Enantio- & chemo-selective preparation of enantiomerically enriched aliphatic nitro alcohols using Candida parapsilosis ATCC 7330

Article abstract of DOI:10.1039/c5ra13593a

Enantiomerically pure β- and γ-nitro alcohols were prepared from their respective nitro ketones by asymmetric reduction mediated by the biocatalyst, Candida parapsilosis ATCC 7330 under optimized reaction conditions (ee up to >99%; yields up to 76%). This

Full text of DOI:10.1039/c5ra13593a

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Enantio- & chemo-selective preparation of
enantiomerically enriched aliphatic nitro alcohols
Cite this: RSC Adv., 2015, 5, 73807
using Candida parapsilosis ATCC 7330
a
ab
*
Sowmyalakshmi Venkataraman and Anju Chadha
Enantiomerically pure b- and g-nitro alcohols were prepared from their respective nitro ketones by
asymmetric reduction mediated by the biocatalyst, Candida parapsilosis ATCC 7330 under optimized
reaction conditions (ee up to >99%; yields up to 76%). This biocatalyst exhibits high chemoselectivity and
reduces the keto group in preference to nitro group along with good enantioselectivity to produce
enantiomerically enriched nitro alkanols. The asymmetric reduction of aliphatic nitro ketones was carried
out in water with ethanol as cosolvent and glucose as cosubstrate using the whole cells of Candida
parapsilosis ATCC 7330 in much lesser time (4 h). For the first time, the biocatalytic asymmetric
reduction of the following ketones is reported here: 1-nitro-butan-2-one, 1-nitro-pentan-2-one,
3-methyl-1-nitro-butan-2-one and 1-cyclohexyl-2-nitroethanone to produce (R)-alcohols [ee up to
79%, yield up to 74%] and 1-nitro-hexan-2-one and 1-nitro-heptan-2-one to produce (S)-alcohols [ee
up to 81%, yield up to 76%].
Received 11th July 2015
Accepted 21st August 2015
DOI: 10.1039/c5ra13593a
www.rsc.org/advances
has resulted in developing biocatalysed reactions for a host of
organic transformations.
Introduction
Enantiomerically pure b- and g-nitro alcohols can be
prepared by using different biocatalysts. Naemura et al. repor-
ted the kinetic resolution of racemic 5-nitro-2-pentanol to
produce the enantiomerically enriched (S)-5-nitro-2-pentanol
using lipase-QL from Alcaligenes sp. with 45% conversion and
22% enantiomeric excess (ee),14,15 which is the chiral building
block for the preparation of spirocyclic pheromone of Andrena
haemorrhoa.16 In another instance, goat liver lipase was used in
the kinetic resolutions of 4-nitro-2-butanol and 5-nitro-2-
pentanol to produce the (S)-enantiomers (up to 72% ee and
up to 40% yields).17 Borah et al. studied the kinetic resolution of
several racemic aliphatic b-nitro alcohols like 1-nitro-hexan-2-
ol, 1-nitro-octan-2-ol etc., using Pseudomonas uorescens lipase
to produce the (S)-alcohol with ee up to 94%.18 The effect of
different organic solvents on the resolution of these aliphatic
b-nitro alcohols using lipase from Pseudomonas sp. to produce
their (S)-alcohols in up to 78% ee19 was also reported. Apart
from lipases,20,21 several other enzymes were also employed for
the preparation of these b-nitro alcohols. Among them, hydroxy
nitrile lyase from Hevea brasiliensis catalyses the Henry reaction.
Using this enzyme, various (S)-aryl and aliphatic nitro alkanols
were prepared with high ee (up to 99%) and yields (up to
77%).22,23 In another report, (R)-enantiomers of nitro alcohols
were preferably formed by a hydroxy nitrile lyase from Arabi-
dopsis thaliana.24 Notably, other biocatalytic methods are also
available for the preparation of aliphatic nitro alcohols using
different enzymes, namely halohydrin dehalogenase from
Agrobacterium radiobacter AD1,25 TGase (protein-glutamine-g-
Organic nitro compounds are important for various applica-
tions in synthetic chemistry as the electron withdrawing nature
of the nitro group make the protons acidic at a-position to
generate and stabilise the carbanion, thereby facilitating the
formation of a new carbon–carbon bond. The nitro group can
also be converted into several other functional groups like
amines, carbonyl compounds among others.1 Enantiomerically
pure b- and g-nitro alcohols are important chiral building
blocks for the preparation of various bioactive molecules like
ephedrine, chloramphenicol, various b-adrenergic receptor
agonists like (S)-propranolol, (S)-pindolol2 and in pheromone
preparations.3
Asymmetric Henry reaction is a widely used method for the
preparation of enantiomerically pure b-nitro alcohols.
Numerous reports are available for the synthesis of these b-nitro
alkanols using different chiral chemocatalysts, namely,
C2-asymmetric bis(oxazoline)-Cu(OAc)2$H2O,4,5 bioxazolines
from tartaric acid,6 zinc-amino alcohols system7 and many
others.8–12 In general, however on an industrial scale, the use of
biocatalysts is preferred over chemocatalysts due to ambient
operating conditions13 and the urgent need for ‘green’ methods
aLaboratory of Bioorganic Chemistry, Department of Biotechnology, Indian Institute of
Technology Madras, Chennai 600 036, India. E-mail: anjuc@iitm.ac.in; Fax: +91 44
2257 4102; Tel: +91 44 2257 4106
bNational Center for Catalysis Research, Indian Institute of Technology Madras,
Chennai 600 036, India
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glutamyl transferase EC 2.3.2.13) from Streptorerticillium gri- nitro ketones thus using the chemoselectivity of the biocatalyst
seoverticillatum26 and others.27
to advantage.
Another strategy for the preparation of these aliphatic nitro
alcohols is the asymmetric reduction of the respective nitro
ketones. The asymmetric reduction of b- and g-nitro ketones is
reported using different biocatalysts. Nakamura et al. studied
the asymmetric reduction of these nitro ketones using baker's
yeast to produce the (S)-nitro alcohols with high ee (up to 99%)
and yields (up to 66%).1,28 The enantiomerically pure (S)-4-nitro-
2-butanol acts as a chiral precursor for the preparation of various
natural products like (+)-brefeldin A, a compound with wide
range of biological applications like antiviral, antifungal, anti-
tumor activities and in the synthesis of (S)-(+)-sulcatol, a pher-
omone.29 The asymmetric reduction of 4-nitro-2-butanone by
baker's yeast to produce the (S)-alcohol with high ee (up to 99%)
and yields (up to 74%) is also reported.30,31 Fantin et al. reported
the asymmetric reduction of b- and g-nitro ketones using
different strains of Yarrowia lipolytica, Saccharomyces cerevisiae
and Rhizopus sp. among others. Among the strains tested, Yar-
rowia lipolytica selectively produced the (S)-enantiomer of
5-nitro-pentan-2-ol (ee: 60–90%; yields: up to 100%) and
(R)-enantiomer of 5-nitro-pentan-3-ol (ee: 26–84%; yields: up to
8%) while different strains of Saccharomyces cerevisiae produced
both enantiomers of 5-nitro-pentan-3-ol (ee: up to 76%; yields:
up to 30%) and 5-nitro-pentan-2-ol (ee: up to >99%; yields: up to
97%).32 Unlike b- and g-nitro ketones, the asymmetric reduction
of aliphatic a-nitro ketones is scarcely reported. One such study
was the asymmetric reduction of 1-nitro-octan-2-one using the
lyophilised cells of Comamonas testosteroni to produce the (S)-1-
nitro-2-octanol in 48 h (ee: >99%; yield 47%).33
Candida parapsilosis ATCC 7330 is established as a versatile
biocatalyst for asymmetric reduction and deracemisation reac-
tions to yield several enantiomerically pure molecules: e.g. aryl
a-hydroxy esters,34 b-hydroxy esters,35 alkyl 2-hydroxy-4-arylbut-
3-ynoates,36 1-phenyl ethanols37 etc., are prepared through
deracemisation from their racemates and aryl-1,2-diols,38 aryl
a-hydroxy amides,39 aryl a-hydroxy esters,40 etc., by asymmetric
reduction of the corresponding prochiral ketones in high
optical purity and yields. The biocatalyst is chemoselective36,40–42
and regioselective.43 The substrate repertoire of the biocatalyst
also includes aliphatic substrates. The enantioselective asym-
metric reduction of ethyl-4-chloro-3-oxobutanoate using this
biocatalyst produces the (S)-hydroxy ester44 (ee > 99%; yield
96%). Notably, a change in the carbon source in the growth
medium, results in a change in the absolute conguration of
the product alcohol.45 Enantiomerically enriched (S)-alkyl
3-hydroxybutanoates (Prelog products) were prepared from the
corresponding prochiral ketones using Candida parapsilosis
ATCC 7330 in water (pH 6.8) with glucose as cosubstrate and
acetonitrile as cosolvent46 with high ee (up to >99%) and yields
(up to 71%). Using the same biocatalyst, the deracemisation of
different racemic alkyl b-hydroxy esters was carried out in a
different reaction medium (pH 8.5 Tris–HCl buffer with acetone
as cosubstrate and DMF/DMSO as cosolvent) to produce their
(R)-hydroxy esters (anti-Prelog products) predominantly with
high ee (up to >99%).47 The present work reports a biocatalytic
Results and discussion
For this study, 1-nitro-2-butanone 1a was the model substrate.
The asymmetric reduction was carried out using the earlier
reported method.44 In this reaction, the substrate 1a in ethanol
was incubated with the wet cells of Candida parapsilosis ATCC
7330 suspended in water with glucose (50 g Lꢀ1) as a cosubstrate.
The reaction was monitored using thin-layer chromatography
and aer 4 h, the product formed was identied as (R)-1-nitro-2-
butanol 2a0 with 42% ee and 95% conversion (Scheme 1).
Screening of cosolvents
To further improve the ee, the asymmetric reduction of 1a was
carried out using different cosolvents. Among the cosolvents
tested, chloroform showed hardly any change in ee (43%). All
the other solvents, viz. dimethyl sulfoxide (ee: 35%), tetrahy-
drofuran (ee: 36%), 1,4-dioxane (ee: 31%), ethyl acetate (ee:
39%) and 2-propanol (ee: 35%) showed comparatively lower ee
than ethanol (ee: 42%) (Table 1). Ethanol was found to be the
most suitable cosolvent for the biotransformations as it also
plays an important role in the regeneration of cofactors. The
importance of the cosolvents in enhancing ee of the product
alcohols in different biotransformations was reported earlier
from our group.41,46 The effect of different solvents reported for
the kinetic resolution of racemic 1-nitro-2-pentanol using
Novozyme 435, showed that the resolution proceeded favour-
ably in diisopropyl ether with good conversion (54%) and ee
(92%).48 In certain instances, a switch in enantioselectivity is
also observed in the product when different solvents are used in
the reaction. In an earlier report on the asymmetric reduction of
ethyl-4-chloro-3-oxobutanoate carried out using baker's yeast in
water as well as in other organic solvents, the (S)-enantiomer (ee
14%, conversion 100%) was produced in water and the
(R)-enantiomer was obtained when the reaction was carried out
with different organic solvents.49 In the present study, the
product nitro alcohols did not show a switch in enantiose-
lectivity with such a change in solvents.
Effect of inhibitors
The low ee in whole cell mediated biotransformations could be
due to the presence of multiple oxidoreductases in the bio-
catalyst with opposite stereochemical preferences.45 This can be
averted by using selective inhibitors to improve the ee of the
Scheme 1 Asymmetric reduction of 1-nitro-2-butanone 1a using
asymmetric reduction for the preparation of nitro alcohols from C. parapsilosis ATCC 7330.
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Table
1 Asymmetric reduction of 1-nitro-2-butanone 1a using
C. parapsilosis ATCC 7330 using different cosolvents
Entry
Cosolvents used
ee (%)
1.
2.
3.
4.
5.
6.
7.
Ethanol
42
35
36
43
31
39
35
Dimethyl sulphoxide
Tetrahydrofuran
Chloroform
1,4-Dioxane
Ethyl acetate
2-Propanol
Scheme 2 Asymmetric reduction of 1-nitro-2-butanone 1a using
C. parapsilosis ATCC 7330 using allyl alcohol as inhibitor.
due to its irreversible inhibitory effect on the yeast alcohol
dehydrogenase enzyme.55 In addition, the use of allyl alcohol as
inhibitor in the reaction had shown only a marginal improve-
product. Inhibitors like allyl alcohol or allyl bromide are known ment in ee of (R)-2a0 (from 42% to 50%) with a drastic decrease
to inhibit the Re-face yeast enzymes and Si-face yeast enzymes in conversion (from 95% to 74%).
respectively.50 Hence in the present study, these inhibitors were
Therefore, the reaction conditions for the asymmetric
used to improve the ee of the product. The concentration of allyl reduction of a-, b- and g-nitro ketones using Candida para-
alcohol used was as per our earlier report.51 Allyl alcohol psilosis ATCC 7330 was optimised to carry out in water (pH 6.8)
(8.6 mM) or allyl bromide (4.13 mM) was added to the cell using ethanol as cosolvent and glucose as cosubstrate in the
suspension (separately) and incubated for 1 h. The product 2a0 absence of inhibitor (Scheme 3). The results obtained are
obtained aer allyl alcohol treatment showed a marginal tabulated in Table 2.
increase in ee of up to 50%, giving the (R)-alcohol with a marked
decrease in conversion (74%) unlike the untreated cells which
produced (R)-alcohol with 42% ee and 95% conversion Scope of substrates
(Scheme 2). The marginal increase in ee of (R)-2a0 could be
attributed by the selective inhibition of (S)-specic enzymes
The ee of 2a0–2e0 (Table 2) alters with increase in chain lengths
of alkyl groups attached to the carbonyl carbon along with the
present in the biocatalyst. The cells treated with allyl bromide
switch in enantioselectivity. As observed with products 2a0 and
produced (R)-2a0 with a low ee (<12%). The drastic decrease in ee
2b0, there is a decrease in ee for 2b0 compared to 2a0 due to the
could be due to the selective inhibition of (R)-specic enzymes52
increase in the enantioselectivity for the (S)-enantiomer.49 The
present in the cells. A series of experiments were also carried out
products 2a0, 2b0 and 2c0 were obtained as (R)-enantiomers
(ee: 8.2–79.5%) which could be due to the presence of alkyl
groups like ethyl, propyl, isopropyl groups on the carbonyl
carbon, where the enzyme prefers to direct the hydride attack
from the Si face resulting in the formation of (R)-alcohols.56 The
as earlier with the model substrate using lower concentrations
of allyl alcohol (1.7–8.6 mM). Under the conditions tested, there
is a marginal increase in the ee from 42% to 50% (maximum ee
with 8.6 mM allyl alcohol treatment) with a gradual decrease in
conversion (up to 74%) was observed. Nevertheless, increasing
increase in ee as well as enantio-reversal were observed with
the period of pretreatment of the biocatalyst with allyl alcohol
products 2d0 and 2e0. Zhou et al. and Keinan et al. reported the
switch in enantioselectivity and improvement in ee upon
(up to 2 h 30 min) did not show any improvement in the ee. In
our earlier study on the asymmetric reduction of ethyl 4,4,4-
increasing the length of the alkyl chain on the carbonyl
triuoro-3-oxobutanoate, the biocatalyst was pretreated with
carbon.56,57 Further increase in the alkyl chain to up to 6 carbons
different concentrations (1.7–8.6 mM) of allyl alcohol up to 2 h
showed a substantial decrease in the ee (59%) of 2f0 (Table 2)
30 min. The formation of (S)-alcohol with a gradual increase in
which may be attributed to steric factors as reported by us
earlier.46 In the case of 2g0 (Table 2) with bulky cyclohexyl group,
the ee observed was considerably low (11%) with an opposite
conguration. This observation is in consensus with a recent
report from our lab which also highlighted the effect of steric
factors on the ee of product alcohol under the conditions
ee from 63% to 84% was observed while increase in allyl alcohol
concentration to 10.3 mM showed a marked decrease in ee
(79%) and conversion (52%).51 In the asymmetric reduction of
ethyl-4-chloro-3-oxobutanoate using allyl alcohol treated
baker's yeast, showed initial inhibition of the Re-face specic
enzymes at lower concentration, which facilitates the increased
studied using this biocatalyst.58
availability of cofactors to the Si-face specic enzymes to
produce (S)-alcohol [change in R, S conguration due to
substituent priority as per CIP rules] and later the increase in
allyl alcohol concentration also inhibits the Si-face enzymes
thus resulting in the low conversion of the (S)-product.53 It is
known that allyl alcohol is a good substrate for alcohol dehy-
drogenase which in turn gets oxidised to produce acrolein,
which acts as a potent inhibitor for the yeast alcohol dehydro-
genase enzymes.54 Hence, in the present study the increase in
Scheme 3 Asymmetric reduction of nitro ketones 1a–i using C. par-
the concentration of allyl alcohol (>8.6 mM) was not attempted
apsilosis ATCC 7330.
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Table 2 Asymmetric reduction of nitroketones 1a–i using C. para-
psilosis ATCC 7330
AD-H (Daicel) column. Hexane : 2-propanol mixture was used as
mobile phase. Mobile phase ratio and ow rate were varied for
different products. Optical rotations were determined on
Rudolph, Autopol IV digital polarimeter. TLC was carried out
using Kieselgel 60 F254 aluminium sheets (Merck 1.05554).
Entry Product
eea (%); conguration Yield (%)
2a0
2b0
42, (R)
8.2, (R)
74
69
Growth conditions of Candida parapsilosis ATCC 7330
Candida parapsilosis ATCC 7330 was purchased from ATCC,
Manassas, VA 20108, USA and maintained at 4 ꢂC in yeast malt
agar medium that contained 5 g Lꢀ1 peptic digest of animal
tissue, 3 g Lꢀ1 malt extract, 3 g Lꢀ1 yeast extract, 10 g Lꢀ1
dextrose and 20 g Lꢀ1 agar. All chemicals used for media
preparation were purchased from Himedia.
2c0
79.5, (R)
74
2d0
2e0
2f0
62, (S)
81, (S)
59, (S)
76
69
76
Candida parapsilosis ATCC 7330 was precultured for 12 h at
25 ꢂC with shaking at 200 rpm in yeast malt broth medium
(contains 5 g Lꢀ1 peptic digest of animal tissue, 3 g Lꢀ1 malt
extract, 3 g Lꢀ1 yeast extract, 10 g Lꢀ1 dextrose). The precultured
broth, 2 mL (4% v/v) was transferred to a 250 mL Erlenmeyer
ask that contained 48 mL of yeast malt broth. The culture was
grown on Orbitek shaker at 25 C and 200 rpm for 14 h.44 The
ꢂ
2g0
2h0
11, (R)
54
cells were harvested by centrifugation at 10 000 rpm for 10 min
at 4 ꢂC and subsequently washed thrice with distilled water and
the wet cells were used for biotransformation.
89, (S)
72
74
Synthesis of a-nitro ketones from racemic b-nitro alcohols
2i0
>99, (S)
The a-nitro ketones (1a–g) were synthesised from racemic
b-nitro alcohols (2a–g) using modied protocol of Elmaaty
et al.60 About 2 g (20 mmol) of chromium trioxide was added to a
three-necked ask containing 9 mL of water and 1 mL of
acetone tted with a mechanical stirrer maintained in ice-bath
under nitrogen atmosphere. To this, 5 mmol of the corre-
sponding b-nitro alcohol was added slowly to the cooled solu-
tion and stirring was continued for another 10 minutes,
followed by the dropwise addition of sulphuric acid (0.8 mL) in
ice-cold condition. The reaction was allowed to stir for another
3 h followed by addition of 50 mL of water. The mixture was
extracted with dichloromethane followed by subsequent
washing with water (200 mL) and 5% sodium carbonate solu-
tion (200 mL). The separated organic layer was dried over
a
Enantiomeric excess was determined using chiral GC column.
In the asymmetric reduction of b- and g-nitro ketones viz., 1h
and 1i, the products formed were the (S)-enantiomers of 2h0 and
2i0 (Table 2) respectively, with high enantioselectivity (ee: up to
>99%) in much lesser time (4 h) unlike the earlier reported
methods (2–4 days).1,32
Experimental
The racemic b-nitro alcohols (2a–g) were synthesised from the anhydrous sodium sulfate and the solvent was evaporated in
corresponding aldehyde and nitromethane using Henry reac- vacuo to get the crude products, which were puried by silica gel
tion.59 The a-nitro ketones (1a–g) were synthesised by the column chromatography using hexane : ethyl acetate (97 : 3) to
oxidation of b-nitro alcohols using chromium trioxide and obtain the desired a-nitro ketones 1a–g, in quantitative yields.
sulphuric acid.60 The b- and g-nitro ketones (1h, 1i) were The puried products obtained were characterised using spec-
prepared using the reported methods.61,62 1H and 13C NMR troscopic techniques and compared with the literature reported
spectra were recorded in CDCl3 solution on a Bruker AVANCE III values and were used for the biotransformation. The spectro-
500 MHz spectrometer operating at 500 MHz and 125 MHz scopic characterisations of 1-nitroheptan-2-one using 1H, 13C
respectively using TMS as an internal standard. Infrared spectra NMR and HRMS analysis are given as follows:
were recorded on a JASCO FT/IR-4200 instrument. The enan-
1-Nitro-heptan-2-one (1e). Light yellow colour solid; mp:
tiomeric excess (ee) was determined by gas chromatography 44 ꢂC (uncorrected), IR (cmꢀ1): 3542 (enolic OH), 2971, 2937,
1
using PerkinElmer CLARUS 600 gas chromatograph tted with 1730, 1552, 1383, 1097; H NMR (CDCl3; 500 MHz; d in ppm):
FID detector using VARIAN Chirasil Dex CB chiral column 0.88 (t, 3H, J ¼ 7 Hz), 1.61–1.67 (quint, 2H, J ¼ 7.5 Hz), 2.53 (t,
(0.25 mm ꢁ 25 mm ꢁ 25 m). Helium was used as the carrier gas 3H, J ¼ 7.5 Hz), 5.26 (s, 2H); 13C NMR (CDCl3; 125 MHz; d in
with a ow rate of 2 mL minꢀ1 with a split factor of 10 : 1. For ppm): 13.75, 22.24, 22.79, 30.94, 40.39, 83.18, 196.18; keto : enol
few compounds ee was determined by HPLC using Chiralpak tautomerism: 88 : 12 (as calculated from 1H NMR spectra);
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HRMS: m/z; (C7H13O3N) Calculated mass: 182.0788 [(M + Na)+]; were repeated in triplicate for consistent results and control
found: 182.0785 [(M + Na)+].
experiments were done in parallel without the whole cells and
also using heat-killed cells under identical conditions.
Typical procedure for the asymmetric reduction of 1-nitro-2-
butanone 1a using C. parapsilosis ATCC 7330 with ethanol or
other cosolvents
Spectroscopic data of products
The puried nitro alcohols were characterised using spectro-
In a 150 mL Erlenmeyer ask, 2.4 g of wet cells of Candida
parapsilosis ATCC 7330 was suspended in 10 mL of sterile
distilled water containing 500 mg of glucose. Aer 10 min, the
substrate, 1-nitro-butan-2-one 1a (5 mg, 0.04 mmol) dissolved in
200 mL of ethanol or other cosolvent under study was added and
the reaction was continued up to 4 h. Aer the reaction time, the
product was extracted thrice with ethyl acetate and the organic
layer was dried over anhydrous sodium sulphate. The solvent
was removed by evaporation using rotary evaporator and the ee
of the product alcohol was determined with a chiral GC column
using conditions mentioned in Experimental section.
1
scopic (IR, H and 13C NMR) techniques and were consistent
with the literature reported values. The absolute conguration
for all the products were assigned by comparing their specic
rotation values with literature reported values (given in paren-
theses). The ee of the products were determined using GC or
HPLC using a chiral column. The details are given below.
(2R)-1-Nitro-butan-2-ol (2a0).11,18 Light yellow oil, specic
rotation: [a]2D4 ꢀ14.5 (c 0.5, CHCl3), {[a]D25 ꢀ24.7 (c 1, CHCl3), ee:
85% (R)}. The peaks are resolved by GC using the chiral column
ꢂ
(injector and detector tꢀem1 perature: 220 C, oven temperature:
70 ꢂC 5 min, 15 ꢂC min , 130 ꢂC 5 min; split: 1 : 10, ow rate:
2.0 mL minꢀ1) with retention times (min): 10.30 (S, minor);
10.37 (R, major).
Typical procedure for the asymmetric reduction of 1-nitro-2-
butanone 1a using C. parapsilosis ATCC 7330 with different
inhibitors
(2R)-1-Nitro-pentan-2-ol (2b0).8,63 Light yellow oil, specic
rotation: [a]2D4 ꢀ1.4 (c 0.5, CHCl3), {[a]D25 +12.2 (c 0.2, CHCl3) ee:
77% (S)}. The peaks are resolved by GC using the chiral column
In a 150 mL Erlenmeyer ask, 2.4 g of wet cells of Candida
parapsilosis ATCC 7330 was suspended in 10 mL of sterile
distilled water containing 500 mg of glucose. To this allyl
alcohol (8.6 mM) or allyl bromide (4.13 mM) was added and the
incubation was carried out for 1 h. The substrate, 1-nitro-butan-
2-one 1a (5 mg, 0.04 mmol) dissolved in 200 mL of ethanol was
added and the reaction was continued up to 4 h. Aer the
reaction time, the product was extracted thrice with ethyl
acetate and the organic layer was dried over anhydrous sodium
sulphate. The solvent was removed by evaporation using rotary
evaporator and the ee of the product alcohol was determined
with a chiral GC column using conditions mentioned in
Experimental section.
ꢂ
(injector and detector temperature: 220 C, oven temperature:
ꢀ1
ꢂ
ꢂ
ꢂ
90 C 5 min, 5 C min , 170 C 2 min; split: 1 : 10, ow rate:
2.0 mL minꢀ1) with retention times (min): 13.57 (S, minor);
13.80 (R, major).
(2R)-3-Methyl-1-nitro-butan-2-ol (2c0).63,64 Light yellow oil,
specic rotation: [a]D24 ꢀ13.1 (c 1, CHCl3), {[a]2D5 ꢀ24.0 (c 1.05,
CHCl3) ee: 86% (R)}. The peaks are resolved by GC using the
chiral column (injector and detector temperature: 220 ꢂC, oven
temperature: 90 ꢂC 5 min, 5 ꢂC minꢀ1, 170 ꢂC 2 min; split: 1 : 10,
ow rate: 2.0 mL minꢀ1) with retention times (min): 12.89
(S, minor); 13.14 (R, major).
(2S)-1-Nitro-hexan-2-ol (2d0).4,18,65 Light yellow oil, specic
rotation: [a]2D4 +5.0 (c 0.5, CHCl3), {[a]D25 ꢀ9.0 (c 1.0, CHCl3), ee:
90% (R)}. The peaks are resolved by GC using the chiral column
General procedure for the asymmetric reduction of nitro
ketones using C. parapsilosis ATCC 7330
ꢂ
(injector and detector temperature: 220 C, oven temperature:
ꢀ1
ꢂ
ꢂ
ꢂ
In a 250 mL Erlenmeyer ask containing 14.4 g of wet biomass 90 C 5 min, 5 C min , 170 C 5 min; split: 1 : 10, ow rate:
of Candida parapsilosis ATCC 7330 suspended in 40 mL of 2.0 mL minꢀ1) with retention times (min): 15.94 (S, major);
sterile distilled water and 2 g (50 g Lꢀ1) of glucose was added 16.12 (R, minor).
and allowed to shake at 200 rpm and 25 ꢂC for 10 min. Aer 10
(2S)-1-Nitro-heptan-2-ol (2e0).18,66 Yellow oil, specic rotation:
min, 98 mg (0.84 mmol) of substrate 1-nitro-butan-2-one 1a [a]2D4 +12.4 (c 1, CHCl3), {[a]2D5 ꢀ18.7 (c 1, CHCl3), ee: 88% (R)}.
dissolved in 2 mL of ethanol was added and the reaction was The peaks are resolved by GC using the chiral column (injector
continued in a water-bath shaker at 200 rpm and 25 ꢂC up to 4 h. and detector temperature: 220 ꢂC, oven temperature: 90 ꢂC 5
ꢀ1
ꢂ
ꢂ
Aer the reaction time, the product was extracted thrice with min, 5 C min , 170 C 5 min; split: 1 : 10, ow rate: 2.0 mL
ethyl acetate and the organic layer was dried over anhydrous minꢀ1) with retention times (min): 17.79 (S, major); 18.00 (R,
sodium sulphate. The solvent was removed by evaporation minor).
using rotary evaporator and the enantiomerically enriched (R)-1-
(2S)-1-Nitro-octan-2-ol (2f0).7,11 Light yellow oil, specic rota-
nitro-2-butanol 2a0 was obtained as light yellow oil aer puri- tion: [a]2D4 +6.5 (c 0.7, CHCl3), {[a]D25 +6.2 (c 1.6, CHCl3), ee: 62%
cation by silica gel column chromatography using (S)}. The peaks are resolved by GC using the chiral column
ꢂ
hexane : ethyl acetate (95 : 5) as eluent. The ee of the product (injector and detector temperature: 220 C, oven temperature:
ꢀ1
ꢂ
ꢂ
ꢂ
was found to be 42% as determined by the GC and the isolated 90 C 5 min, 5 C min , 170 C 5 min; split: 1 : 10, ow rate:
yield was found to be 74% (Scheme 3; Table 2).
2.0 mL minꢀ1) with retention times (min): 19.59 (S, major);
The same procedure was followed for other aliphatic a-, b- 19.79 (R, minor).
and g-nitro ketones 1b–i (Scheme 3) for the asymmetric
(2R)-1-Cyclohexyl-2-nitroethanol (2g0).66,67 Light yellow oil,
reduction using Candida parapsilosis ATCC 7330. The reactions specic rotation: [a]2D4 ꢀ1.6 (c 0.7, CHCl3), {[a]D25 ꢀ14.7 (c 1.03,
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CHCl3), ee: 73% (R)}. The peaks are resolved by HPLC using
CHIRALPAK AD-H column at 0.8 mL minꢀ1 ow rate using
hexane : 2-propanol (97 : 3) as mobile phase with retention
times (min): 21.21 (S, minor); 22.89 (R, major).
(2S)-4-Nitrobutan-2-ol (2h0).29,68 Light yellow oil, specic
rotation: [a]2D4 +30.1 (c 1.7, CHCl3), {[a]D25 +40.6 (c 1.15, CHCl3),
ee: 99% (S)}. The peaks are resolved by HPLC using CHIRALPAK
AD-H column at 1 mL minꢀ1 ow rate using hexane : 2-prop-
anol (95 : 5) as mobile phase with retention times (min): 18.29
(R, minor); 21.41 (S, major).
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(2S)-5-Nitropentan-2-ol (2i0).17,30 Light yellow oil, specic
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99% (S)}. The peaks are resolved by GC using the chiral column
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ꢂ
(injector and detector temperature: 220 C, oven temperature:
ꢀ1
ꢂ
ꢂ
ꢂ
90 C 5 min, 5 C min , 170 C 5 min; split: 1 : 10, ow rate: 10 A. Toussaint and A. Pfaltz, Eur. J. Org. Chem., 2008, 2008,
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Acknowledgements
One of the authors, Sowmyalakshmi Venkataraman gratefully
´
acknowledges the Indian Institute of Technology (IIT) Madras, 25 G. Hasnaoui-Dijoux, M. Majeric Elenkov, J. H. Lutje
India for the fellowship. We thank the Sophisticated Analytical
Instrumentation Facility (SAIF), IIT Madras for the IR and NMR
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