Regio- and enantio-selective oxidation of diols by Candida parapsilosis ATCC 7330

Article abstract of DOI:10.1039/c4ra08146c

Selectivity between primary and secondary alcohols was observed in oxidation using whole cells of Candida parapsilosis ATCC 7330, where the secondary alcohol was preferentially oxidized. In racemic sec alcohols, the 'R' enantiomer was selectively oxidized to the corresponding keto alcohol (yield = 18-54%) leaving the 'S' diol (yield = 31-69% and enantiomeric excess from 14% to >99%). A biphasic system consisting of isooctane-water (48 : 2 v/v) was used as a medium for biotransformation at 25 °C. This is the first report of the regio- and enantio-selective oxidation of diols using C. parapsilosis ATCC 7330.

Full text of DOI:10.1039/c4ra08146c

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Regio- and enantio-selective oxidation of diols by
Candida parapsilosis ATCC 7330
Cite this: RSC Adv., 2014, 4, 60526
a
ab
*
Thakkellapati Sivakumari and Anju Chadha
Selectivity between primary and secondary alcohols was observed in oxidation using whole cells of Candida
parapsilosis ATCC 7330, where the secondary alcohol was preferentially oxidized. In racemic sec alcohols,
the ‘R’ enantiomer was selectively oxidized to the corresponding keto alcohol (yield ¼ 18–54%) leaving the
‘S’ diol (yield ¼ 31–69% and enantiomeric excess from 14% to >99%). A biphasic system consisting of
isooctane–water (48 : 2 v/v) was used as a medium for biotransformation at 25 ꢀC. This is the first report
of the regio- and enantio-selective oxidation of diols using C. parapsilosis ATCC 7330.
Received 5th August 2014
Accepted 13th October 2014
DOI: 10.1039/c4ra08146c
www.rsc.org/advances
negative infections,16 androgen receptor modulators,17 and
isotopically labelled structurally diverse a-amino acids, which are
useful for research on amino acid and protein metabolism.18
Introduction
Optically pure substituted 1-phenylethane-1,2-diols are useful
in the synthesis of pharmaceuticals, liquid crystals, agrochem-
icals,1 inhibitors of fatty acid amide hydrolase,2 antiviral
agents,3 anti-cancer compounds,4,5 (R)-selegiline (which is
widely used, together with L-DOPA, in the treatment of Parkin-
son's disease as well as Alzheimer's disease),6 and scyphostatin
(which is a potent inhibitor of neutral sphingomyelinase).7
Chemical syntheses of optically pure diols by dihydroxylation of
olens using ligands such as cinchona alkaloid derivatives have
been reported but the ligands need to be synthesized.8 Hydro-
genation by a rhodium catalyst involves a multi-step synthesis
of the ligand9 and asymmetric borane reduction by Corey–
Bakshi–Shibata (CBS) reagent involves protection and depro-
tection of primary alcohols.10 Biocatalytic preparations of opti-
cally pure diols have been reported for Geotrichum candidum IFO
5767 via stereoinversion,11 asymmetric reduction of the corre-
sponding phenylglyoxals,12 chemo-enzymatic synthesis using
immobilized lipase from Pseudomonas cepacia13 and enantio-
selective hydrolysis by epoxide hydrolase from the potato
Solanum tuberosum.14 Optically pure diols can also be prepared
by regio- and enantio-selective oxidation of secondary alcohols
in diols.
Preparation of 2-hydroxy-1-phenylethanones has been reported
using chemical oxidizing agents. Chemical oxidation of alcohols
is a well-established reaction in organic chemistry, but reagents
for selective oxidation are still limited.19 Several chemical
reagents have been used to selectively oxidize secondary alcohols
in the presence of primary alcohols, but they are not enantio-
selective.20 In the case of biocatalysts, selective oxidation of
primary alcohols over secondary alcohols has been reported
using Sphingomonas sp. HXN-200 for 3-O-benzylglycerol, phenyl-
1,2-ethanediol, p-chlorophenyl-1,2-ethanediol and p-methyl-
phenyl-1,2-ethanediol.21 Selective oxidation of secondary alcohols
over primary alcohols for n-octane-1,2-diol, hexane-1,5-diol and
2,3-octanediol has also been reported using the alcohol dehy-
drogenase (ADH-‘A’) from Rhodococcus ruber DSM 44541 over-
expressed in Escherichia coli,22 and in 1,n-alkane diols (n ¼ 2–6)
by R. ruber DSM 44541.23
Candida parapsilosis ATCC 7330 has been used for dera-
cemization of sec alcohols, asymmetric reduction of prochiral
ketones and separation of DL-N-protected amino acid
esters.24–28 Mechanistically, the deracemization using C. para-
psilosis ATCC 7330 proceeds via stereo-inversion, i.e., enantio-
selective oxidation followed by reduction [a keto intermediate
was observed using high-performance liquid chromatography
(HPLC)].29 Another mechanistic investigation of such dera-
cemization using a deuterated substrate also showed the
formation of an undeuterated product, which is possible when
the oxidation of an enantiomer is followed by its reduction.30
These investigations have led us to carry out the enantio-
selective oxidation of allylic alcohols and 4-phenylbutan-2-ols
was established.31
Regio- and enantio-selective oxidation of secondary alcohols
in diols gives keto alcohols together with optically pure diols. In
the present study, substituted 2-hydroxy-1-phenylethanones are
formed by the regio- and enantio-selective oxidation of secondary
alcohols of diols. These ketones are starting materials for the
syntheses of inhibitors of Tie-2 and VEGFR-2 receptor tyrosine
kinases,15 antibacterial LpxC inhibitors for the treatment of Gram
aLaboratory of Bioorganic Chemistry, Department of Biotechnology, IIT Madras,
Chennai 600 036, India. E-mail: anjuc@iitm.ac.in; Fax: +91 44 2257 4102; Tel: +91
44 2257 4106
To address the selectivity in the oxidation between primary
and sec alcohols, 1-phenyl-1,2-ethanediol was selected as a
substrate for oxidation using the same biocatalyst in the current
bNational Center for Catalysis Research, Indian Institute of Technology Madras,
Chennai 600 036, India
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work. Regio- and enantio-selective oxidation of the sec alcohol because this mixture optimized the oxidation of primary alco-
group in preference to the primary alcohol was observed. In this hols using the same biocatalyst, according to our unpublished
molecule, only the ‘R’ diol is oxidized to the keto alcohol, data. The reaction was monitored every 4 h for up to 48 h. The
leaving the ‘S’ diol intact. This study also presents the regio- and substrate concentration used was 0.03 mM (which amounts to 4
enantio-selective oxidation of other diols [2ꢁ11], where the sec mg). A portion of acetone (500 ml, i.e., 1% of the nal volume of
alcohol group is oxidized in preference to the primary alcohol 50 ml of the hexane–water mixture) and 2.6 g of wet cells of C.
group, except for 7 and 8 where one of the two sec alcohol parapsilosis ATCC 7330 were also used. (Because the substrate is
groups present in the molecule was selectively oxidised. This is a solid, acetone (cosolvent) was used to dissolve the substrate
the rst report for the biocatalytic regio- and enantio-selective and was added to the reaction medium.) The alcohol was not
oxidation of diols [1ꢁ11] from the corresponding racemic oxidised in water and buffer in 48 h. In the mixture of hexane–
alcohols using whole cells of C. parapsilosis ATCC 7330. An water, a 13.22% conversion to the corresponding keto alcohol
earlier kinetic resolution of (1R,2S)-1-phenyl-1,2-propanediol to 1a was observed in 24 h. Furthermore, different ratios of
obtain the keto (S)-alcohol was reported using resting cells of hexane–water (i.e., (48 : 2 v/v), (45 : 5 v/v) and (25 : 25 v/v) were
Saccharomyces cerevisiae.32 Similarly, asymmetric oxidation of tested and it was found that 48 : 2 (v/v) was optimum. But the
(1S,2S)-1,2-diphenylethane-1,2-diol was reported using N-bro- conversion was only 13.22% in a reaction time of 24 h.
mosuccinimide in the presence of a chiral copper catalyst.33 In
Reaction time. In order to improve the conversion to the keto
the present study, the starting material is a mixture of all four alcohol, ‘isooctane’ was used to replace the hexane. The reac-
diastereomers, which is certainly an advantage as it eliminates a tion time was monitored for a 48 : 2 (v/v) mixture of isooctane to
step in the purication of the starting material, and gives the water from 0 to 30 h at a constant substrate concentration of
product keto (S)-alcohol with high enantiomeric excess (ee) and 0.03 mM (4 mg) with 500 ml of acetone (i.e., 1% of the nal
yield, except in the case of diol 8. For the regio- and enantio- volume of 50 ml of the 48 : 2 (v/v) isooctane–water mixture), and
selective oxidation of racemic diols [1–11], chemical catalyst with 2.6 g of wet fungal cells. For this mixture, a maximum
based methods are not reported so far in the literature.
conversion of 36% was observed at 24 h and the same solvent
system was used for further studies.
Cosolvent screening. To increase the conversion to the cor-
responding keto alcohol, different cosolvents, such as 1,4-
Results and discussion
1-Phenylethane-1,2-diol 1 (Scheme 1, Table 1) was the model dioxane, acetonitrile (ACN), dimethylsulfoxide, tetrahydrofuran
substrate for regio- and enantio-selective oxidation of diols and dimethylformamide, were used instead of acetone. Very low
using whole cells of C. parapsilosis ATCC 7330. For the reaction,
a 14 h culture was used based on our previous report.34 Different
parameters such as type of reaction medium, reaction time,
cosolvent screening, substrate concentration and acetone
(cosolvent) concentration were optimized in order to maximize
conversions of 3–15% were observed for the cosolvents other
than acetone. From this it is clear that acetone was useful not
only for dissolving the substrate but also for cofactor regener-
ation.38 Acetone was used as cosolvent for further reactions.
Substrate concentration. At 24 h, the conversion was moni-
the conversion to the corresponding keto alcohol. Formation of tored at varying substrate concentrations, i.e., from 0.02–0.07
product was conrmed using HPLC using a reverse-phase C18 mM, with 500 ml of acetone, (i.e., 1% of the nal 50 ml volume of
column.
the 48 : 2 (v/v) isooctane–water mixture), and 2.6 g of wet fungal
cells. A maximum conversion of 41% was observed at 0.04 mM
substrate (which amounts to 6 mg).
Optimization studies
Acetone concentration. Furthermore, the amount of acetone
(cosolvent) was optimized by testing volumes between 50 ml and
1000 ml in a nal volume of 50 ml. The maximum conversion of
45% was observed with 200 ml of acetone (i.e., 0.4% of the nal
reaction volume of 50 ml).
Reaction medium. A major challenge in most biocatalysed
reactions is that they are generally water-based, and high
loading of organic substrates is difficult in this medium. (A few
enzymes such as lipases are active in organic and other
unconventional solvents.35,36) It is therefore very important to
study the effects of solvents on these biocatalysed reactions.
Initially different solvents, namely, water, buffer, and a 48 : 2 v/v
mixture of hexane and water37 were tested for the biotransfor-
mation. The mixture of hexane and water was investigated
Under the previously mentioned optimum reaction condi-
tions, yield experiments were carried out for 0.52 mM (72 mg)
substrate. Conversion of 1-phenylethane-1,2-diol 1 to the cor-
responding 2-hydroxy-1-phenylethanone 1a was 45.35% with an
isolated yield of 38.22% (27.12 mg). The unreacted optically
pure (S)-1-phenylethane-1,2-diol 1b was also isolated (yield
46.07% i.e., 33.17 mg, ee 97%). The synthesis of optically pure
(S)-1-phenylethane-1,2-diols were reported previously using the
same biocatalyst via asymmetric reduction of the corresponding
phenylglyoxals,12 indicating the presence of multiple oxidore-
ductases in C. parapsilosis ATCC 7330 and highlighting the fact
that different oxidoreductases act on different substrates under
different reaction conditions.
Scheme 1 Regio- and enantio-selective oxidation of p-substituted 1-
phenylethane-1,2-diols 1–6 by C. parapsilosis ATCC 7330.
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Table 1 Regio- and enantio-selective oxidation of p-substituted 1-phenylethane-1, 2-diols 1–6 by C. parapsilosis ATCC 7330
Conversion to keto Isolated yield of keto Unreacted diola (%) Isolated yield of ee (%) Reaction Speciꢀc rotation
alcohola (%) 1a–6a alcoholb (%) 1a–6a
(from HPLC) 1b–6b diolb (%) 1b–6b 1b–6b time (h) [a]3D0
1b–6b
C
Entry
R
1c
2
H
45.35 ꢂ 2.01
38.22 ꢂ 1.90
51.12 ꢂ 0.16
54.11 ꢂ 0.32
36.3 ꢂ 0.06
54.65 ꢂ 2.01
40.94 ꢂ 0.07
38.30 ꢂ 0.22
56.34 ꢂ 0.25
46.07 ꢂ 2.31
32.38 ꢂ 0.23
31.90 ꢂ 0.01
47.12 ꢂ 0.48
97
>99
>99
88
24
24
24
24
+64.86 (C1, CHCl3)12
+60.17 (C0.5, CHCl3)12
+68.45 (C1.12, CHCl3)12
+38.19 (ref. 12)
p-OCH3 59.06 ꢂ 0.07
3
p-CH3
p-Br
61.70 ꢂ 0.22
43.66 ꢂ 0.25
4c
(C1.0, CHCl3)12
5c
6
p-Cl
p-NO2
45.95 ꢂ 0.06
38.48 ꢂ 0.96
54.05 ꢂ 0.06
46.69 ꢂ 0.33
98
—
24
72
+50.96 (C1.6, CHCl3)12
—
—
—
100
—
a
Conversion was checked by HPLC. b Isolated yields were calculated for 0.52 mM (72 mg) substrate as starting material. c The keto alcohol [4.05–
6.34%] was enantio-selectively reduced to the (S) diol in the case of 1, 4 and 5, and this is why an increase in ee is observed.
The benet of regio- and enantio-selective oxidation over tolylethane-1,2-diol 3 gave a 61.70% conversion to the corre-
asymmetric reduction of the prochiral ketones is two-fold—the sponding 2-hydroxy-1-p-tolylethanone 3a, with a yield of 54.11%
corresponding oxidized keto alcohol is also formed with a yield (38.44 mg) while the unreacted optically pure (S)-diol 3b gave a
of 50% along with the optically pure diol.
yield of 31.9% (22.97 mg). The presence of the p-Br group in the
Note that only a few chemical reagents are available for the case of 1-(4-bromophenyl)ethane-1,2-diol 4 decreased the conver-
selective oxidation of sec alcohols in the presence of primary sion to the corresponding 1-(4-bromophenyl)-2-hydroxyethanone
alcohols, i.e., in a diol. Selective oxidation of the secondary 4a to 43.66%, giving a yield of 36.3% (25.89 mg). The unreacted
alcohol in 1-phenylethane-1,2-diol 1 was reported using SiO2- optically pure (S)-diol 4b gave a yield of 47.12% (33.93 mg).
supported ruthenium chloride and 3-(dichloroiodo)benzoic Similarly, in the case of the p-Cl substituent 1-(4-chlorophenyl)
acid with a yield of 26% in 3 h, but here benzaldehyde was also ethane-1,2-diol 5, the conversion to the corresponding 1-(4-
isolated with a yield of 74% (which is because of over-oxidation chlorophenyl)-2-hydroxyethanone 5a was 45.95%, and the yield
to a keto acid followed by decarboxylation).39 In another was 38.48% (27.38 mg). The unreacted optically pure (S)-diol 5b
report, oxidation to a keto alcohol used a resin/2,2,6,6- gave a yield of 46.69% (33.62 mg). The presence of the electron
tetramethyl-1-piperidinyloxyl (TEMPO) for 24 h with a yield withdrawing group p-NO2 in the case of 1-(4-nitrophenyl)ethane-
of 44%.40 Biocatalytic oxidation of 1 has not been reported so 1,2-diol 6 did not result in the formation of the corresponding
far. Biocatalytic oxidative kinetic resolution of 1 was reported oxidized keto alcohol 6a even aer 72 h.
using glycerol dehydrogenase for 64 h (conversion 50%, ee
Preparation of 2a, 3a and 5a from the corresponding diols by
>99%).41 Another report for the preparation of 1b was by oxidation using silica-encapsulated phosphotungstic acid
stereoinversion using C. parapsilosis CCTCC M203011 for 60 h (H3PW12O40) as a recyclable heterogeneous photocatalyst was
(yield 90%, ee 99.02%).42 Compound 1b was also prepared by reported to occur in 1 to 1.5 h with yields of 84–90%. The
biocatalytic asymmetric dihydroxylation of styrene for 43 h catalyst needed to be synthesised, and the use of a metal, which
(yield 76%, ee 99.9%) using styrene monooxygenase from E. is not environmentally benign, was unavoidable.44 In another
coli JM101 and the epoxide hydrolase from Sphingomonas sp. report, the oxidation of 2 and 5 to the corresponding hydroxy
HXN-200.43 Thus, it can be seen that the method reported here ketones 2a and 5a uses tetrapropylammonium perruthenate
is better in terms of yield for the production of keto alcohol 1a (TPAP), and involves three steps and in this case the primary
and in terms of the reaction time for the production of opti- alcohol had to be protected for the selective oxidation of the
cally pure (S)-diol 1b.
secondary alcohol.45 The synthesis of 3a was also reported using
Effect of substitution on aromatic ring. To study the effect of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone for 5 h with a yield
substitution on the aromatic ring, different para-substituted 1- of 67%, but here the corresponding keto aldehyde was also
phenylethane-1,2-diols 2–6 (Scheme 1, Table 1) were subjected formed in an 8 : 1 ratio.46 Oxidation of 4 to the corresponding
to oxidation using the previously optimized conditions. In all hydroxy ketone 4a was reported using 3,3-diiodo-2,2,6,6-
the cases regio- and enantio-selective oxidation was seen. tetramethoxy-4,4-biphenyldicarboxylic acid (DIDA)/Oxone® for
Electron donating groups, namely, p-OMe and p-Me on the 13 h, which gave a yield of 82%; note that the synthesis of DIDA
aromatic ring increased the conversion to the corresponding involves multiple steps.47 Thus, the present method is better
keto alcohol. The presence of the p-OMe group in the case of 1- than the others: it is relatively ‘green’ for the preparation of
(4-methoxyphenyl)ethane-1,2-diol 2 increased the conversion to hydroxy ketones from the corresponding diols, requires neither
the corresponding 2-hydroxy-1-(4-methoxyphenyl)ethanone 2a synthesis of a catalyst nor protection and deprotection, and
to 59.06% with a yield of 51.12% (36.36 mg). The corresponding there is no over-oxidation to keto aldehyde/ketoacid.
unreacted optically pure (S)-diol 2b was isolated with a 32.38%
(S)-1-(4-Methoxyphenyl)ethane-1,2-diol 2b was shown to be
yield (23.31 mg). Similarly, a p-Me group in the case of 1-p- synthesized via an asymmetric reduction using Yamadazyma
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farinosa IFO 10896 for 48 h (yield 95%, ee >99%).45 Lithiated N-
boc thiazolidine which is obtained by multi step synthesis, has
also been reported for the synthesis of 2b [yield 55%, ee 66%].48
Synthesis of 3b (yield 96%, ee >99%) by asymmetric reduction of
the corresponding keto alcohol using an N-phenylamine–
borane complex was reported, and it involved protection and
deprotection of the primary alcohol.10 (S)-1-p-Tolylethane-1,2-
diol 3b was also prepared within 12 h using lipase (yield 45%,
ee 77%) in three steps.49 Ten hour syntheses of (S)-1-(4-
bromophenyl)ethane-1,2-diol 4b (yield 39%, ee 91%) and (S)-1-
(4-chlorophenyl)ethane-1,2-diol 5b (yield 47%, ee 72%) were
reported using Ps. cepacia lipase.49 Syntheses of 4b (yield 62%,
ee 94%) and 5b (yield 67%, ee 98%) were reported using 25
mol% of a polymer supported chiral sulfonamide, NaBH4/
Me3SiCl for 1.5 h under reux conditions.50 Signicantly, the
current study presents a simple method for the preparation of
optically pure diols with good ee and yields, together with the
corresponding keto alcohols under mild reaction conditions at
room temperature.
Substrate scope. To expand the substrate scope, different
substrates, in particular 7–11 (Chart 1, Table 3), were subjected
to the biotransformation. In the case of 1-phenylpropane-1,2-
diol 7 {for which the ratio of erythro [(R,S) and (S,R)] : threo
[(R,R) and (S,S)] was 78 : 22 based on nuclear magnetic reso-
nance (NMR)}, the reaction time was increased signicantly
from 24
h to 72 h. The conversion to (S)-2-hydroxy-1-
phenylpropan-1-one 7a was 49.67% (yield 42.34%, 30.08 mg,
ee 99%). The other product was the corresponding unreacted
diol 7b (with an 87 : 13 ratio of erythro : threo based on NMR)
[yield 41.59%, 29.95 mg, ee 90.26% (S,R-major)]. Here the
increase in erythro and decrease in threo in the case of 7b
compared to the starting diol 7 indicates that one of the threo
forms is oxidised. This oxidation was conrmed using NMR and
HPLC,51 and it was the (S,S) isomer that was oxidised completely
to the (S)-keto alcohol. The overall formation of the (S)-keto
alcohol 7a is because of the oxidation of erythro (R,S) and threo
(S,S) (conrmed by HPLC). Unreacted diol 7b is a mixture of
erythro [(S,R)-major (ee 90.26%), (R,S)-minor] and threo (R,R). An
in situ generated dioxirane (from the fructose derived ketone)
was used previously to carry out a two hour synthesis of 7a using
threo 1-phenylpropane-1,2-diol with an ee of 69%, and from
erythro with an ee of 23%, where the conversion as calculated
from the results of 1H-NMR was 20% and 34% respectively. The
corresponding 2-hydroxy-1-phenyl-1-propanone and 1-hydroxy-
1-phenyl-2-propanone are formed in an 84 : 16 ratio for threo
and 89 : 11 ratio for ethryo.52 Another preparation of 7a used
(1R,2S)-1-phenyl-1,2-propanediol and resting cells of S. cer-
evisiae at 30 ꢀC: the product was recovered in 7 days (yield 64%,
ee 93%), and the corresponding diketone was also formed.32 In
the present method, (S)-2-hydroxy-1-phenylpropan-1-one 7a was
formed in 72 h with a good ee [99%], and without any side
product. In the same reaction time of 72 h, the presence of a
phenyl group instead of a methyl group as in the case of 1,2-
diphenylethane-1,2-diol 8 [(meso : ꢂ) : (88 : 12) ratio based on
NMR], reduced the conversion to the corresponding keto
alcohol 8a to 24.41% [yield 18.06% (12.88 mg), ee 14% (R)]. The
unreacted diol 8b [(meso : ꢂ) : (78 : 22) ratio from NMR] gave a
Chart 1 Regio- and enantio-selective oxidation of diols 7–11 by C.
parapsilosis ATCC 7330.
yield of 68.92% [49.62 mg, ee 14% (R,R)]. This decrease in meso
[(S,R) or (R,S)], and corresponding increase in racemic [(R,R) and
(S,S)] in the case of 8b, compared to the starting diol 8 indicates
that the meso form gets oxidised to the corresponding (R)-keto
alcohol 8a. Reduced yields for 8 as compared to 7 were also
reported in the case of catalytic asymmetric dihydroxylation of
olens,53 possibly because of steric hindrance.
Introduction of a double bond between the phenyl ring and
diol in the case of 9 (Chart 1, Table 3) reduced the reaction time
from 24 h to 7 h, as was also reported for allylic alcohols.31
Conversion to an unsaturated keto alcohol 9a [yield 22.02%
(15.66 mg)] and the corresponding saturated keto alcohol 1-
hydroxy-4-phenyl-2-butanone was 63.14% [yield 30.45% (21.92
mg)]. Because the unsaturated keto alcohol 9a and saturated
keto alcohol could not be separated, yields were calculated
based on NMR [ratio of unsaturated and saturated keto alcohols
was 1 : 1.4]. The unreacted optically pure diol 9b, [yield 30.74%
(22.13 mg), ee >99%] was also formed.
Oxidation of 9 to 9a has not been reported so far in the
literature. Earlier, a synthesis of 9b that took 24 h was reported
using the same catalyst via deracemization (yield 86%, ee
>99%).54 A multi-step synthesis of 9b (yield 60%, ee 98%) was
also reported using CBS-oxazaborolidine.55
Introduction of a triple bond between the phenyl ring and
the diol in the case of 10 showed very low conversion (5.62%)
aer 24 h. On extending the reaction time to 72 h, no oxidized
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Table 2 Cell viability
(racemic keto alcohol for HPLC analysis) were provided by Pula
Mahajabeen. Substrates 9, 54 and 10 54,60 were synthesized by
Thangavel Saravanan. All substrates were characterized using
1H-NMR and 13C-NMR.
Reaction time (h)
Cell viabilitya (%)
24
48
66
80
77
39
21
8
Microorganism maintenance
C. parapsilosis ATCC 7330 was bought from American Type
Culture Collection, Manassas, VA 20108, USA and maintained at
4 ꢀC in yeast malt agar (HiMedia). C. parapsilosis ATCC 7330 was
grown and harvested as per the earlier reported procedure and
used for the biotransformation procedures.66
a
Experiments were performed in triplicate and values given are average.
product was detected. A similar observation was reported for the
oxidative kinetic resolution of rac-4-pentyn-2-ol, and rac-1-
octyn-3-ol using R. ruber DSM 44541.56 In the case of 11, which
has an ether linkage in addition to the absence of conjugation,
the oxidized product was not seen even aer 72 h. The presence
of an ether linkage reduced the rate of oxidation in the case of
oxidation of phenoxymethanol using the three enzyme system
that includes 2-phenylethanol dehydrogenase, phenyl acetal-
dehyde dehydrogenase and NADH oxidase.57
Cell viability. The viability of C. parapsilosis ATCC 7330 cells
was checked using the reaction medium isooctane–water (48 : 2
v/v) for up to 80 h using the conventional agar plate method.58
The results are presented in Table 2.
Immobilization studies. Preliminary studies using immobi-
lized C. parapsilosis ATCC 7330 cells were carried out. Immo-
bilized cells were prepared using sodium alginate according to
the reported procedure59 and used for the biotransformation
under the experimental conditions that were optimized for the
present study. Each of the rst two cycles showed 100% activity,
but the activity decreased to 87% in the 3rd cycle, and decreased
drastically to 2% in the 4th cycle.
General procedure for regio- and enantio-selective oxidation
of diols by C. parapsilosis ATCC 7330
Isooctane–water (48 : 1 v/v) was mixed ultrasonically for 5 min
using a Vibra-Cell ultrasonic processor (pulse 5 s on, 5 s off,
amplitude 38) prior to the reaction, to make the reaction
medium homogeneous. Wet cells (2.6 g) of C. parapsilosis ATCC
7330 suspended in 1 ml of water were added to the isooctane–
water mixture to give a total volume of 50 ml. 1-Phenylethane-
1,2-diol 1 (0.04 mM, 6 mg) dissolved in 200 ml acetone (i.e.,
0.4% of the nal 50 ml volume) was added to the above cell
suspension, incubated at 25 ꢀC, and mixed at 150 rpm for 24 h.
For yield experiments, 0.52 mM (72 mg) of substrate 1 was used
(in parallel, in 12 different asks, i.e., 6 mg ꢃ 12 asks). Aer
reacting for 24 h, the contents of all the asks were combined
and extracted with ethyl acetate (3 ꢃ 50 ml), then dried over
anhydrous sodium sulfate and concentrated using a rotary
evaporator. Conversion to the corresponding keto alcohol 1a
was checked using HPLC with a C18 column (Table 1). Enan-
tiomeric excess for the unreacted diol 1b was determined by
using HPLC with an OB-H column. Isolated yields for the
products were determined by column chromatography using
hexane–ethyl acetate (98 : 2, ml mlꢁ1) as eluent.
The same procedure was followed for the other alcohols 2–6
(Table 1, Scheme 1) and 7–11 (Table 3, Chart 1) with subsequent
change in the reaction time for the regio- and enantio-selective
oxidation using C. parapsilosis ATCC 7330. Reactions were done
in triplicate for consistent results and control experiments were
carried out in parallel without whole cells and also using heat
killed cells under identical conditions. The absolute congu-
ration for all the optically pure alcohols was determined to be
(S), except for 8, which gave (R,R).
Experimental
All substituted benzaldehydes and selenium, palladium on
carbon were purchased from Spectrochem. Sodium borohydride
and calcium chloride dihydrate were purchased from Merck.
Sodium alginate was purchased from SRL. Yeast malt agar and
yeast malt broth components (glucose, soya peptone, yeast
extract powder and malt extract powder) were purchased from
Himedia. HPLC analysis of diols was carried out on a Jasco PU-
1580 liquid chromatograph with a photodiode array detector.
Conversion was checked using a reverse-phase C18 column, with
ACN–water (60 : 40 v/v) as the mobile phase. Resolution was
carried out using Daicel OJ-H, OB-H, OD-H and AD-H chiral
columns. A hexane–isopropanol mixture was used as the mobile
phase. The proportion of solvents varied for different diols.
Optical rotations were recorded on a Rudolph Research Autopol
IV digital polarimeter. The characterization of racemic and
enantiomerically pure diols was carried out by 1H-NMR and 13C-
NMR and spectra were recorded in deuterated chloroform on a
Bruker Avance III 500 MHz spectrometer operating at 500 MHz
and 125 MHz.
General procedure for the biotransformation using
immobilized C. parapsilosis ATCC 7330 cells
Sodium alginate (100 ml, 2% w/v) and calcium chloride (CaCl2)
aqueous solution (100 ml, 2% w/v) were autoclaved prior to the
biotransformation.
Wet C. parapsilosis ATCC 7330 cells (2.6 g) suspended in 2.8
ml of distilled water were added to 13 ml of the sodium alginate
and stirred for 1 h to make a homogeneous mixture. This
homogeneous cell suspension was added dropwise to the pre-
Synthesis of substrates
All substituted 1-phenyl-1,2-ethane diols 1–8, 12 11 61 were chilled CaCl2 solution (2% w/v), which resulted in the forma-
prepared using reported methods. Substrates 4, 7 and 8 tion of beads. The beads were kept in the CaCl2 solution for 12
60530 | RSC Adv., 2014, 4, 60526–60533
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Table 3 Regio- and enantio-selective oxidation of diols 7–11 by C. parapsilosis ATCC 7330
Conversion to keto Isolated
Unreacted diol (%)
Isolated
ee (%)
Reaction
ꢀ
Entry alcohola (%) 7a–11a yieldb (%) 7a–11a (from HPLC) 7b–11b yieldb (%) 7b–11b 7b–11b time (h) Specic rotation [a]2D5
C
7
8
49.67 ꢂ 1.81
24.41 ꢂ 3.08
42.34 ꢂ 1.62
18.06 ꢂ 2.64
50.33 ꢂ 1.81
75.59 ꢂ 3.08
41.59 ꢂ 1.75
68.92 ꢂ 1.68
>99d, 90 72
ꢁ90.69 (C1, CHCl3)62 7a (S)
+27.49 (C3.2, CHCl3)63 7b (S,R)
ꢁ10.86 (C1, CH3COCH3)64 8a (R)
+12.77 (C1, EtOH)65 8b (R,R)
+28.65 (C1, CHCl3)54 9b
—
14e, 14
72
9
10
11
63.14 ꢂ 2.93c
5.62 ꢂ 2.35
—
52.47 ꢂ 0.57c
36.86 ꢂ 2.93
94.38 ꢂ 2.35
100
30.74 ꢂ 0.59
>99
—
—
7
24
24
—
—
—
—
—
a
Conversion was checked by HPLC. b Isolated yields were calculated for 72 mg substrate as starting material. c Ratio of unsaturated keto alcohol 9a
and the corresponding saturated keto alcohol was 1 : 1.4, based on NMR. ee of 7a. e ee of 8a.
d
h, and then washed with distilled water [3 ꢃ 100 ml] and stored
1,2-Diphenylethane-1,2-diol 8b. O-JH column [hexane–2-
propanol ¼ 90 : 10, 1.0 ml minꢁ1; retention times 10.53 min
ꢀ
at 8 C before biotransformation.
Immobilized cells were added to the isooctane–water (48 : 1
v/v) (ultrasonically mixed as described previously) using 1 ml of
distilled water. 1-Phenylethane-1,2-diol 1 (0.04 mM, 6 mg) dis-
solved in acetone (200 ml, 0.4% of the nal volume) was added to
the immobilized cells suspended in isooctane–water and incu-
bated at 25 ꢀC, 150 rpm for 24 h. Aer the 24 h, the immobilized
cells were removed by ltration and the products were extracted
with ethyl acetate (3 ꢃ 10 ml), and analyzed by HPLC. Beads were
washed with distilled water (3 ꢃ 30 ml) and used for the next cycle.
(S,S-minor), 11.84 min (R,R-major)].
(E)-4-Phenylbut-3-ene-1,2-diol 9b. OD-H column [hexane–2-
propanol ¼ 90 : 10, 1.0 ml minꢁ1; retention times 13.67 min
(S-major), 15.55 min (R-minor)].
Conclusion
Various 1,2-diols (1–11) were regio- and enantio-selectively
oxidized using whole cells of C. parapsilosis ATCC 7330. Regio-
and enantio-selective oxidation of diols 1–9 gave corresponding
keto alcohols (1a–9a) and optically pure (S) diols 1b–9b, except
for 8, which gave (R)-keto alcohol 8a and (R,R)-diol 8b. In the
Spectral data
Spectral data for the products 1a,67 2a,67 3a,67 4a,67 5a,67 7a,68 8a,64 case of 7, the benzylic hydroxy group was preferentially oxidized
9a,69 1b,12 2b,12 3b,12 4b,12 5b,12 7b,63 8b 53 and 9b 54 are in over the methyl attached hydroxy group. Enantio-selective
agreement with the values reported in the literature.
oxidation of sec alcohols in diols (2–5, 9) is reported for the
rst time in this study. Excellent enantio-selectivities were
HPLC resolution details for all compounds are given next.
2-Hydroxy-1-phenylpropan-1-one 7a. AD-H column [hexane– observed for the optically pure alcohols (up to >99%) except for
2-propanol ¼ 95 : 05, 0.5 ml minꢁ1; retention times 15.37 min 6 and 11, which did not show any oxidized product even aer 72
(R-minor), 17.51 min (S-major)].
h. Those compounds whose two alcohol groups are both sec
2-Hydroxy-1,2-diphenylethanone 8a. AD-H column [hexane– alcohols, i.e., 7 and 8, took a longer time to react compared to
2-propanol ¼ 90 : 10, 1.0 ml minꢁ1; retention times 9.73 min the compounds having one primary and one secondary alcohol.
(S-minor), 13.55 min (R-major)].
Introduction of a double bond in (E)-4-phenylbut-3-ene-1, 2-diol
1-Phenylethane-1,2-diol 1b. OB-H column [hexane–2-prop- 9 reduced the reaction time signicantly to 7 h. Introduction of
anol ¼ 90 : 10, 0.5 ml minꢁ1; retention times 14.01 min a triple bond, in 4-phenylbut-3-yne-1,2-diol (10), showed very
(R-minor), 17.81 min (S-major)].
low conversion. Compounds 2, 3 and 9 showed conversions of
1-(4-Methoxyphenyl)ethane-1,2-diol 2b. OD-H column more than 50%, which may be because of the reaction of more
[hexane–2-propanol ¼ 98 : 02, 0.8 ml minꢁ1; retention times than one enzyme (since the biocatalyst is a whole cell). The
68.29 min (R-minor), 73.03 min (S-major)].
1-(4-Methylphenyl)-1,2-ethanediol
present study can also be scaled up to 1 g and above using a
3b. OB-H column reactor. For reactor scale-up, separate optimization of the
[hexane–2-propanol ¼ 90 : 10, 0.5 ml minꢁ1; retention times previous parameters such as substrate concentration is needed
22.08 min (R-minor), 25.72 min (S-major)].
for free cells. Immobilized cells also can be used for reactor
1-(4-Bromophenyl)ethane-1,2-diol 4b. OD-H column scale-up as they are reusable.
[hexane–2-propanol ¼ 98 : 02, 0.8 ml minꢁ1; retention times
67.80 min (R-minor), 74.19 min (S-major)].
Acknowledgements
1-(4-Chlorophenyl)ethane-1,2-diol 5b. OD-H column
[hexane–2-propanol ¼ 98 : 02, 0.8 ml minꢁ1; retention times T. Sivakumari thanks the Council of Scientic and Industrial
53.36 min (R-minor), 59.41 min (S-major)].
Research (CSIR, India) for her Fellowship. We thank the
1-Phenylpropane-1,2-diol 7b. AD-H column [hexane–2-prop- Sophisticated Analytical Instrumentation Facility (SAIF), IIT
anol ¼ 98 : 02, 1.0 ml minꢁ1; retention times 40.85 min (S,R- Madras and the Department of Biotechnology, Government of
major), 43.79 min (R,S)-minor)].
India, New Delhi, India.
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