 (C3 ), 120.7 (C5 ), 126.0 (C6 ),
ꢀ
ꢀ
ꢀ
128.2 (C4 ), 133.4 (C1 ), 156.4 (C2 ); determination of the conversion
by HPLC: Spherisorb, 0.8 mL/min; 5% 2-propanol/hexane; 20 ◦C;
tR (2a) 6.3 min, tR (2b) 7.1 min; determination of the ee by HPLC:
Chiralcel OB-H, 0.8 mL/min; 5% 2-propanol/hexane; 20 ◦C; tR (S)
10.3 min, tR (R) 16.9 min. [˛]D25 = −17.3 (c = 0.75, CHCl3) for the
(S)-enantiomer in >99% ee. Absolute configuration was assigned by
comparing the retention times with the one previously reported
in the literature [16,27].
(S)-1-(2-Nitrophenyl)ethanol (3b). 1H NMR (CDCl3, 300.13 MHz):
1.55 (3H, d, J = 6.3 Hz, H2), 5.40 (1H, q, J = 6.3 Hz, H1), 7.41 (1H, dt,
ꢀ
ꢀ
J = 8.1 and 1.2 Hz, H4 ), 7.63 (1H, dt, J = 8.1 and 1.5 Hz, H5 ), 7.82 (1H,
dd, J = 8.1 and 1.2 Hz, H6 ), 7.88 (1H, dd, J = 8.1 and 1.5 Hz, H3 ); 13C
ꢀ
ꢀ
ꢀ
ꢀ
NMR (CDCl3, 75.5 MHz): 24.2 (C2), 65.2 (C1), 124.2 (C3 ), 127.5 (C6 ),
ꢀ
ꢀ
ꢀ
ꢀ
128.0 (C4 ), 133.5 (C5 ), 140.9 (C1 + C2 ); determination of the con-
version by HPLC: Spherisorb, 0.8 mL/min; 10% 2-propanol/hexane;
20 ◦C; tR (3a) 6.3 min, tR (3b) 8.9 min; determination of the ee by
HPLC: Chiralpak AS, 0.8 mL/min; 5% 2-propanol/hexane; 20 ◦C; tR
(S) 24.0 min, tR (R) 22.1 min. [˛]D25 = +18.1 (c = 0.23, MeOH) for the
(S)-enantiomer in 92% ee. Absolute configuration was assigned by
comparing the retention times with the one previously reported in
the literature [16,28].
2.2. Typical procedure for the preparation of racemic alcohols
2–6b
(S)-1-(3-Methoxyphenyl)ethanol (4b). 1H NMR (CDCl3,
300.13 MHz): 1.47 (3H, d, J = 6.6 Hz, H2), 3.81 (3H, s, OCH3),
ꢀ
4.83 (1H, q, J = 6.6 Hz, H1), 6.81 (1H, dd, J = 8.5 and 1.2 Hz, H6 ),
To a solution of the corresponding ketone 2–6a (200 mg) in dry
MeOH (4.0 mL) was slowly added sodium borohydride (4 equiv.)
at 0 ◦C under nitrogen atmosphere. The reaction was stirred at
room temperature during 3 h and its progress monitored by TLC
analysis (20% EtOAc/hexane) until complete disappearance of the
starting ketone. Solvent was evaporated under reduced pressure
and the resulting suspension was redissolved in H2O and extracted
with EtOAc (3 × 10 mL). Organic phases were combined and dried
over anhydride Na2SO4. After solvent distillation under reduced
pressure, the resulting crude product was purified by flash chro-
matography [10% EtOAc/hexane for 2b (74% yield), 4b (74% yield),
and 20% EtOAc/hexane for 3b (97% yield), 5b (95% yield) and 6b
(95% yield)] [26].
6.93 (2H, m, H2 and H4 ), 7.26 (1H, t, J = 8.5 Hz, H5 ); 13C NMR
ꢀ
ꢀ
ꢀ
ꢀ
(CDCl3, 75.5 MHz): 25.0 (C2), 55.1 (OCH3), 70.0 (C1), 110.8 (C2 ),
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
112.7 (C4 ), 117.6 (C6 ), 129.3 (C5 ), 147.6 (C1 ), 159.6 (C3 ); deter-
mination of the conversion by HPLC: Spherisorb, 0.8 mL/min;
5% 2-propanol/hexane; 20 ◦C; tR (4a) 5.8 min, tR (4b) 8.4 min;
determination of the ee by HPLC: Chiralcel OB-H, 0.8 mL/min;
10% 2-propanol/hexane; 20 ◦C; tR (S) 13.7 min, tR (R) 18.8 min.
[˛]D25 = −25.1 (c = 0.85, MeOH) for the (S)-enantiomer in 87% ee.
Absolute configuration was assigned by comparing the retention
times with the one previously reported in the literature [16,29].
(S)-1-(3-Nitrophenyl)ethanol (5b). 1H NMR (CDCl3, 300.13 MHz):
1.47 (3H, d, J = 6.6 Hz, H2), 4.96 (1H, q, J = 6.6 Hz, H1), 7.47 (1H, t,
ꢀ
ꢀ
J = 7.8 Hz, H5 ), 7.67 (1H, d, J = 7.8 Hz, H6 ), 8.04 (1H, ddd, J = 8.3,
ꢀ
ꢀ
2.3. General method for the biocatalyzed reduction of ketones
1–9a using L. theobromae
2.1 and 0.9 Hz, H4 ), 8.18 (1H, t, J = 1.8 Hz, H2 ); 13C NMR (CDCl3,
ꢀ
ꢀ
ꢀ
75.5 MHz): 25.2 (C2), 69.1 (C1), 120.2 (C4 ), 122.1 (C2 ), 129.3 (C5 ),
ꢀ
ꢀ
ꢀ
131.6 (C6 ), 147.8 (C1 ), 148.1 (C3 ); determination of the conversion
by HPLC: Spherisorb, 0.8 mL/min; 5% 2-propanol/hexane; 20 ◦C; tR
(5a) 9.2 min, tR (5b) 12.0 min; determination of the ee by HPLC:
Chiralcel OB-H, 0.8 mL/min; 10% 2-propanol/hexane; 20 ◦C; tR (S)
13.2 min, tR (R) 14.8 min. [˛]D25 = −14.5 (c = 1.0, CHCl3) for the (S)-
enantiomer in 45% ee. Absolute configuration was assigned by
comparing the retention times with the one previously reported
in the literature [16,30].
Erlenmeyers containing 50 mL of commercial potato dextrose
(PD) culture medium were previously autoclaved at 121 ◦C for
15 min and inoculated under aseptic condition with two 7 mm disk
of the microorganism (7 days old in PDA). After 7 days in static
conditions at 28 ◦C, 10 mg of the corresponding ketone 1–9a was
added to the Erlenmeyer and shaken (200 rpm) at 28 ◦C. Aliquots of
1 mL were collected after 1, 3 and 6 days reaction from each flask
and extracted with ethyl acetate (3 × 2 mL). The organic solvent
was evaporated under reduced pressure. The crude products were
analyzed by HPLC or GC in order to determine the conversion and
the optical purity of alcohols 1–9b. Experiments were performed
in triplicate.
(S)-1-(4-Nitrophenyl)ethanol (6b). 1H NMR (CDCl3, 300.13 MHz):
1.50 (3H, d, J = 6.6 Hz, H2), 5.01 (1H, q, J = 6.6 Hz, H1), 7.53 (2H, d,
J = 8.7 Hz, H2 and H6 ), 8.17 (2H, d, J = 8.7 Hz, H3 and H5 ); 13C NMR
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
(CDCl3, 75.5 MHz): 25.3 (C2), 69.5 (C1), 123.6 (C2 and C6 ), 126.0 (C3
ꢀ
ꢀ
ꢀ
and C5 ), 147.0 (C1 ), 153.1 (C4 ); determination of the conversion

B.A. Barros-Filho et al. / Journal of Molecular Catalysis B: Enzymatic 65 (2010) 37–40
39
Scheme 1. Bioreduction of prochiral aromatic ketones 1–6a by Lasiodiplodia theobromae.
by HPLC: Spherisorb, 0.8 mL/min; 10% 2-propanol/hexane; 20 ◦C;
tR (6a) 6.5 min, tR (6b) 7.7 min; determination of the ee by HPLC:
Chiralpak AS, 0.8 mL/min; 20% 2-propanol/hexane; 20 ◦C; tR (S)
13.7 min, tR (R) 15.8 min. [˛]D25 = −10.5 (c = 1.2, CHCl3) for the (S)-
enantiomer in 42% ee. Absolute configuration was assigned by
comparing the retention times with the one previously reported
in the literature [16,31].
(R)-1-Cyclohexylethanol (7b). Determination of the conversion
by GC analysis: HP-1, 70 ◦C (4 min) 20 ◦C/min, 150 ◦C; tR (7a)
3.7 min, tR (7b) 4.2 min. Determination of the ee by GC: Chiralsil DEX
CB, 90 ◦C (5 min), 2.5 ◦C/min, 105 ◦C, 5 ◦C/min, 120 ◦C, 20 ◦C/min,
180 ◦C; tR (S) 13.0 min, tR (R) 13.7 min. Absolute configuration was
assigned by comparing the retention times with the one previously
reported in the literature [16,32].
(R)-Octan-2-ol (8b). Determination of the conversion by GC anal-
ysis: HP-1, 70 ◦C (4 min) 20 ◦C/min, 150 ◦C; tR (8a) 3.0 min, tR (8b)
3.3 min. Determination of the ee by GC: RT␤DEXse, 90 ◦C (5 min),
2.5 ◦C/min, 105 ◦C, 5 ◦C/min, 130 ◦C (2 min), 20 ◦C/min, 180 ◦C; tR (S)
14.7 min, tR (R) 16.4 min. Absolute configuration was assigned by
comparing the retention times with the one previously reported in
the literature [16,33].
(R)-Methyl 3-hydroxy-4-chloroacetoacetate (9b). Determination
of the conversion by GC analysis: HP-1, 70 ◦C (4 min), 20 ◦C/min,
200 ◦C; tR (9a) 3.6 min, tR (9b) 4.2 min. Determination of the ee by
GC: RT␤DEXse, 90 ◦C (5 min), 2.5 ◦C/min, 105 ◦C, 5 ◦C/min, 130 ◦C
(2 min), 20 ◦C/min, 180 ◦C; tR (S) 20.3 min, tR (R) 20.6 min. Absolute
configuration was assigned by comparing the retention times with
the one previously reported in the literature [16,34].
Table 1
Lasiodiplodia theobromae biocatalyzed reduction of aromatic ketones 1–6a in potato
dextrose medium at 28 ◦C and 200 rpma.
Entry
Ketone
Time (days)
c (%)b
eeP (%)b
Config. alcohol
1
2
3
4
5
6
7
8
9
10
11
12
13
1a
1a
2a
2a
3a
3a
4a
4a
5a
5a
5a
6a
6a
1
3
1
3
3
6
1
3
1
3
6
1
3
≥99
≥99
≥99
≥99
≥99
≥99
91
97
≥99
≥99
93
92
92
87
84
45
43
S
S
S
S
S
S
S
S
S
S
S
S
S
94
≥99
≥99
≥99
≥99
≥99
38
42
35
a
For more detailed reaction conditions see Section 2.
Conversion (c) and enantiomeric excesses (ee) were determined by HPLC.
b
3-methoxyacetophenone (2a and 4a) and 2-, 3- or 4-nitro ace-
tophenone (3a, 5a and 6a) as candidates. For all the substrates very
high activities were found, reaching conversions over 90%. Respect-
ing the growing cells selectivity, very different enantiomeric excess
values were reached in the preparation of the alcohols depending
on the substrate structure (ee values from 35% until 99%). Ortho-
substituted ketones 2a and 3a were completely reduced to the
(S)-alcohols in high enantiomeric excesses (entries 3–6). A loss in
the enantiomeric excess of (S)-2-methoxy-1-phenylethanol (2b)
was observed at longer reaction times, maybe caused by the exis-
tence of concurrent oxidation-reduction processes (>99–93% ee,
entries 3 and 4).
3. Results and discussion
The presence of an electrondonating group (OMe) in the meta
position of the aromatic ring (4a) led to higher enantiomeric
excesses (up to 80% ee, entries 7 and 8) in comparison with the exis-
tence of an electron-withdrawing functionality (NO2) in the same
position. Thus, for ketone 5a moderate enantiomeric excesses val-
ues were attained (38–45% ee, entries 9–11), being observed for
both substrates a slight decrease of the stereopreference at longer
reaction times. Finally, the bioreduction of para-nitro acetophe-
none allowed the recovery of (S)-6b with similar optical purity
as obtained with the meta derivative (entries 12 and 13). The
In order to explore the substrate specificity and selectivity
of this new enzymatic system, growing cells of L. theobromae
were employed in the bioreduction of both aromatic and aliphatic
ketones, and the results have been compared with the ones pre-
viously obtained in our research groups using the basidiomycete
Lentinus strigellus as biocatalyst [16]. Initially, the bioreductions
of acetophenone (1a) and five different derivatives substituted in
the aromatic ring (2–6a) were analyzed employing a PD medium
(Scheme 1). Aliquots were regularly taken and analyzed after 1, 3
and 6 days, being the most representative results summarized in
Table 1. All the aromatic ketones were stereoselectively reduced by
L. theobromae, and the hydride attack occurred in the Re face of the
corresponding aromatic ketones 1–6a, showing a Prelog selectivity
as occurs with L. strigellus.
Acetophenone (1a) was stereoselectively reduced to almost
enantiopure (S)-1-phenylethanol (1b) achieving a complete con-
version after 1 day of reaction (entry 1). Higher reaction times
(entry 2) increased the ee of (S)-1b, being possible to obtain this
alcohol in enantiopure form after 3 days. These excellent results
encouraged us to study the biocatalyzed reduction of different aro-
matic ketones, selecting monosubstituted ketones such as 2- or
Scheme 2. Bioreduction of prochiral aliphatic ketones 7a and 8a, and ketoester 9a
by Lasiodiplodia theobromae.

40
B.A. Barros-Filho et al. / Journal of Molecular Catalysis B: Enzymatic 65 (2010) 37–40
Table 2
been illustrated finding an excellent activity after 1 day of reac-
tion. Growing cells of L. theobromae have nicely worked in aqueous
medium under very mild experimental conditions allowing the
production of (S)-aromatic and (R)-aliphatic alcohols in moderate
to excellent enantiomeric excesses. Further investigations are cur-
rently ongoing, trying to expand the specificity and exploring new
catalytic activities of this biocatalytic agent.
Lasiodiplodia theobromae biocatalyzed reduction of compounds 7–9a in potato dex-
trose medium at 28 ◦C and 200 rpma.
Entry
Compound
Time (days)
c (%)b
eeP (%)b
Config. alcohol
1
2
3
4
5
6
7
8
9
7a
7a
7a
8a
8a
8a
9a
9a
9a
1
3
6
1
3
6
1
3
6
96
99
98
90
97
96
35
37
40
60
72
78
40
40
40
R
R
R
R
R
R
R
R
R
Acknowledgments
≥99
96
97
The authors thank the Brazilian and Spanish agencies CAPES-
DGU (Process: 149/07), CNPq, FUNCAP, PRONEX, Ministerio de
Ciencia e Innovación (MICINN, Project CTQ 2007-61126) for fel-
lowships and financial support. G. de G. and V. G.-F. thank MICINN
for personal grants (Juan de la Cierva and Ramon y Cajal program,
respectively). We are also in debt to Dr. Francisco Marto Viana
(EMBRAPA Agroindústria Tropical) for providing the fungus strain.
a
For more detailed reaction conditions see Section 2.
Conversion (c) and enantiomeric excesses (ee) were determined by GC.
b
reduction and oxidation of the ketones were the unique activities
observed during the processes, without observing any reactivity of
the growing cells towards the nitro functionality for compounds
3a, 5a and 6a.
References
Comparing these results with the ones obtained in the investi-
gation using L. strigellus as biocatalyst [16] L. theobromae catalyzes
the bioreduction of the same aromatic ketones with higher reaction
rates (91–99% conversion) in the same reaction times. However, the
best enantioselectivities were obtained when using L. strigellus. In
this case, the chiral alcohols 1–6b were produced in optical puri-
ties higher than 90% while the optical activities achieved with L.
theobromae stayed in the interval of 42–99% ee.
At this point, we decided to study the bioreduction of aliphatic
ketones, considering different structural motifs such as cyclohexyl
methyl ketone (7a) and 2-octanone (8a), and the biocatalyzed
reduction of a ␤-ketoester such as methyl 4-chloroacetoacetate
(9a) in order to also study the chemoselectivity properties of our
enzyme (Scheme 2). Surprisingly, anti-Prelog products (R-alcohols)
were recovered from the bioreductions of 7a and 8a, while the
expected Prelog alcohol (R)-9b was observed when ketone 9a was
reduced in presence of this enzymatic system.
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Table 2 summarized the results obtained for these three
compounds. As occurred in the reduction of aromatic ketones, con-
version values were in all cases very high (90–99%), however in
these reactions (R)-alcohols were achieved, all of them in low to
moderate enantiomeric excesses. Cyclic ketone 7a was almost com-
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In summary, we have presented L. theobromae as a promising
biocatalyst for the production of optically active alcohols. Thus,
the bioreduction of a set of aromatic and aliphatic ketones has