Chemoselective biohydrogenation of α,β- and α,β,γ,δ-unsaturated ketones by the marine-derived fungus Penicillium citrinum CBMAI 1186 in a biphasic system

Article abstract of DOI:10.1016/j.molcatb.2015.01.017

To broaden the range of applicability of a reduction reaction mediated by the marine fungal strain Penicillium citrinum CBMAI 1186 to organic chemical processes, the ability of whole mycelia to grow in biphasic mixtures with organic solvents was tested with acetone, ethyl acetate, n-butanol, dichloromethane, n-hexane and toluene. n-Hexane was the least toxic solvent according to the amount of mycelial mass grown in an artificial sea water medium mixed with each solvent. Therefore, whole hyphae of P. citrinum CBMAI 1186 were used as biocatalysts in the chemoselective biotransformation of the carbon-carbon double bond in α,β-, di-α,β-, and mono-α,β,γ,δ-unsaturated ketones (3a, 3c-f) in a biphasic system of phosphate buffer and n-hexane (9:1). Only the di-α,β,γ,δ-unsaturated ketone (3b) was not biocatalyzed under these conditions. In general, there were good conversions of saturated ketones by the enoate reductase enzymes of P. citrinum CBMAI 1186.

Full text of DOI:10.1016/j.molcatb.2015.01.017

Accepted Manuscript
Title: Chemoselective biohydrogenation of ␣,␤- and
␣,␤,␥,␦-unsaturated ketones by the marine-derived fungus
Penicillium citrinum CBMAI 1186 in a biphasic system
´
´
Author: Irlon M. Ferreira Eloa B. Meira Isac G. Rosset Andre
L.M. Porto
PII:
S1381-1177(15)00034-X
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2015.01.017
MOLCAB 3103
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date:
Revised date:
Accepted date:
30-1-2014
25-1-2015
26-1-2015
Please cite this article as: I.M. Ferreira, E.B. Meira, I.G. Rosset,
A.L.M. Porto, Chemoselective biohydrogenation of rmalpha,rmbeta- and
rmalpha,rmbeta,rmgamma,rmdelta-unsaturated ketones by the marine-derived
fungus Penicillium citrinum CBMAI 1186 in a biphasic system, Journal of Molecular
Catalysis B: Enzymatic (2015), http://dx.doi.org/10.1016/j.molcatb.2015.01.017
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Chemoselective biohydrogenation of α,β- and α,β,γ,δ-unsaturated ketones
by the marine-derived fungus Penicillium citrinum CBMAI 1186 in a
biphasic system
Irlon M. Ferreira, Eloá B. Meira, Isac G. Rosset, André L. M. Porto*
Laboratório de Química Orgânica e Biocatálise, Instituto de Química de São Carlos,
Universidade de São Paulo, Av. João Dagnone, 1100, Ed. Química Ambiental, Jd. Santa
Angelina, 13563-120, São Carlos, SP, Brazil.
*Corresponding author: Tel.: +55 16 33738103; Fax: + 55 16 33739952; E-mail:
almporto@iqsc.usp.br
Abstract
To broaden the range of applicability of a reduction reaction mediated by the
marine fungal strain Penicillium citrinum CBMAI 1186 to organic chemical processes,
the ability of whole mycelia to grow in biphasic mixtures with organic solvents was
tested with acetone, ethyl acetate, n-butanol, dichloromethane, n-hexane and toluene. n-
Hexane was the least toxic solvent according to the amount of mycelial mass grown in
an artificial sea water medium mixed with each solvent. Therefore, whole hyphae of P.
citrinum CBMAI 1186 were used as biocatalysts in the chemoselective
biotransformation of the carbon-carbon double bond in α,β-,di-α,β-, and mono-α,β,γ,δ-
unsaturated ketones (3a, 3c-f) in a biphasic system of phosphate buffer and n-hexane
(9:1). Only the di-α,β,γ,δ-unsaturated ketone (3b) was not biocatalyzed under these
conditions. In general, there were good conversions of saturated ketones by the enoate-
reductase enzymes of P. citrinum CBMAI 1186.
Page 1 of 26
1

Keywords: Biocatalysis; Enoate reductases; α,β-Unsaturated ketones; Marine
microorganisms.
1. Introduction
In recent years, biocatalysis has emerged as a viable alternative to conventional
chemical methods, owing to its high product selectivity, mild process conditions, low cost
and little environmental damage; in short, inexpensive production matching green
chemistry with high efficiency [1-3].
A disadvantage of biotransformation as a tool for organic synthesis is the low
solubility of most organic compounds in the reaction mixtures because the process is
generally carried out in an aqueous medium. To overcome this limitation, biphasic systems
have been increasingly used in biotransformation reactions [4].
A very important criterion on which to choose a non-aqueous solvent is its
biocompatibility ratio, expressed as the logarithm of the partition coefficient ratio for a
standard two-phase system, logP. Solvents with logP >4 tend to be less toxic to a
biological system [5]. Biphasic systems consisting of an organic and an aqueous phase
represent valuable tools for the biotransformation of hydrophobic substrates. Furthermore,
such system can be used to avoid inhibition effects by substrate and/or product and can
help to guide equilibrium reactions into the desired direction enhancing stereoselectivity. A
crucial step is the choice of the organic solvent, which depends on several factors,
including toxic or inhibitory effects of the solvent on the cells and substrate, and product
solubility [6].
Biocatalytic reductions of α,β-unsaturated ketones, mediated by the bacterium
Micrococcus luteus CCT 2283 in a biphasic system of water/[(BMIn)PF6], provided better
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diastereoselectivities and enantioselectivities than in pure water [7]. In another study,
strains of the yeast Saccharomyces cerevisiae employed in biphasic systems for the
biotransformation of unsaturated ketones achieved better conversions than in pure water
[8,9].
Few studies have focused on the biotransformation of organic compounds by
fungal mycelia in a biphasic system. Some examples are Candida krusei SW2026, used as
a biocatalyst in the efficient reduction of ethyl 2-oxo-4-phenylbutyrate, and Rhizopus
nigricans, used in the biotransformation of 16α,17-epoxyprogesterone carried out in a
[BMIm][PF6]-water two-phase system [10,11].
However, no studies involving organic solvents in biphasic systems with marine-
derived fungi have been reported. Recently, we have investigated the use of whole mycelia
of Penicillium citrinum CBMAI 1186 immobilized on biopolymers for the reduction of
α,β-unsaturated ketones [12]. Herein, we decided to explore the multienzymatic system of
this marine-derived fungus in the chemoselective biohydrogenation of carbon-carbon
bonds in α,β-, α,β,γ,δ-, and di-α,β,γ,δ-unsaturated ketones by employing a biphasic system.
2. Experimental
2.1 General methods
All manipulations involving the marine-derived fungus P. citrinum CBMAI 1186
were carried out under sterile conditions in a Veco laminar flow hood. A Technal TE-421
orbital shaker was used in the biocatalytic experiments. For gas chromatography–mass
spectrometry, a Shimadzu GC2010 Plus gas chromatography system coupled to a mass-
selective detector (Shimadzu MS2010 Plus) in electron ionization mode (70 eV) was used.
FTIR spectra were recorded on a Bomen MB-100 spectrometer samples were prepared as
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thin films on KBr disks (solid samples) or liquid film (liquid samples) in the 4000-400
1
cm−1 region. H NMR and 13C NMR spectra were recorded on an Agilent Technologies
500/54 Premium Shielded or Agilent Technologies 400/54 Premium Shielded
spectrometer, with CDCl3 as the solvent and TMS as the internal standard unless otherwise
noted. The chemical shifts are given in ppm and coupling constants (J) in Hz.
2.2 Chemical reagents
Acetone (1a) (99%), 1-(thiophen-3-yl)ethanone (1b) (99%), acetophenone (1c)
(99%), benzaldehyde (2a) (99%) and 4-phenyl-3-buten-2-one (99%) (3g) were purchased
from Sigma-Aldrich. Cinnamaldehyde (2b) (98%) was purchased from Vetec (Brazil), and
sodium hydroxide (97%) and hydrochloric acid (37%) were purchased from Quemis
(Brazil). All compounds were used without further purification. The salts used for
preparation of the artificial sea water were purchased from Vetec and Synth (Brazil).
Deuterated chloroform was purchased from Cambridge Isotope Laboratories. The solvents
acetone, ethyl acetate, n-butanol, dichloromethane, n-hexane and toluene were purchased
from Vetec and Synth (Brazil).
2.3 Preparation of the α,β-unsaturated ketones (3a-3f)
The unsaturated ketones were prepared via aldol condensation (Table 1). For
products 3a and 3b, acetone 1a (10 mmol) and the corresponding aldehydes 2a or 2b (20
mmol) were used, respectively. The products 3c-e were prepared from the appropriate
ketone 1b or 1c (10 mmol) and the corresponding aldehyde 2a or 2b (11 mmol). Finally,
the product 3f was synthesized from acetone 1a (10 mmol) and aldehyde 2b (5 mmol). All
the aldol condensation reactions were carried out in a two-neck, round-bottomed flask (250
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mL) containing 50 mL ethanol by slow addition of 5 mL NaOH (6 mol.L-1) to the
reactants. The reactions were stirred at room temperature for 6 h, after which an aqueous
10 % HCl solution (10 mL) was added, yielding yellow precipitates. The precipitates were
filtered and washed with iced distilled water. The products were obtained in good yields
and identified using 1H NMR, 13C NMR, IR and GC-MS spectroscopic data
(Supplementary content).
Table 1 here
2.4. Marine-derived fungus Penicillium citrinum CBMAI 1186
Marine-derived fungus P. citrinum CBMAI 1186 was isolated from the marine alga
Caulerpa sp., collected by Prof. R. G. S. Berlinck of São Sebastião city, on the coast of the
State of São Paulo, Brazil. The fungus used in this research was identified using both
conventional and molecular methods at the Chemical, Biological and Agricultural
Pluridisciplinary Research Center (CPQBA) at the University of Campinas, SP, Brazil
[13]. A type culture of P. citrinum CBMAI 1186 is deposited in the Brazilian Collection of
Environmental
and
Industrial
Microorganisms
(CBMAI
-
http://webdrm.cpqba.unicamp.br/cbmai/). Stock cultures of the fungus were stored on solid
culture medium [K2HPO4 (1.0 g·L−1), MgSO4·7H2O (0.5 g·L−1), KCl (0.5 g·L−1),
FeSO4·7H2O (0.01 g·L−1), CoCl2·6H2O (0.001 g·L−1), ZnSO4·7H2O (0.0067 g·L−1)],
glucose (15.0 g·L−1), malt (15.0 g·L−1) and agar (15.0 g·L−1) in Petri dishes maintained at 4
°C in the refrigerator [14].
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2.5 Preparation of culture medium and artificial sea water
Composition of culture medium: 20 g.L-1 malt extract (Acumedia) in artificial sea
water. Composition of artificial sea water: CaCl2.2H2O (1.36 g.L-1), MgCl2.6H2O (9.68
g.L-1), KCl (0.61 g.L-1), NaCl (30.0 g.L-1), Na2HPO4 (0.014 mg.L-1), Na2SO4 (3.47 g.L-1),
NaHCO3 (0.17 g.L-1), KBr (0.1 g.L-1), SrCl2.6H2O (0.040 g.L-1), and H3BO3 (0.030 g.L-1)
at pH 8 [14].
2.6. Testing the growth of marine-derived fungus P. citrinum CBMAI 1186 in
organic solvents
To study the effects of organic solvents on the growth of P. citrinum CBMAI 1186,
three slices of solid medium from the stock culture (0.5 × 0.5 cm) bearing fungal mycelia
were cut and grown at 32 ºC in Erlenmeyer flasks (250 mL) containing 95 mL of artificial
sea water (pH 7.0) supplemented with 5 mL of each organic solvent (acetone, ethyl acetate,
n-butanol, dichloromethane, n-hexane or toluene). After 6 d of incubation on a rotary
shaker (130 rpm, 32 °C), the mycelia were filtered on a Buchner funnel, then dried and
weighed.
2.7. Optimizing preparation of n-hexane in a biphasic system used in the
biocatalytic reduction of an α,β-unsaturated ketone (3a) by the marine-derived fungus P.
citrinum CBMAI 1186
Small slices of solid medium (0.5 × 0.5 cm) bearing the mycelium of P. citrinum
CBMAI 1186 were cut from the stock solid culture and used to inoculate 600 mL of a
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liquid culture medium contained in Erlenmeyer flasks (1 L). The mycelia were incubated
in the medium based on artificial sea water at 32 ºC for 6 d in an orbital shaker (130 rpm),
after which they were harvested by Buchner filtration and suspended in 100 mL of the
biphasic reaction mixtures in Erlenmeyer flasks (250 mL). The biocatalytic reductions
were carried out with 5.0 g (wet weight) of mycelium and 50 mg α,β-unsaturated ketone
3a, previously dissolved in dimethylsulfoxide (400 µL), in a mixture of n-hexane (0, 5, 10,
20, 40, 60, 80 and 100 mL) and phosphate buffer solution (Na2HPO4/KH2PO4, pH = 7, 0.1
mol.L-1), yielding 100 mL of solution. The mixtures were incubated for 6 d in an orbital
shaker at 32 ºC and 130 rpm. The reactions were monitored using TLC every 24 h. Finally,
1.0 mL samples were extracted with 1.0 mL EtOAc by mixing on a vortex and centrifuging
at 6000 rpm for 6.0 min in a HERMLE Z-200 centrifuge and analyzed using GC-MS.
2.8. Biocatalytic reduction of α,β-unsaturated ketones (3a-g) by the marine-derived
fungus P. citrinum CBMAI 1186 in a biphasic system
The mycelia of the fungus P. citrinum CBMAI 1186 were harvested via Buchner
filtration and suspended in 100 mL of the biphasic reaction mixtures contained in
Erlenmeyer flasks (250 mL). The biocatalytic reductions were carried out with 5.0 g (wet
weight) of mycelium and 50 mg α,β-unsaturated ketone (3a-g), previously dissolved in
dimethylsulfoxide (400 µL), in a mixture of 10 mL n-hexane and 90 mL phosphate buffer
solution (Na2HPO4/KH2PO4, pH = 7, 0.1 mol.L-1). The mixtures were incubated for 6 d in
an orbital shaker at 32 ºC and 130 rpm. The reactions were monitored using TLC every 24
h. Products were isolated and identified in the medium (See Section 2.9).
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2.9. Isolation of products from biotransformation by P. citrinum CBMAI 1186 in
the n-hexane-aqueous biphasic system
After 6 d of reaction, the mycelium was filtered on a Buchner funnel and the filtrate
was extracted with EtOAc (3×50 mL). The organic phase was dried over anhydrous
Na2SO4, filtered, evaporated under vacuum and analyzed using GC-MS. The EtOAc
extracts obtained were purified using flash CC over silica gel to yield the pure products.
The spectroscopic data of the isolated products were in agreement with those reported in
the literature.
2.10. Absolute configuration
The optical rotation of the purified product 4-phenylbutan-2-ol (5) was measured in
a PerkinElmer (Waltham, MA, USA) 241 polarimeter with a 1 dm cuvette at the sodium
D-line. The absolute configuration of alcohol 5 was determined by comparing the sign of
the specific rotation measured for the product with that reported in the literature [15].
25
Experimental data for (S)-(+)-4-phenylbutan-2-ol (5) were as follows: [α]D +1.8 (c 1.2,
CHCl3, 36% ee) and literature [α]D25 +3.8 (c 1.84, CHCl3, 15% ee) [15]. The enantiomeric
excess was determined with a Shimadzu LC-10AD HPLC system with UV detector (190-
254 nm) and a Chiralcel® OD-H chiral column (0.46 cm × 25 cm; 5 µm); mobile phase
hexane/2-propanol (90:10) and flow rate 0.5 mL·min-1.
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3. Results and Discussion
3.1 Screening of organic solvents in biphasic systems with marine-derived fungus P.
citrinum CBMAI 1186
Little was known about the tolerance of the marine-derived fungus P. citrinum
CBMAI 1186 to organic solvents. A preliminary study was therefore carried out on the
growth of this fungus in the presence of various organic solvents (Table 2). The latter were
chosen to cover a wide range of logP values and polarities. The experiments were carried
out in duplicate, and the results are summarized in Table 2.
The results show that the solvent of lowest toxicity to the growth of the mycelium of
P. citrinum CBMAI 1186 was n-hexane, which is consistent with the empirical rule that
the toxicity of the solvent is expressed as the logarithm of the partition coefficient (logP).
A solvent whose logP is greater than 4.0 tends to show little toxicity in microbial cells,
whereas those with low values often inhibit mycelial growth. n-Hexane has a logP of 3.98
and was clearly the least inhibitory to the growth of P. citrinum CBMAI 1186. n-Hexane
was then used in the biphasic system for the reduction reactions.
Some studies have demonstrated that non-polar solvents, such as n-hexane, do not
displace the water molecules on the enzyme, making them good candidates to carry out the
reaction in a biphasic system for biocatalysis [16]. The solvents that inhibited the growth
of this marine-derived fungus most strongly were n-butanol (Entry 4, Table 2) and
dichloromethane (Entry 5, Table 2). Some organic solvents in the medium interfere with
hyphal growth, nuclear division and differentiation (when it occurs), leading eventually to
cellular lysis and death [17].
It is worth noting that the two-phase system containing acetone in the culture
medium (5%) showed significant growth of the fungus. This result is the opposite of what
was expected because acetone has a logP of -0.24. The unusual behavior of acetone may
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be due to its miscibility in water and the fact that it can be biotransformed by the fungus
[18].
Table 2 here
3.2 Bioreduction of ketone 3a with the mycelium of the marine fungus P. citrinum
CBMAI 1186 in a biphasic system with n-hexane as organic solvent
Among the solvents tested in the biphasic system, the one showing the lowest
inhibition of growth of the marine fungus P. citrinum CBMAI 1186 was chosen for the
reduction of ketone 3a. n-Hexane was used in a biphasic system for the biohydrogenation
of (1E,4E)-1,5-diphenylpenta-1,4-dien-3-one (3a). The efficient and chemoselective
reduction of α,β-unsaturated ketones has attracted the interest of several researchers. In this
experiment, we used the reduction of a compound containing two symmetrical α,β-
unsaturated double bonds (ketone 3a) to assess the biohydrogenation chemoselectivity.
The reaction was conducted with 50 mg of 3a, dissolved in 50 µL of DMSO, in 8
different mixtures of n-hexane and phosphate buffer (pH = 7, 0.1 mol·L-1) to optimize the
rate of the biocatalytic process (the reduction of carbon-carbon double bonds) and to
observe the selectivity of the biohydrogenation (Table 2).
The biotransformation of compound 3a led exclusively to the formation of
compound 4a after 6 d of reaction with the mycelia of P. citrinum CBMAI 1186 at 32 °C
and 132 rpm. Compound 4a is produced by the action of the enoate reductases (ERs) on
the two carbon-carbon double bonds in the α,β-unsaturated ketone 3a. The double bond is
known to be effectively reduced by fungal ERs as it is made susceptible to the nucleophilic
attack of a hydride delivered by the reduced flavin cofactor in the presence of an electron-
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withdrawing substituent, which is also able to form hydrogen bonds with certain amino
acid residues within the active site of the enzyme [19].
The hydrogenation of the carbon-carbon double bonds in the absence of n-hexane
gave product 4a in 62% yield. In addition, 10 to 20% n-hexane yielded excellent results in
the biohydrogenation of ketone 3a (Table 3, entries 2 and 3), with a selective reduction of
both carbon-carbon double bonds and 90% conversion (46% isolated yield) according to
GC-MS analysis. The hydrogenation of the carbon-carbon double bonds in preference to
the carbonyl was not affected by varying the concentration of n-hexane under these
conditions. Invariably, we observed only the formation of compound 4a.
Table 3 here
When the n-hexane exceeded 40% of the medium, a significant reduction was
observed in the enzymatic hydrogenation of ketone 3a by the fungus P. citrinum CBMAI
1186 (Table 3, entry 4). When 100% n-hexane was used with the mycelium, no conversion
was observed. This decrease in the rate of conversion of ketone 3a may be due to various
phenomena, including inactivation and denaturation of the enoate reductase enzymes
responsible for this biotransformation. It was noted that the larger the amount of n-hexane,
the less dispersed the mycelium of the fungus in the reaction medium was. The reactions
conducted at zero or a low concentration of n-hexane showed less than optimal rates of
conversion to 4a. Therefore, the use of n-hexane in proper amounts was crucial to the good
performance of the reaction. Recently, Schaefer et al. [20] also observed the
chemoselective reduction of the double bonds of ketone 3a in good conversion (82%) after
optimization of the experimental conditions using baker’s yeast. The best reaction
conditions were 66.7 g·L−1 of Fleischmann BY, 8.3 × 10−3 mol·L-1 of substrate, pH 6.5 at
Page 11 of 26
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35 °C in a biphasic system with phosphate buffer and n-hexane (70:30), and with DMSO
(2.5%) as additive.
Under the above optimum reaction conditions, the α,β-unsaturated ketones 3b-g were
biotransformed with the whole mycelium of the fungus P. citrinum CBMAI 1186 in a
biphasic system of phosphate buffer and n-hexane (9:1) for the chemoselective reduction
of their carbon-carbon double bonds. These results show the importance of using a
biphasic system in reactions catalyzed by the fungus P. citrinum CBMAI 1186 to increase
the efficiency of biohydrogenation.
3.3 Bioreduction of ketone 3b-g with the mycelium of the marine-derived fungus P.
citrinum CBMAI 1186 in a biphasic system of phosphate buffer and n-hexane (9:1)
To explore the selective conjugate reduction of α,β-unsaturated ketones with whole
cells of the fungus P. citrinum CBMAI 1186 in the biphasic system of phosphate buffer
and n-hexane (9:1), (1E,3E,6E,8E)-1,9-diphenylnona-1,3,6,8-tetraen-5-one 3b was selected
as the substrate (Table 4, entry 1). In contrast with the previous result, no
biotransformation of this compound was observed. We must recall that this is the first
recorded attempt at biotransformation of compound 3b using fungal cells. Elongation of
the unsaturated chain prevented reduction of the double bond, most likely because the size
of the substrate hindered its interaction with the catalytic site of the enzyme.
To explore further the biohydrogenation of α,β-unsaturated ketones in this biphasic
system of phosphate buffer and n-hexane (9:1) with P. citrinum CBMAI 1186 as a
biocatalyst, (E)-3-phenyl-1-(thiophen-3-yl) prop-2-en-1-one (3c) and (2E,4E)-5-phenyl-1-
(thiophen-2-yl)penta-2,4-dien-1-one (3d) were used in the next experiments. These
substrates are interesting because they allow comparison with some synthetic methods
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already reported for the reduction of 3c to the α,β-hydrogenated product 4c, also obtained
here by biocatalytic reduction with P. citrinum CBMAI 1186.
Table 4 here
Feng et al. [21] reported the reduction of α,β-unsaturated enone 3c with N,N-
dimethylbenzylimidazole (1.5 equiv) as a source of hydride ion and Mg(ClO4)2 (10 mol%)
as a catalyst, in toluene for 24 h at 80 °C, resulting in 86% yield. Xu et al. [22] used a
Hantzsch ester as a source of hydride to reduce the carbon-carbon double bond of enone
derivatives by refluxing in xylene at 40 h, again resulting in 82% yield. These chemical
methods are in some cases less efficient and they are less eco-friendly than biocatalytic
methods. The chemoselective biohydrogenation of ketone 3c was performed by P. citrinum
CBMAI 1186 in the biphasic system, yielding 99% of product 3c according to GC-MS
(65% isolated yield) (Table 4, entry 2). When (2E,4E)-5-phenyl-1-(thiophen-2-yl)penta-
2,4-dien-1-one (3d) was subjected to the bioreduction reaction under the same conditions,
(E)-5-phenyl-1-(thiophen-2-yl)pent-4-en-1-one (4d) was obtained in a good yield of 69%
(Table 4, entry 3).
The bioreduction of (2E,4E)-1,5-diphenylpenta-2,4-dien-1-one (3e) was also
investigated to further assess the extent of the chemoselectivity of enoate reductases in the
multienzymatic biological system of the mycelia of the marine-derived fungus P. citrinum
CBMAI 1186. The γ,δ-unsaturated product, (E)-1,5-diphenylpent-4-en-1-one (4e), was
then obtained in an excellent conversion of 97% (Table 4, entry 4). To the best of our
knowledge, the chemoselective reduction of the α,β-carbon-carbon double bond in 3e is
not achievable by using a conventional nucleophilic hydride as a reducing agent. This is
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the first report of chemoselective reduction by the enzymatic reaction of systems with
α,β,,δ-unsaturated bonds.
The chemoselective and regioselective reduction of the α,β-unsaturated bonds of
compounds similar to 3e in excellent yield is an arduous task by synthetic means as the
transfer of hydride to compounds similar to 3e is not so selective. In addition, catalytic
metal compounds, such as InCl3 [23], are used in the presence of NaBH4 or TiCl4 with a
Hantzsch ester [24] or rhodium with trimethylsilane [25]. The reduction of compound 3e
by the wet mycelia of the microorganism P. citrinum CBMAI 1186, as reported in this
article, in addition to being innovative in biocatalysis, was highly efficient, giving high
yields with a very simple and eco-friendly procedure.
The proposed mechanism of ER-mediated reactions (Figure 1) first consists of the
transfer of a hydride ion from the flavin prosthetic group of the enzyme to the sp2 carbon
atom in the β-position with respect to the activating substituent, the latter being involved in
the formation of hydrogen bonds with amino acid residues in the active site of the enzyme.
A tyrosine residue delivers a proton, which can be derived from exchange with the
medium, to the carbon atom, usually on the opposite stereoheterotopic face of the alkene,
affording anti-hydrogen addition [26-27]. This proposed mechanism was based on the
mechanism for reactions mediated by Old Yellow Enzymes (OYEs).
Figure 1 here
We also assessed the performance of the ER enzymes of the marine-derived fungus
P. citrinum CBMAI 1186 in reducing (3E,5E)-6-phenylhexa-3,5-dien-2-one (3f) (Table 4,
entry 5). The result was highly significant for substrate 3f because this substrate was
converted to the α,β-saturated ketone 4f (76%) by reduction of the α,β-C=C double bond,
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whereas the γ,δ-C=C double bond and the C=O bond remained intact. The merit of these
experiments is the high chemoselectivity and regioselectivity of the C=C bond reduction.
A different reaction course was observed for (E)-4-phenylbut-3-en-2-one (3g). In
this case, after the sixth day of biotransformation, the reduction of enone 3g yielded two
main products (4g and 5). The ketone 3g was completely metabolized to 4-phenylbutan-2-
one (4g) in 62% (57% isolated yield) and 4-phenylbutan-2-ol (5) in 37% (30% isolated
yield) of the reaction mixture (GC-MS). A trace amount (1%) of the reaction mixture was
obtained as the allyl alcohol (E)-4-phenylbut-3-en-2-ol (6) (Scheme 1). Alcohol (S)-5 was
obtained in 36% ee. The low predominance of the (S)-isomer of alcohol 5 (20% ee) was
also observed by Skrobiszewski et al. [28]. This compound constituted only 6% of the
reaction mixture after 6 days of reaction mediated by whole cells of Pleurotus ostreatus.
Formation of alcohol 5 was also observed by Janeczko et al. [29] during
biotransformation of ketone 3g when submitted to the action of different fungal and yeast
strains. Hydrogenation of double bond was then followed by a reduction of resulting
ketone 4 which in the case of Yarrowia lipolytica KCh 71yielded after 7 h the (S)-alcohol 5
with 31% of conversion and 27% ee. Longer incubation time resulted in a increase of the
(R)-enantiomer and led to obtain (R)-alcohol (5) with 91% of conversion and ee > 99%
which was the result of enantioselective oxidation of (S)-enantiomer of alcohol 5.
Scheme 1 here
In summary, we have developed a novel approach to the transition metal-free
conjugate reduction of α,β-unsaturated and α,β,γ,-unsaturated ketones to the
corresponding saturated ketones with the mycelium of the marine-derived fungus P.
citrinum CBMAI 1186 in a biphasic system containing 10% n-hexane. However, in our
Page 15 of 26
15

investigations, the course of the transformation differed depending on the substrate
structure.
4. Conclusion
Biotransformation with the mycelium of the marine-derived fungus P. citrinum
CBMAI 1186 as the source of hydride in the biphasic system of phosphate buffer and n-
hexane (9:1) has provided a novel method for the chemoselective reduction of the α,β-
carbon-carbon double bond in α,β-unsaturated ketones. This is very hard to achieve using a
nucleophilic hydride from conventional reducing agents. Certainly, this study constitutes
the first use of a biphasic system to achieve chemoselective biohydrogenation in high
yields by the marine-derived fungus P. citrinum CBMAI 1186. We believe this procedure
will find useful applications in biosynthetic methods.
Acknowledgments
I. M. Ferreira thanks the Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq) for the scholarship. A.L.M. Porto thanks the CNPq and the Fundação
de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for financial support. The
English in this article was reviewed by Timothy Roberts, MSc., a native English speaker,
and American Journal Experts.
Supplementary data
Supplementary data (1H NMR, 13C NMR spectra and GC-MS) associated with this
article can be found, in the online version, at http://dx.doi.org/xxxx
Page 16 of 26
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18

Table 1. Preparation of the unsaturated ketones 3a-f by aldol condensation.
NaOH
O
O
O
R1
+
H
R2
EtOH, 6 h, r.t.
R1
R2
1a-c
2a-b
3a-f
Entry
1
Ketone
Aldehyde
Product
Yield (%)
65
O
O
O
H
1a
3a
2a
O
O
O
H
2
82
78
83
73
1a
2b
3b
O
O
O
H
O
S
3
4
S
1b
2a
3c
O
O
H
H
S
S
1b
3d
2b
O
O
O
O
5
6
3e
2b
2b
1c
O
O
H
89
1a
3f
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Table 2. Growth of the mycelium of the marine-derived fungus P. citrinum CBMAI 1186
in the presence of organic solvents.
Entry
Solvent (5%)
artificial sea watera
acetoneb
ethylacetateb
n-butanolb
dichloromethaneb
n-hexaneb
logPc
-
Dry weight (mg)
1
2
3
4
5
6
7
783
193
16
-0.24
0.73
0.88
0.93
3.98
2.73
12
12
724
32
tolueneb
a100 mL of artificial sea water (See experimental section).
b95 mL of artificial sea water at pH 8 plus 5 mL of the respective organic solvent (See
experimental section 2.7).
clogarithm of the partition coefficient [18].
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Table 3. Study of the proportion of hexane in mixtures with an aqueous medium
(phosphate buffer) for the bioreduction of (1E,4E)-1,5-diphenylpenta-1,4-dien-3-one (3a)a.
Penicillium citrinum
O
O
CBMAI 1186
biphasic system
32 ºC, 132 rpm, 6 d
4a
3a
Entry
n-hexane (%)
conv. (%)b
1
2
3
4
5
6
5
75
90
90
80
36
29
10
20
40
60
80
aReaction conditions: 5.0 g of wet mycelia and 50 mg of ketone 3a (6 d, 32 °C, 130 rpm).
bconv. = concentration of product 4a determined by GC-MS analysis.
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Table 4. Bioreduction of ketone 3b-f with the mycelium of the marine-derived fungus P.
citrinum CBMAI 1186 in a biphasic system of phosphate buffer and n-hexane (9:1).
Penicillium citrinum
O
O
H
CBMAI 1186
biphasic system
R1
R2
R1
R2
H
32 ºC, 132 rpm, 6 d
3b-f
4b-f
Entry
1
Substrate
Product
Conv. (%)
0
O
Nr
3b
O
O
2
3
99
S
S
3c
4c
O
O
69
97
76
S
S
3d
4d
O
O
4
5
3e
4e
4f
O
O
3f
Nr: No reaction.
Page 22 of 26
23

Penicilium citrinum
CBMAI 1186
O
OH
O
OH
biphasic system
+
+
32 ºC, 132 rpm, 6 d
4g
3g
5
6
57%
not isolated
30%
Scheme 1. Biotransformation of 3g by the marine-derived fungus P. citrinum CBMAI
1186 in the biphasic system of phosphate buffer and n-hexane (9:1).
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24

O
H
H
O
P. citrinum
H
H
CBMAI 1186
- H-FMNH2
Figure 1. Mechanism of possible substrate reactive binding modes for ER-mediated
reduction of C=C double bonds by OYE.
Page 24 of 26
25

Graphical Abstract
Chemoselective biohydrogenation of α,β- and α,β,γ,δ-unsaturated ketones by the
marine-derived fungus Penicillium citrinum CBMAI 1186 in a biphasic system
Irlon M. Ferreira, Eloá B. Meira,Isac G. Rosset, André L. M. Porto*
Penicillium citrinum
O
H
O
CBMAI 1186
biphasic system
32 ºC, 132 rpm, 6 days
H
97
conversion =
%
Representative
examples:
H
H
O
O
H
H
S
92
%
76
conversion =
conversion =
%
H
H
O
H
H
90
conversion =
%
Page 25 of 26
26

 Highlights
 Chemoselective biohydrogenation of unsaturated ketones by whole mycelia of
Penicillium citrinum CBMAI 1186;
 Reduction of α,β- and α,β,γ,δ-unsaturated ketones employing a biphasic system;
 Study the effect of organic solvents on the cell growth of P. citrinum CBMAI 1186;
 Marine fungus presented active enoate reductases.
Page 26 of 26
27