Mechanistic investigation of the Candida albicans CCT 0776 stereoinversion system and application to obtain enantiopure secondary alcohols

Article abstract of DOI:10.1016/j.tetasy.2009.10.003

Phenylethanol deracemization with Candida albicans CCT 0776 whole cells yields the (R)-enantiomer in over 99% enantiomeric excess and 98% yield. The deracemization process involves, in the first step, a fast, highly (S)-selective oxidation (NADP and O₂ dependent) and, in the second step, a slower partially (S)-selective reduction (NADH dependent) of the intermediate ketone. The process was extended to other 2-alkanols, 1,2-diols and 1,3-diols and, with the exception of 1,3-diol (unreactive), the enantiomeric excess and yield of the deracemization were about 99% and 62-98%, respectively.

Full text of DOI:10.1016/j.tetasy.2009.10.003

Tetrahedron: Asymmetry 20 (2009) 2635–2638
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Mechanistic investigation of the Candida albicans CCT 0776 stereoinversion
system and application to obtain enantiopure secondary alcohols
*
Simone M. Mantovani, Célio F. F. Angolini, Anita J. Marsaioli
Chemistry Institute, University of Campinas—UNICAMP, PO Box 6154, 13084-971 Campinas, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Phenylethanol deracemization with Candida albicans CCT 0776 whole cells yields the (R)-enantiomer in
over 99% enantiomeric excess and 98% yield. The deracemization process involves, in the first step, a fast,
highly (S)-selective oxidation (NADP and O2 dependent) and, in the second step, a slower partially (S)-
selective reduction (NADH dependent) of the intermediate ketone. The process was extended to other
2-alkanols, 1,2-diols and 1,3-diols and, with the exception of 1,3-diol (unreactive), the enantiomeric
excess and yield of the deracemization were about 99% and 62–98%, respectively.
Received 14 September 2009
Accepted 2 October 2009
Available online 2 November 2009
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
tion of 1-hydroxy-2-octanone. Herein, the exact stereoinversion
mechanism and driving force have been investigated using 1,2-
Chiral secondary alcohols as used in the pharmaceutical, flavor
and fragrance industries can be produced by biocatalytic methods
via asymmetric reduction of prochiral ketones, kinetic resolution of
racemic esters, and enantioselective esterification and/or oxida-
tion.1–3 In most cases the deracemized secondary alcohol is
obtained in about 50% yield. However, one attractive approach is
the deracemization of racemic alcohols, which has the advantage
of producing the desired enantiomer in almost quantitative yield,
thus breaking the 50% yield barrier.4,5
phenylethanol and this process was applied to additional alcohols
of synthetic interest.
2. Results and discussion
For a better understanding of C. albicans CCT0776 cofactor
requirements during the phenylethanol 1 stereoinversion process,
a biotransformation reaction was monitored by GC-FID with a chi-
ral stationary phase column. After 2.5 h-reaction time, the racemic
mixture turned into an almost pure (R)-enantiomer (99% ee, 48%
conversion) and acetophenone 2 (50% yield) indicating that derace-
mization was catalyzed by redox enzymes (alcohol oxidase or alco-
hol dehydrogenase).15,16 Longer bioreaction times provided better
yields reaching a maximum of 96% yield of the (R)-enantiomer
after 24 h (Table 1).
Biocatalytic deracemizations can be enzymatic or chemo-enzy-
matic processes with enzymes or whole cells. Whole cell processes
do not depend on the addition of cofactors while the stereoinver-
sion of one enantiomer can be an advantage when the deracemiza-
tion involves oxidation and reduction processes. These have been
applied to
a
-hydroxyesters/lactones,6 b-hydroxyesters7 and ben-
zylic8 and aliphatic9 alcohols, as well as vic-diols.10
In order to verify the deracemization mechanism, ketone 2 was
used as a substrate to reveal that the (S)-alcohol was preferentially
produced in low ee. This alcohol was oxidized to ketone 2 in a
highly (S)-selective reaction, therefore after many cycles, the (R)-
alcohol was produced in high enantiomeric excess and high yields
(Table 2, Scheme 1). This mechanism was confirmed by using
enantiomerically pure alcohols which showed that the (R)-alcohol
was not reactive, while the (S)-alcohol was completely oxidized in
4.5 h.
According to the recent stereoinversion experiments, molecular
oxygen could be involved in cofactor regeneration of an alcohol
oxidase.17 Following this line of reasoning we searched for the par-
ticipation of an alcohol oxidase by monitoring deracemization of
( )-1 in aerobic and anaerobic conditions (Table 2). Little biotrans-
formation of ( )-1 was observed under anaerobic conditions
The most accepted mechanistic rationale in stereoinversion is
the participation of two oxidoreductases with opposite stereopre-
ferences.11 In addition, oxidoreductases from different strains do
not have the same efficiency and coenzyme requirements, furnish-
ing the desired product in different yields and ee values. Therefore,
knowledge of the cofactor requirement can provide valuable infor-
mation on the diversity of microbial enzymes, and can be useful for
the alteration of coenzyme requirements by the approach of site-
direct mutagenesis to change or extend the coenzyme specificity
of enzymes and make the enzymes more available.12,13
In a previous investigation we successfully used Candida albi-
cans CCT 0776 to deracemize 1,2-octanediol14 through the forma-
* Corresponding author. Tel.: +55 (19) 3521 3067; fax: +55 (19) 3521 3023.
E-mail address: anita@iqm.unicamp.br (A.J. Marsaioli).
0957-4166/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2009.10.003

2636
S. M. Mantovani et al. / Tetrahedron: Asymmetry 20 (2009) 2635–2638
Table 1
OH
Deracemization by C. albicans CCT 0776 of 1
Time (h)
% ee
% 1
Configuration
—
oxidorreductase (S)-specific
NADPH depedent
0
0
66
89
99
99
99
99
100
70
58
48
60
70
96
(A)
(S)-1
(R)-1
0.67
1.33
2.5
4.5
16
(R)
(R)
(R)
(R)
(R)
(R)
O
OH
2
(B)
24
reductase partially (S)-selective
NADH-dependent
Table 2
Monitoring the reduction of 2 under aerobic and anaerobic conditions
Scheme 2. Stepwise stereoinversion mechanism. Step (A)—kinetic resolution
furnishing 50% of (R)-1 and 50% of 2. Step (B)—selective reduction of 2 furnishing
100% of (R)-1.
Time (h)
% ee
% 1
Configuration
Aerobic condition
2.5
4.5
16
26
18
73
99
7.5
7.0
12
20
(S)
(R)
(R)
(R)
Table 3
Cofactor consumption in
24
1 stereoinversion by C. albicans whole cell monitored
spectrophotometrically at 340 nm
Anaerobic condition
2.5
4.5
16
92
93
91
92
6.7
9.6
15
23
(S)
(S)
(S)
(S)
Substrate
NAD+
Absorbance
NADH
NADP+
NADPH
24
( )-1
(R)-1
(S)-1
2
0.1
0.1
0.1
—
1.1
0.9
0.9
—
—
—
—
0.6
—
—
—
1.5
suggesting that the transformation of 1 into 2 is performed by an
alcohol oxidase rather than an alcohol dehydrogenase. Therefore,
oxygen has to be present during the multiple deracemization oxi-
dation reduction cycles. To further confirm such evidence, we
investigated the reduction of 2 under aerobic and anaerobic condi-
tions; the configuration of the predominant alcohol did not change
from (S) to (R) with reaction progress under anaerobic conditions
(Table 2, Scheme 2).
Table 4
Monitoring oxidation of 1 mediated by C. albicans cell-free extract using GC–MS
Cofactor
Time (h)
Conversion
( )-1
(R)-1
(S)-1
The oxidoreductases (alcohol dehydrogenases and alcohol oxi-
dases) involved in the stereoinversion mechanisms usually require
NAD(P)+ or NAD(P)H as cofactors. The cofactor requirements were
studied using C. albicans cell-free extracts and monitoring the reac-
tion progress spectrophotometrically at 340 nm. The reduction of 2
revealed a higher consumption of NADH than NADPH, indicating
the participation of a NADH-dependent enzyme (Table 3). The
cofactor requirement in the oxidation of 1 was also investigated,
revealing that it is mediated by a NADP+ dependent enzyme
(Table 3).
While monitoring the free cell extract oxidation reaction pro-
gress we were interested by the relative absorbance curves indicat-
ing that ( )-1 was more reactive than (S)-1 and (R)-1, and that (S)-1
reacted in a similar manner to (R)-1. This required a detailed time–
course study focusing on the mass balance of alcohol 1 and ketone
2, which was achieved by GC–MS (Table 4).
NAD+
2.5
16
—
19
—
—
—
37
NADP+
2.5
16
26
47
—
—
81
96
specific NADP+ dependent enzyme leaving unreacted (R)-1; second,
the slow reduction of 2 by a NADH-dependent reductase showed
almost no enantioselectivity therefore (R)-1 is produced in quanti-
tative chromatographic yield and enantiomeric excess of >98%
after 24 h.
Next, we extended the application of our biocatalyst to the ste-
reoinversion of secondary alcohols of interest (Table 5). Deracemi-
zation of 1,2-diol 3 with C. albicans whole cells produced (S)-3 in
98% ee and 81% yield. The stereochemical course of the inversion
is analogous to that observed with racemic 1. However in compar-
ison to 1, the presence of the vicinal primary hydroxyl group in 3
led to an apparent inverse final configuration of the secondary
alcohol. In fact (R)-1 and (S)-3 have the same spatial arrangement.
Furthermore, the deracemization of 4, which is a 1,3-diol, could
furnish the other enantiomer of 1,2,4-butanetriol. However, no
These results clearly indicate that conversion of (S)-1 into 2 was
more efficient than (R)-1 in the presence of NADP+ (Table 4). Con-
sequently the stereoinversion of ( )-1 into (R)-1 can be summa-
rized as a two-step process: first a kinetic resolution involving
the fast oxidation of the (S)-enantiomer to ketone 2 by an enantio-
OH
OH
OH
O
oxidation
(S)-enantioespecific
(S)-1
(R)-1
2
(R)-1
(S) partially selective reduction
Scheme 1. Reduction of 2 by C. albicans CCT 0776 whole cells.

S. M. Mantovani et al. / Tetrahedron: Asymmetry 20 (2009) 2635–2638
2637
Table 5
Deracemization of secondary alcohols and diols by whole cells of C. albicans CCT 0776
4.3. Substrates
Substrateb
% Alcohol
eeproduct
Time (h)
Absolute configuration
The substrates ( )-1-phenylethanol (( )-1), (R)-1-phenyletha-
nol ((R)-1), (S)-1-phenylethanol ((S)-1), acetophenone (2), ( )-2-
nonanol (( )-5), (S)-2-nonanol ((S)-5), ( )-2-undecanol (( )-6)
and (S)-2-undecanol ((S)-6) were purchased from Sigma–Aldrich.
Substrates 3 and 4 were synthesized.
( )-3
( )-4a
( )-5
( )-6
81
>95
62
98
0
>99
>99
120
120
0.5
(S)
—
(R)
(R)
65
1.0
a
No biotransformation was observed with this substrate.
( )-3 = ( )-4-benzyloxy-1,2-butanediol; ( )-4 = ( )-1-benzyloxy-2,4-butane-
diol, ( )-5 = ( )-2-nonanol, ( )-6 = ( )-2-undecanol.
b
4.3.1. General procedure for the benzylation of alcoholic
hydroxyl groups—I
To a suspension of sodium hydride (555 mg, 13.87 mmol) in dry
THF (12 mL), at 0 °C, the alcohol (1 g, 13.87 mmol) was added
slowly. Then the ice bath was removed and the resulting suspen-
sion was kept under stirring for 30 min at room temperature after
which benzyl bromide (2.37 g, 13.87 mmol) was added in a single
portion. The reaction was stirred for 48 h at room temperature and
then was diluted with ether. After washing with a saturated NaCl
solution, the aqueous phase was extracted with ether/ethylacetate
(1:1), and the organic layer was dried over MgSO4 and evaporated.
biotransformation was observed suggesting that the enzymes in-
volved in this oxidation do not transform 1,3-diols.
Considering entries 5 and 6 we have additionally observed
deracemization of alcohols ( )-5 and ( )-6 in about 60% yield and
99 ee (Table 5) with the corresponding ketones in about 40% yield.
Consequently, these results cannot be attributed exclusively to a
kinetic resolution but to the presence of an additional process
inverting (S)-5 and (S)-6 to (R), which is less efficient than with
( )-1.
4.3.2. General procedure for the protection of hydroxyl groups
with tert-butyldimethylsilyl chloride—II
3. Conclusion
To a solution of alcohol (1.84 g, 8.4 mmol) in CH2Cl2 (50 mL),
imidazole (572 mg, 8.4 mmol) and tert-butyldimethylsilyl chloride
(1.26 g, 8.4 mmol) were added. The resulting suspension was stir-
red for 12 h at room temperature. The reaction mixture was
washed with saturated NaCl and NaHCO3 solutions. The organic
layer was dried over MgSO4 and the solvent was evaporated.
In conclusion, we have shown for the first time that whole cells
of C. albicans CCT 0776 can deracemize alcohols 1, 3, 5, and 6, by
sequential (S)-selective NADP+ dependent oxidation (fast), and
(S)-selective NADH dependent reduction (slow) which proceed
under aerobic conditions. This process was extended to the pro-
duction of secondary alcohols 5 and 6 and to 1,2-diols, although
in some cases it is not so efficient.
4.3.3. General procedure for the dihydroxylation of alkene
groups—III
These results are coherent with mechanisms proposed for other
microorganisms15 and reveal a novel application of C. albicans CCT
0776 whole cells in the deracemization of secondary alcohols with
a commercially available strain.
To a solution of alkene (1.3 g, 8.02 mmol) in a mixture of ace-
tone/water (50 mL, 2.5:1) under stirring, 4-methylmorpholine N-
oxide (1.13 g, 9.62 mmol) and a solution of osmium tetroxide
(0.4 mL) in tert-butanol (2.5%) were added. The solution was stir-
red for 24 h. The addition of Na2SO3 (10%, 4 mL), stirring for
30 min, and extraction with ethyl acetate produced the desired
alcohol after solvent evaporation.
4. Experimental
4.1. Analytical
4.3.4. Synthesis of ( )-4-benzyloxy-1,2-butanediol 3
Synthesis of 1-benzyloxy-3-butene 7: 3-Buten-1-ol (1.0 g, 13.87
mmol) was protected with benzyl bromide following procedure I
for the benzylation of the hydroxyl groups. Purification of the crude
product by flash chromatography (hexane/EtOAc, 7:3) afforded 7 as
a colorless oil, 2.1 g (12.9 mmol, 93.4%). 1H NMR (300.06 MHz,
CDCl3) d in ppm: 2.39 (q, 2H), 3.45 (t, 2H), 4.52 (s, 2H), 5.10 (t, 2H),
5.81 (m, 1H), 7.27 (m, 5H); 13C NMR (75.45 MHz, CDCl3) d ppm:
34.2 (CH2), 69.6 (CH2), 72.9 (CH2), 116.3 (CH2), 127.5 (CH), 127.6
(CH), 128.3 (CH), 135.2 (CH), 138.4 (C).
GC–MS analyses were performed with an Agilent 6890 Series GC
System and mass spectra were recorded with a Hewlett–Packard
5973 Mass Selective Detector (70 eV) using a HP-5MS fused silica
capillary column (crosslinked 5% phenyl methyl siloxane,
30 m  0.25 mm ID  0.25
lm film thickness) and helium as a car-
riergas (1 mL/min). Chiral GCwasperformedonanAgilent6850Ser-
ies GC System with a FID detector, using hydrogen as a carrier gas
(10 psi) and a chiral capillary column, Chirasil-Dex CB b-cyclodex-
trin (Chrompack, 25 m  0.25 mm ID  0.25
lm film thickness).
Synthesis of ( )-4-benzyloxy-1,2-butanediol 3: Compound
7
(1.3 g, 8.02 mmol) was dihydroxylated following procedure III.
Purification of the crude product by flash chromatography (hex-
ane/EtOAc, 2:8) afforded 3 as a colorless oil, 1.25 g (6.41 mmol,
80%). 1H NMR (300.06 MHz, CDCl3) d in ppm: 1.74 (m, 2H), 3.48
(dd, 1H), 3.60 (dd, 1H), 3.64 (m, 2H), 3.90 (m, 1H), 4.50 (m, 2H),
7.30 (m, 5H); 13C NMR (75.45 MHz, CDCl3) d ppm: 32.7 (CH2),
66.5 (CH2), 68.1 (CH2), 71.1 (CH), 73.2 (CH2), 127.5 (CH), 127.7
(CH), 128.3 (CH),137.6 (C); EI/MS m/z: 196 (1), 107 (40), 91
(100), 79 (10).
4.2. Microorganisms and culture conditions
The microorganism C. albicans CCT 0776 was acquired from the
CCT collection (CCT—Coleção de Culturas Tropical, Fundação Trop-
ical de Pesquisas André Tosello—Brasil) and was grown in Erlen-
meyer flasks (500 mL) containing 100 mL of culture medium
containing yeast extract (3.0 g/L), malt extract (3.0 g/L), peptone
(5.0 g/L) and D-glucose (1.0 g/L). After this stage, the cells were har-
vested by centrifugation (5000 rpm, 20 min and 18 °C).
The free cell extracts were prepared by suspending ca. 5.0 g of
the collected cells in a phosphate buffer, pH 7.0, which were dis-
rupted by maceration using liquid nitrogen. The cell debris was re-
moved by centrifugation for 15 min at 6000g and 4 °C, and the
supernatant was used as a biocatalyst.
4.3.5. Synthesis of ( )-1-benzyloxy-2,4-butanediol 4
Synthesis of 1-tert-butyldimethylsilyloxy-3-butene 8: Compound
1 (1.0 g, 13.87 mmol) was protected with tert-butyldimethylsilyl
chloride following procedure II. Purification of crude product by
flash chromatography (hexane/EtOAc, 2:8) afforded 8 as a colorless

2638
S. M. Mantovani et al. / Tetrahedron: Asymmetry 20 (2009) 2635–2638
oil, 2.4 g (12.87 mmol, 93%). The product was used directly to ob-
tain 4-tert-butyldimethylsilyloxy-1,2-butanediol 9.
NaCl. For substrates ( )-1, ( )-5, and ( )-6, the conversion was
based on residual alcohols in comparison with the internal stan-
dard (benzophenone, 0.1 mg mLÀ1), and the enantiomeric excesses
were determined by GC-FID with a chiral phase. The absolute con-
figurations were obtained by coinjection of racemic 1, 5, and 6 with
the appropriate (S)-enantiomers.
For substrate ( )-3, the conversion was based on residual alco-
hols in comparison with the internal standard (pentadecane,
0.05 mg/mL) and analysis by GC–MS. The enantiomeric excess
was determined by 1H NMR analysis of (S)-methoxyphenylacetic
acid [(S)-MPA] derivatives.18 The assignment of absolute configura-
tion was also made by 1H NMR analysis in comparison with the (S)-
MPA derivative of (S)-3.
Synthesis of 4-tert-butyldimethylsilyloxy-1,2-butanediol 9: Com-
pound 5 (1.2 g, 6.44 mmol) was dihydroxylated following procedure
III. Purification of the crude product by flash chromatography
(hexane/EtOAc, 8:2) afforded 9 as a colorless oil, 1.07 g (4.9 mmol,
76%). 1H NMR (300.06 MHz, CDCl3) d in ppm: 0.00 (s, 3H), 0.08 (s,
3H), 0.90 (s, 9H), 2.19 (s, 2H), 2.62 (s, 2H), 3.59 (m, 1H), 3.86 (m,
2H); 13C NMR (75.45 MHz, CDCl3) d ppm: -5.6 (CH3), -5.5 (CH3),
18.0 (C), 25.8 (CH3), 34.8 (CH2), 61.7 (CH2), 66.6 (CH2), 71.8 (CH);
EI/MS m/z: 189 (11), 145 (57), 133 (14), 115 (15), 105 (24), 89 (20),
75 (100).
Synthesis of 1-benzyloxy-4-tert-butyldimethylsilyloxy-2-butanol
10: Compound 9 (361.6 mg, 1.64 mmol) was protected with benzyl
bromide following procedure I. Purification of the crude product by
flash chromatography (hexane/EtOAc, 6:4) afforded 10 as a color-
less oil, 406.3 mg (1.31 mmol, 80%). 1H NMR (300.06 MHz, CDCl3)
d ppm: 0.07 (s, 3H), 0.09 (s, 3H), 0.90 (s, 9H), 1.70 (q, 2H), 3.50
(m, 1H), 3.81 (m, 2H), 4.09 (p, 2H), 4.51 (s, 2H), 7.37 (m, 5H); 13C
NMR (75.45 MHz, CDCl3) d ppm: À5.39 (CH3), À5.4 (CH3), 18.3
(C), 25.9 (CH3), 67.0 (CH2), 67.9 (CH2), 70.4 (CH), 73.2 (CH2),
127.6 (CH), 128.4 (CH), 129.6 (CH), 138.2 (C); EI/MS m/z: 310 (1),
203 (1), 179 (1), 161 (9), 131 (3), 91 (100), 75 (10).
Acknowledgments
The authors are grateful for the financial support from PETRO-
BRAS, Conselho Nacional de Desenvolvimento Científico e Tec-
nológico (CNPq) and the Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior (Capes). We also thank Prof. Carol Collins
for helpful suggestions in style and grammar.
Synthesis of ( )-1-benzyloxy-2,4-butanediol 4: Compound 10
(128 mg, 0.41 mmol) was deprotected with an acidic resin (Dowex
50Wx8) quantitatively in the presence of methanol. Filtration and
evaporation of the crude product afforded 4 as a colorless oil. 1H
NMR (300.06 MHz, CDCl3) d in ppm: 1.70 (m, 2H), 3.53 (m, 2H),
3.87 (t, 2H), 4.12 (m, 1H), 4.56 (s, 2H), 7.43 (m, 5H); 13C NMR
(75.45 MHz, CDCl3) d in ppm: 35.0 (CH2), 61.9 (CH2), 71.2 (CH),
73.0 (CH2), 74.6 (CH2), 128.0 (CH), 128.5 (CH), 129.0 (CH), 138.2
(C); EI/MS m/z: 196 (1), 151 (1), 121 (5), 107 (27), 105 (5), 91
(100), 89 (3), 75 (20).
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Whole cells (2–4 g) were stirred in a 125-mL Erlenmeyer flask
at 150 rpm containing 40 mL of phosphate buffer, pH 7.0, and
maintained at 28 °C. Then, 20
to the medium (final substrate concentration 1
l
L of the racemic diol were added
l
L mLÀ1). The bio-
18. Trost, B. M.; Belletire, J. L.; Godlesk, S.; McDougal, P. G.; Balkovec, J. M. J. Org.
Chem. 1986, 51, 2370–2374.
conversion was monitored from time to time by extracting aliquots
from the bioreaction with organic solvent after saturation with