Microbial biotransformations in water/organic solvent system. Enantioselective reduction of aromatic β- and γ-nitroketones

Article abstract of DOI:10.1016/S0957-4166(98)00096-2

The production of single enantiomers of γ and β-nitroalcohols by microbial bioreduction has been studied. A restricted screening among 14 yeasts was performed using 1-phenyl-4-nitro-1-butanone 1 as substrate. Pichia minuta (CBS 1708) and Pichia etchellsii

Full text of DOI:10.1016/S0957-4166(98)00096-2

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 9 (1998) 1389–1394
Microbial biotransformations in water/organic solvent system.
Enantioselective reduction of aromatic β- and γ-nitroketones
Francesco Molinari,a Ernesto G. Occhiato,b Fabrizio Aragozzini a and Antonio Guarna b,∗
aDipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Sezione Microbiologia Industriale, Università degli Studi
di Milano, via Celoria 2, I-20133 Milano, Italy
bDipartimento di Chimica Organica ‘U. Schiff’ and Centro dei Composti Eterociclici C.N.R., Università di Firenze, Via G.
Capponi 9, I-50121 Firenze, Italy
Received 9 February 1998; accepted 3 March 1998
Abstract
The production of single enantiomers of γ- and β-nitroalcohols by microbial bioreduction has been studied.
A restricted screening among 14 yeasts was performed using 1-phenyl-4-nitro-1-butanone 1 as substrate. Pichia
minuta (CBS 1708) and Pichia etchellsii (CBS 2011) gave the highest enantiopreference for (S)-alcohol 3
formation (e.e. 85% and 80%, respectively), while Kluyveromyces marxianus (CBS 397) was the only strain able
to preferentially furnish the (R) enantiomer (e.e. 70%). These three microorganisms, along with baker’s yeast,
were then employed in reactions performed in water/organic solvent systems using different solvents (hexane,
benzene and dibutyl ether) affording alcohol 3 in high enantiomeric excesses (>95–97%). These strains were also
employed to reduce 1-phenyl-3-nitro-1-propanone 2, maintaning the same stereobias observed with γ-nitroketone
1 and showing high enantioselectivity in both simple aqueous (>85–97%) and biphasic media (>97%). © 1998
Elsevier Science Ltd. All rights reserved.
1. Introduction
Enantiopure nitroalcohols are useful building blocks in asymmetric synthesis because they can be
easily converted into many other chiral compounds through the formation of new C–C bonds at the
position α to the nitro group.1 Furthermore, nitro and hydroxy groups can be transformed into other
functionalities.1 Optically active secondary nitroalcohols have been obtained by microbial reduction
of nitroketones using baker’s yeast,2–10 providing preferentially the corresponding (S)-nitroalcohols in
accordance with Prelog’s rule.11
Recently we have reported on the baker’s yeast (Saccharomyces cerevisiae, type II, Sigma) reduction of
aromatic γ-nitroketones which, with respect to the reduction of aliphatic γ-nitroketones performed under
∗
Corresponding author. E-mail: guarna@chimorg.unifi.it
0957-4166/98/$19.00 © 1998 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(98)00096-2

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the same conditions, provided low yields of nitroalcohols and, in most cases, incomplete enantioselectiv-
ity, after a 7 day reaction time.12 These results could be due to either the activity of a single enzyme with
low enantiodiscrimination or the presence of various dehydrogenases which, having different selectivity,
would catalyze unwanted redox equilibria causing racemization of the formed product.
Since it is known that the use of organic co-solvents in microbial reductions considerably reduces the
rate of undesired reactions, particularly racemization processes,13,14 we decided to evaluate if baker’s
yeast and other microorganisms could be used to catalyze the bioreduction of aromatic nitroketones in
water/organic solvent systems in order to produce single enantiomers of γ- and β-nitroalcohols with high
enantiomeric excess. Moreover, since the production of both the enantiomers is an important synthetic
goal, we also tried to identify microorganisms able to produce nitroalcohols having an (R) absolute
configuration.
With this in mind, 14 microorganisms14,15 were chosen for a first screening carried out in an aqueous
medium to evaluate the reduction of aromatic γ-nitroketone 1 (Scheme 1). Then, the most promising
strains, along with baker’s yeast, were used for the reduction of the same substrate in water/organic
solvent systems and also employed for the bioreduction of β-nitroketone 2 (Scheme 1). The reduction
of the latter compound in particular affords a precursor which could be easily transformed into chiral
aminoalcohols which are potential biologically active compounds.16
Scheme 1.
2. Results and discussion
The results of the bioreduction screening on 1 in an aqueous medium, carried out in presence of glucose
or ethanol as co-substrates, are reported in Table 1.
As in the reduction carried out with baker’s yeast (results summarized in entry 15),12 all the tested
strains afforded the (S)-nitroalcohol, with the exception of K. marxianus (CBS 397) (entry 10) which
provided the nitroalcohol with absolute (R) configuration in 50–70% e.e. Among the screened strains,
P. minuta (entry 9) and P. etchellsii17 (entry 12) furnished nitroalcohol 3 with enantiomeric excesses
(80–85%) comparable or even superior to that obtained by using baker’s yeast. All the other microorgan-
isms failed to give better results, and in some cases (entries 13 and 14) almost racemic products were
obtained. It is noteworthy that the reduction rates with all the screened strains were much higher than that
observed with baker’s yeast, with conversions to nitroalcohol 3 being almost complete after 24 hours. The
use of different co-substrates such as ethanol or glucose, which could affect the stereochemical outcome
of the bioreductions,14 did not determine dramatic differences in the enantiomeric excesses (and molar
conversions) with any of the microorganisms in these experiments, perhaps with the exception of C.
boidinii (entry 3).
The two microorganisms which furnished the (S)-alcohol with the highest enantiomeric excess (P.
minuta and P. etchellsii), the strain able to give the (R) enantiomer (K. marxianus), and S. cerevisiae
type II were at this point used for the reduction of 1 in water/organic solvent systems, using different
organic co-solvents as reported in Table 2. The four yeasts maintained the stereobias observed in water,
but the enantiomeric excesses were much higher, up to 97%, using hexane, benzene or dibutyl ether

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Table 1
Microbial reduction of 1 to alcohol 3 in watera
Table 2
Microbial reduction of 1 to alcohol 3 in water/organic solvent systemsa
as co-solvents. In particular, (R)-nitroalcohol (+)-3 was obtained with high enantiomeric purity with K.
marxianus. The conversion rates were to some extent lower than those observed in the aqueous medium
and the reductions were stopped after 48 hours (when the conversions were about 80%). The reduction
carried out with baker’s yeast was very slow, having a conversion lower than 20% after 48 hours.
However, despite the low reduction rates observed with S. cerevisiae, the high enantiomeric excesses
obtained make this yeast worthy of use for the reduction of aromatic nitroketone 1 in the presence of an
organic solvent.
The reduction of β-nitroketone 2 was first performed using baker’s yeast as reported for compound
1.12 The (S)-alcohol 418 was obtained in 85% e.e. (Table 3, entry 4) and the reduction was complete
after 3 days. Also P. minuta, K. marxianus and P. etchellsii were employed in the reduction in water of
the nitroketone 2 (Table 3, entries 1–3) displaying, under the same conditions, higher enantioselectivity

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Table 3
Microbial reduction of 2 to alcohol 4a
(e.e. >97%) than in the reduction of γ-nitroketone 1. The greater differentiation between the large (the
phenyl ring) and the small (the nitroalkyl chain) groups in 2, due to the reduced length of the nitroalkyl
chain, could explain, according to Prelog’s rule, the higher enantioselectivity in the reduction of the
β-nitroketone.12
The reduction of 2 was also carried out in the same two-liquid phase systems used for the reduction
of 1. Virtually enantiopure alcohol 4 (>97% e.e.) was obtained with the four microorganisms, with
conversions similar to those observed in the previous experiments. It is noteworthy that K. marxianus
maintained the same stereobias observed in the reduction of 1, providing the (R)-alcohol both in water
and in water/organic solvent systems.
The extension of P. minuta, P. etchellsii and K. marxianus mediated reductions of 1 and 2 to a possible
synthetic use was also checked. In particular, no significant differences from the results obtained in
the above-described screenings were observed when performing the reduction on a mmol scale, and
enantiopure (+)-3 and (+)-4 and their corresponding enantiomers were isolated and characterized.
In conclusion, this work demonstrates that the use of readily available yeasts in water and/or in
non-conventional media allows for the production of different chiral γ- and β-nitroalcohols with high
enantiomeric excess. Since the strains used in this work are suitable to be applied to a large scale
synthesis of nitroalcohols, their use appears to be a general and useful method to provide enantiopure
chiral precursors for asymmetric synthesis.
3. Experimental
3.1. Material and methods
Enantiomeric excesses were determined by GC analysis of the Mosher’s esters19 obtained by reaction
of 3 and 4 with (S)-(+)-α-methoxy-α-trifluoromethylphenylacetyl chloride (MPTA-Cl) using a chiral
capillary column (diameter 0.25 mm, length 25 m thickness 0.25 µ, DMePeBeta-CDX, Mega, Legnano,
Italy). The absolute configuration of 3 was determined by GC analysis of its Mosher’s ester in comparison
with that of (S)-(−)-3 obtained as reported.12 The same method was used for the determination of
the absolute configuration of 4, using for comparison (S)-(−)-4 obtained by baker’s yeast reduction.
Saccaromyces cerevisiae type II was purchased from Sigma.

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3.2. Microorganisms, media and culture conditions
All the yeasts were grown at 27°C on malt broth at pH 6.0 for 36–48 h on a reciprocating shaker (100
strokes/min). Fresh cells from submerged cultures were centrifuged and washed with 0.1 M phosphate
buffer (pH 6.0). Washed cells were directly used for biotransformations.
3.3. Biotransformation conditions
The reduction of 1 using P. etchellsii is reported as an example. Without co-solvent. A solution of
nitroketone (75 mg) in ethanol (1.5 mL) was added to a stirred suspension (50 mL) of fresh cells of P.
etchellsii (2.5 g) resuspended in 0.1 M phosphate buffer (pH 6.0) at 28°C. After 48 h the conversion was
about 80%. Sodium chloride was added and the suspension extracted with diethyl ether. The organic layer
was dried over sodium sulphate, filtered and concentrated to give crude 3. The purification was carried
out as already described to afford pure (S)-(−)-1-phenyl-4-nitro-1-butanol [(−)-3] (57 mg) in 75% yield
and 70% e.e.12 With organic co-solvent. A solution of nitroketone (75 mg) in ethanol (1.5 mL) was added
to a suspension of fresh cells P. etchellsii (2.5 g) resuspended in a mixture of 0.1 M phosphate buffer (15
mL, pH 6.0) and organic solvent (35 mL). The mixture was stirred at 28°C for 48 h. Usual work-up and
purification gave pure (S)-(−)-1-phenyl-4-nitro-1-butanol [(−)-3] (56 mg) in 75% yield and 97% e.e.12
25
[α]D −50.6 (c 0.52, CHCl3).
3.4. (R)-(+)-1-Phenyl-4-nitro-1-butanol (+)-3
Prepared as described above using K. marxianus in a phosphate buffer/hexane two-liquid phase system.
Starting from 75 mg of nitroketone 1, alcohol (+)-3 (57 mg) was obtained in 75% yield and 95% e.e.
25
[α]D +49.6 (c 0.50, CHCl3). Spectroscopic and analytical data are identical to those reported for (−)-
3.12
3.5. (S)-(−)-1-Phenyl-3-nitro-1-propanol (−)-4
Prepared as described above using P. minuta without co-solvent. Starting from 75 mg of nitroketone
2 enantiopure alcohol (S)-(−)-4 (39 mg) was obtained in 52% yield after usual work-up followed by
25
chromatography (eluant Et2O, Rf 0.71) and bulb to bulb distillation (250°C, 5×10−1 mbar). Oil. [α]D
1
−32.3 (c 0.92, CHCl3); e.e. >97%; H NMR (CDCl3) δ: 7.37–7.29 (m, 5H), 4.83 (dd, J=7.9, 5.5 Hz,
1H), 4.68–4.37 (m, 2H), 2.44–2.33 (m, 2H); 13C NMR (CDCl3) δ: 143.5 (s, 1C), 129.5 (d, 2C), 128.9 (d,
2C), 126.2 (d, 1C), 72.9 (d, 1C), 71.8 (t, 1C), 36.7 (t, 1C); MS m/z (%) 133 (13), 107 (81), 105 (100), 70
(70), 77 (49); IR (neat) 3606, 1551, 1363 cm−1. Elemental anal. calcd for C9H11NO3: C, 59.66; H, 6.12;
N, 7.73. Found C, 59.34; H, 6.00; N, 7.51.
3.6. (R)-(+)-1-Phenyl-3-nitro-1-propanol (+)-4
Obtained as reported above for the reduction of 1 with K. marxianus without co-solvent. Starting from
75 mg of nitroketone 2, alcohol (+)-4 (42 mg) was obtained in 55% yield and >97% e.e. [α]D25 +32.5 (c
0.83, CHCl3). Spectroscopic and analytical data are identical to those reported for (−)-4.

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Acknowledgements
We thank the Ministry of University and Scientific and Technological Research (MURST) and CNR
(Italy) for financial support. E.G.O. thanks Università di Firenze for a two-year post-doctoral fellowship.
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