Enantioselective ene-reduction of E-2-cyano-3-(furan-2-yl) acrylamide by marine and terrestrial fungi and absolute configuration of (R)-2-cyano-3-(furan-2-yl) propanamide determined by calculations of electronic circular dichroism (ECD) spectra

Article abstract of DOI:10.1002/chir.23078

This work reports the green organic chemistry synthesis of E-2-cyano-3(furan-2-yl) acrylamide under microwave radiation (55?W), as well as the use of filamentous marine and terrestrial-derived fungi, in the first ene-reduction of 2-cyano-3-(furan-2-yl) acrylamide to (R)-2-cyano-3-(furan-2-yl)propanamide. The fungal strains screened included Penicillium citrinum CBMAI 1186, Trichoderma sp. CBMAI 932 and Aspergillus sydowii CBMAI 935, and the filamentous terrestrial fungi Aspergillus sp. FPZSP 146 and Aspergillus sp. FPZSP 152. A compound with an uncommon CN-bearing stereogenic center at the α-C position was obtained by enantioselective reactions mediated in the presence of the microorganisms yielding the (R)-2-cyano-3-(furan-2-yl) propanamide 3a. Its isolated yield and e.e. ranged from 86% to 98% and 39% to 99%, respectively. The absolute configuration of the biotransformation products was determined by time-dependent density functional theory (TD-DFT) calculations of electronic circular dichroism (ECD) spectra. Finally, the tautomerization of 2-cyano-3-(furan-2-yl) propanamide 3a to form an achiral ketenimine was observed and investigated in presence of protic solvents.

Full text of DOI:10.1002/chir.23078

Received: 21 February 2019
DOI: 10.1002/chir.23078
Revised: 9 April 2019
Accepted: 11 April 2019
R E G U L A R A R T I C L E
Enantioselective ene‐reduction of E‐2‐cyano‐3‐(furan‐2‐yl)
acrylamide by marine and terrestrial fungi and absolute
configuration of (R)‐2‐cyano‐3‐(furan‐2‐yl) propanamide
determined by calculations of electronic circular dichroism
(ECD) spectra
David E.Q. Jimenez1,2 | Juliana C. Barreiro2
| Fernando M. dos Santos Jr3 |
Suzan P. de Vasconcellos4 | André L.M. Porto2 | João M. Batista Jr5
1 Departamento de Química, Universidade
Abstract
Federal do Amapá, Macapá, Brazil
This work reports the green organic chemistry synthesis of E‐2‐cyano‐3(furan‐
2 Laboratório de Química Orgânica e
Biocatálise, Instituto de Química de São
Carlos, Universidade de São Paulo ‐ USP,
São Carlos, Brazil
3 Departamento de Química Orgânica,
Instituto de Química, Universidade
Federal Fluminense, Niterói, Brazil
2‐yl) acrylamide under microwave radiation (55 W), as well as the use of fila-
mentous marine and terrestrial‐derived fungi, in the first ene‐reduction of 2‐
cyano‐3‐(furan‐2‐yl) acrylamide to (R)‐2‐cyano‐3‐(furan‐2‐yl)propanamide.
The fungal strains screened included Penicillium citrinum CBMAI 1186,
Trichoderma sp. CBMAI 932 and Aspergillus sydowii CBMAI 935, and the fila-
mentous terrestrial fungi Aspergillus sp. FPZSP 146 and Aspergillus sp. FPZSP
152. A compound with an uncommon CN‐bearing stereogenic center at the
α‐C position was obtained by enantioselective reactions mediated in the pres-
ence of the microorganisms yielding the (R)‐2‐cyano‐3‐(furan‐2‐yl)
propanamide 3a. Its isolated yield and e.e. ranged from 86% to 98% and 39%
to 99%, respectively. The absolute configuration of the biotransformation prod-
ucts was determined by time‐dependent density functional theory (TD‐DFT)
calculations of electronic circular dichroism (ECD) spectra. Finally, the
tautomerization of 2‐cyano‐3‐(furan‐2‐yl) propanamide 3a to form an achiral
ketenimine was observed and investigated in presence of protic solvents.
4 Departamento de Ciências
Farmacêuticas, Universidade Federal de
São Paulo‐UNIFESP, Diadema, Brazil
5 Instituto de Ciência e Tecnologia,
Universidade Federal de São Paulo‐
UNIFESP, São José dos Campos, Brazil
Correspondence
Juliana C. Barreiro, Laboratório de
Química Orgânica e Biocatálise, Instituto
de Química de São Carlos, Universidade
de São Paulo ‐ USP, Av. João Dagnone,
1100, Ed. Química Ambiental, Jd. Santa
Angelina, 13563‐120, São Carlos, SP,
Brazil.
KEYWORDS
Email: juliana.barreiro@gmail.com
biocatalysis, electronic circular dichroism, enoate reductase, Knoevenagel condensation, marine‐ and
terrestrial‐derived fungi
Funding information
Conselho Nacional de Desenvolvimento
Científico e Tecnológico; Coordenação de
Aperfeiçoamento de Pessoal de Nível
Superior; Fundação de Amparo à Pesquisa
do Estado de São Paulo, Grant/Award
Numbers: 07089‐2/2015, 18257‐0/2014,
23794‐0/2016 and 25222‐9/2014
Chirality. 2019;1–9.
wileyonlinelibrary.com/journal/chir
© 2019 Wiley Periodicals, Inc.
1

2
JIMENEZ ET AL.
1 | INTRODUCTION
2 | MATERIALS AND METHODS
The use of enzymes (homogenous or heterogeneous cat-
alysts) as mediators in organic reactions is widely
known as biocatalysis, and it has become a powerful
2.1 | General methods
All manipulations involving the filamentous fungi were
carried out under sterile conditions in a Veco laminar
flow hood. A Technal TE‐421 orbital shaker was used
for biocatalytic experiments.
tool
for
development
of
organic
synthetical
protocols. Moreover, the presence of biocatalysts can
promote reactions that introduce a chiral center in
the molecule with high chemoselectivity, regioselec-
tivity, and enantioselectivity at mild reaction condi-
tions.1-6 Numerous organic reactions have reported the
use of biocatalytic protocols, such as hydrolytic reac-
tions of esters and amides,7 transesterification reac-
tions,8 oligosaccharide syntheses,9-11 acylation of
chlorohydrins,12 nitrile biodegradations,13 Baeyer‐
Villiger oxidations,14 aldol condensations,15 polymeriza-
tions,16 industrial applications,17 and Knoevenagel
condensations.18
A special attention has been given for the Knoevenagel
condensation reactions as an effective tool to increase the
carbon chain, as well as its functionalization19 being
applied in the synthesis of bioactive organic com-
pounds,20 polymers,21 and synthetic intermediates.22 On
this reaction, an adduct of carbon‐carbon double bond
are formed by reaction between a carbonyl compound
and a compound containing an active methylene group.19
The reactivity of the active methylene groups is directly
attached to electron magnetic groups, for example, CN,
COOR, COR (R = aliphatic, aromatic, amide, ester,
nitrile).23
Our research group have been employed with success
the biocatalytic protocols for different purposes such as
the asymmetric reduction of isatin,24 the biodegradation
of pentachlorophenol,25 the acylation of chlorohydrins,26
the resolution of cyanohydrins,27 the bioreduction of
α,β,γ,δ‐unsatured ketones,28 and the hydrogenation of
bis‐α,β‐unsatured enones.29 Recently, Jimenez et al18
reported for the first time the biocatalytic ene‐reduction
of aromatic benzylidenemalononitriles.
On the basis of these findings, this paper presents a
new reaction step for the synthesis of 2‐cyano‐3‐(furan‐
2‐yl) acrylamide by microwave radiation followed by
bioreduction of the Knoevenagel adduct using marine‐
derived and terrestrial fungi. In addition, the
absolute configuration of (R)‐2‐cyano‐3‐(furan‐2‐yl)
propanamide 3a obtained by biotransformation was
determined by time‐dependent density functional theory
(TD‐DFT) calculations of electronic circular dichroism
(ECD) spectra.
Gas chromatography‐mass spectrometry (GC‐MS), a
Shimadzu GC2010 Plus gas chromatography system
coupled to a mass‐selective detector (Shimadzu MS2010
Plus) in electron ionization mode (70 eV) equipped with
a DB‐5MS fused silica column (Agilent J & W Advances
30 × 0.25 × 0.25 μm) was used. Helium was used as a drag
gas with a constant flow of 0.75 mL/min at 46.5 kPa, and
the injection volume was 0.5 μL of solution, with concen-
trations of 1.0 to 3.0 mg/mL. The method of analysis ini-
tially consisted of maintaining the column at 90°C for
4 minutes and then on a heating ramp of 6°C/min until
reaching the temperature of 280°C, which column was
maintained for 5 minutes. The injections were performed
in split mode at a 1:20 ratio. Ion fragments were detected
in the range of 40 to 500 Da.
Liquid chromatography (LC) analyses were performed
in a Shimadzu equipment model LC‐20 AT (Shimadzu
Co., Japan) with a SPD‐M20A detector (λ=215 nm) con-
nected to
a
CHIRALPAK AD‐H chiral column
(0.46 × 25 cm). The mobile phase was composed of hex-
ane and ethanol (60:40) with a flow rate of 0.5 mL/min;
the oven temperature was set at 30°C and the injection
volume of 10 μL.
Fourier Transform Infrared (FT‐IR) spectra were
recorded on a Shimadzu IRAffinity spectrometer, and
the samples were prepared as KBr disks in the 4000 to
400 cm−1 region.
1H Nuclear Magnetic Resonance (NMR) and 13C NMR
spectra were recorded on an Agilent Technologies 500/54
Premium Shielded or Agilent Technologies 400/54 Pre-
mium Shielded spectrometer, with CD3OD and CDCl3
as deuterated solvents and tetramethylsilane (TMS) as
the internal standard. Unless otherwise noted, the chem-
ical shifts given in ppm, and coupling constants (J) values
were reported in Hz. The chemical shifts (δ) were
expressed in parts per million (ppm) and referenced to
the internal standard (TMS) signal and the deuterated
solvents used, ie, CDCl3 (δH 7.26, δC 77.16) and CD3OD
(δH 4.87, 3.31, δC 49.00).
The microwave irradiation experiment was performed
using a Discover System from CEM Corporation at a
2.45‐GHz frequency with maximal power output of about
200 W. with internal magnetic stirring. For the reactions,
the power of the microwave reactor used was 55 W
(30 min at 80 °C).
Finally, the tautomerization of 2‐cyano‐3‐(furan‐2‐yl)
propanamide 3a to form an achiral ketenimine
was observed and investigated in presence of protic
solvents.

JIMENEZ ET AL.
3
The electronic circular dichroism (ECD) spectrum of
each enantiomer eluting from the chiral LC system was
measured in a Jasco CD‐2095 detector by trapping in a
2.5‐cm quartz cell through a switching valve. The spectra
were average computed over three instrumental scans,
and the intensities were presented in terms of ellipticity
values (mdeg). The ECD spectra were baseline corrected
by subtraction from a measurement obtained for the
same solvent used n‐hexane and EtOH (60:40).
2.4 | Synthesis of racemic 2‐cyano‐3‐
(furan‐2‐yl)propanamide 3a
The E‐2‐cyano‐3‐(furan‐2‐yl)acrylamide 2a (50 mg,
0.308 mmol) was placed into the reaction vial with capac-
ity of 25 mL and dissolved in MeOH. The NaBH4 (80 mg,
2 mmol) was then added at 0°C. The reaction was moni-
tored by TLC (in a mixture containing hexane and ethyl
acetate 8:2). The TLC plates were revealed with subli-
mated iodine‐impregnated on silica gel. After 10 minutes,
all E‐2‐cyano‐3‐(furan‐2‐yl)acrylamide 2a was reduced to
the product 3a. The reaction was extracted with ethyl ace-
tate (3×10 mL). The aqueous phases were discarded and
the organic phases combined and concentrated by vac-
uum until total evaporation of the solvent and the residue
was purified by wash with heat hexane. The product 3a
2.2 | Chemical reagents
Furfural (99%) 1a, cyanoacetamide (99%) 1b were pur-
chased from Sigma‐Aldrich and used without any further
purification. The salts used for preparation of the artificial
sea, NaOH (97%), acetone, absolute ethanol, and THF
were purchased from Vetec and Synth (Brazil). Malt
extract was purchased from Acumedia (São Paulo, Bra-
zil). The deuterated solvents, CD3OD (99.9%) and CDCl3
(99.8%), were purchased from Cambridge Isotope Labora-
tories. The thin layer chromatography (TLC) utilized was
DC‐Fertigfolien ALUGRAM XTra SIL G/UV254 (layer:
0.20‐mm silica gel 60 with fluorescent indicator UV254).
1
was characterized by GC–MS, LC, FT‐IR, and H NMR
and 13C NMR (Supporting Information). Scheme 2 illus-
trates the reduction of the Knoevenagel adduct 2a with
NaBH4.
2.5 | Composition of culture media and
artificial seawater
Composition of culture medium: 20 g/L malt extract
(Acumedia) in artificial seawater. Composition of artifi-
cial seawater: CaCl2.2H2O (1.36 g/L), MgCl2.6H2O
(9.68 g/L), KCl (0.61 g/L), NaCl (30.0 g/L), Na2HPO4
(0.014 mg/L), Na2SO4 (3.47 g/L), NaHCO3 (0.17 g/L),
KBr (0.1 g/L), SrCl2.6H2O (0.040 g/L), and H3BO3
(0.030 g/L) at pH 8 adjusted with KOH solution
(0.1 mol/L).
2.3 | Knoevenagel condensation under
microwave irradiation
A mixture of furfural 1a (1.0 mmol, 96.08 mg),
cyanoacetamide 1b (1.0 mmol, 84.08 mg), and 2 drops
of NaOH (1 M) in water (3 mL) were put in the micro-
wave reactor (55 W) for 30 minutes at 80°C. The reaction
was monitored by TLC. After completion, the reaction
was extracted with ethyl acetate (3×10 mL). The com-
bined organic phases were concentrated under vacuum
until total evaporation of the solvent, and the
Knoevenagel adduct 2a was recrystallized in a mixture
of ethanol and THF (1:1) to yield 95% of the pure com-
2.6 | Isolation and preservation of the
marine‐derived fungus
Marine‐derived fungus Penicillium citrinum CBMAI 1186
was isolated from the marine alga Caulerpa sp.;
Trichoderma sp. CBMAI 932 was isolated from the
sponge Geodia corticostylifera, and Aspergillus sydowii
CBMAI 935 was isolated from the marine sponge
Chelonaplysilla erecta. The alga and sponges were col-
lected by Prof. R.G.S. Berlinck in São Sebastião City, on
the coast of the State of São Paulo, Brazil.30 The fungi
were identified using both conventional and molecular
1
pound. The product 2a was characterized by H NMR
and 13C NMR, FT‐IR, LC, and GC–MS (Supporting Infor-
mation). Supplementary Material Scheme 1 illustrates the
synthesis of the Knoevenagel adduct 2a under microwave
irradiation.
SCHEME
condensation under microwave irradiation
1
Synthesis of adduct 2a by Knoevenagel
SCHEME 2 Synthesis of reduced compound 3a (2‐cyano‐3‐
(furan‐2‐yl)propanamide) in the presence of NaBH4

4
JIMENEZ ET AL.
methods at the Chemical, Biological and Agricultural
Pluridisciplinary Research Center (CPQBA) at the Uni-
versity of Campinas, SP, Brazil. The fungi P. citrinum
CBMAI 1186, Trichoderma sp. CBMAI 932 and A. sydowii
CBMAI 935 were deposited in the Brazilian Collection of
Environmental and Industrial Microorganisms (CBMAI –
http://webdrm.cpqba.unicamp.br/cbmai/). The terrestrial
fungi Aspergillus sp. FPZSP 146 and Aspergillus sp. FPZSP
152 were collected by Prof. Suzan P. de Vasconcellos from
the Unit of Organic Composting Production (UOCP)
belonging to the São Paulo Zoo Park Foundation
(Fundação Parque Zoológico de São Paulo – FPZSP).
2.9 | Isolation of product 3a obtained by
marine‐derived and terrestrial fungi
After 3 days of reaction, the liquid was filtered in a
Buchner apparatus and the mycelial mass obtained was
transferred to a 50 mL Erlenmeyer flask and suspended
in 20 mL of water and ethyl acetate (1:1). This biphasic
mixture was submitted to magnetic stirring for 30 minutes
and filtered again by a Buchner funnel. The filtrate was
extracted with ethyl acetate (3×50 mL). The organic
phase was dried over anhydrous Na2SO4, filtered, and
evaporated under vacuum. The extract obtained from
the filtrate was purified by chromatography column
(CC) over silica gel (230‐400 μm) and eluting with n‐
hexane and ethyl acetate (7:3). Yield obtained for the
product 3a with the fungi Penicillium citrinum CBMAI
1186, Trichoderma sp. CBMAI 932, Aspergillus sydowii
CBMAI 935, Aspergillus sp. FPZSP 146, and Aspergillus
sp. FPZSP 152 were 98%, 92%, 86%, 90%, and 94%, respec-
tively. The compound 3a was characterized by GC‐MS,
2.7 | Microorganism cultivation
The marine fungi P. citrinum CBMAI 1186, Trichoderma
sp. CBMAI 932, and A. sydowii CBMAI 935 were culti-
vated on Petri plates containing 2% of malt solid culture
medium using artificial seawater. The terrestrial fungi
Aspergillus sp. FPZSP 146 and Aspergillus sp. FPZSP
152 were cultivated on Petri plates containing 2% of
malt solid culture medium using distilled water with
the following composition: 20 g/L malt extract in dis-
tilled water. The pH was adjusted to 7. Culture
mediums were sterilized in autoclave (Phoenix, 121°C,
20 min, 1.5 kPa) and incubated for 7 days at 32°C and
3 days at 32°C, for the marine and terrestrial fungi,
respectively.
1
LC, FT‐IR, H NMR and 13C NMR.
2.10 | Calculations
All density functional theory (DFT) and TD‐DFT calcula-
tions were carried out at 298 K in the gas phase using
Gaussian 09 Software.31 Calculations were performed
for the arbitrarily chosen (R)‐2‐cyano‐3‐(furan‐2‐yl)
propanamide 3a. The conformational search was carried
out at the molecular mechanics level of theory with the
Monte Carlo algorithm employing the MM+ force field
incorporated in HyperChem 8.0.10 software package.
Nine conformers with relative energy (rel E.) within
6 kcal mol−1 of the lowest‐energy conformer were
selected and further geometry optimized at the
B3LYP/6‐31G(d) level. The six conformers with rel
E. < 1.9 kcal mol−1, which corresponded to more than
95% of the total Boltzmann distribution, were selected
for UV and ECD spectral calculations. Vibrational analy-
sis at the B3LYP/6‐31G(d) level resulted in no imaginary
frequencies, confirming the considered conformers as
real minima. Geometry optimization and frequency cal-
culations were also performed at the B3PW91/6‐311G(d,
p) level. TD‐DFT was then employed to calculate excita-
tion energy (in nm) and rotatory strength R in dipole
velocity form (Rvel in cgs units: 10−40 esu2 cm2), at the
CAM‐B3LYP/TZVP and wB97XD/aug‐cc‐pVDZ levels.
The calculated rotatory strengths from the first 30 sin-
glet → singlet electronic transitions were simulated into
an ECD curve using Gaussian bands with bandwidth σ
0.25 eV. The predicted wavelength transitions were mul-
tiplied with a scaling factor of 1.05, determined by the
2.8 | Bioreduction of E‐2‐cyano‐3‐(furan‐2‐
yl)acrylamide by filamentous fungi
The mycelia of the fungi P. citrinum CBMAI 1186,
Trichoderma sp. CBMAI 932, A. sydowii CBMAI 935,
Aspergillus sp. FPZSP 146, and Aspergillus sp. FPZSP
152 were harvested via Buchner filtration. In addition,
5 g (wet weight) of mycelium from each fungi was
suspended in 100 mL of phosphate buffer solution
(Na2HPO4/KH2PO4, pH=7, 0.1 mol/L) and distributed
in 250 mL Erlenmeyer flasks. The E‐2‐cyano‐3‐(furan‐2‐
yl)acrylamide 2a (50 mg, 0.308 mmol) was previously dis-
solved in dimethylsulfoxide (300 μL) and added to the
culture media. The reactions were incubated for 3 days
in an orbital shaker at 32°C and 130 rpm. The reaction
was monitored by collecting 2.0 mL aliquot, which was
extracted by stirring with ethyl acetate (2.0 mL) followed
by centrifugation (6.000 rpm, 6 minutes) and analyzed by
TLC and GC‐MS every 24 hours. The product 3a was iso-
lated and identified (Section 2.9). All biotransformations
were performed in triplicates.

JIMENEZ ET AL.
5
best agreement between experimental and calculated UV
spectra. The final spectra were generated as the
Boltzmann average of the lowest energy conformers iden-
tified and plotted using Origin 8 software.
SCHEME 4 Bioreduction of E‐2‐cyano‐3‐(furan‐2‐yl) acrylamide
2a by fungi
3 | RESULTS AND DISCUSSION
used for the bioreduction of E‐2‐cyano‐3‐(furan‐2‐yl)
acrylamide 2a. All reactions were monitored over 3 days
by TLC. After the extraction and purification process,
the products were identified by LC analysis.
The
malononitrile and the biotransformation of 2‐(furan‐2‐
ylmethyl)malononitrile into 2‐cyano‐3‐(furan‐2‐yl)
bioreduction
of
2‐(furan‐2‐ylmethylene)
The biotransformation reaction of compound 2a by
marine‐derived and terrestrial fungi promoted the forma-
tion of a single compound 3a with 86% to 97% of yield
(Table 1), which was obtained by action of ene reductases
(ERs) on the activated C═C double bond. The mecha-
nism established for the ERs enzyme action occurs by
nucleophilic attack of a hydride ion from the flavin cofac-
tor onto a β‐carbon atom of the C═C double bond. Subse-
quently, a tyrosine residue delivers a proton to the α‐
carbon atom, typically on the alkene opposite face,
affording anti‐hydrogen addition.32,33
propanamide by marine fungi P. citrinum CBMAI 1186
have already been demonstrated by Jimenez et al18
(Scheme 3).
Such biotransformation followed a multienzymatic
sequence of cascade reactions,18 in which two steps were
involved. On the first step, the bioreduction of compound
1c to compound 1d occurred possibly by the action of
enoate reductases, whereas on the second step, the hydro-
lysis of the nitrile group on compound 1d yielding the
compound 3a was catalyzed by nitrile hydratase present
in the culture media. The concomitant bioreduction and
biotransformation occurred on the CN group demon-
strate the importance of cascade reactions driven by
multienzymatic media present on the fungi in order to
obtain chiral compounds.
TABLE 1 Bioreduction of E‐2‐cyano‐3‐(furan‐2‐yl) acrylamide 2a
with marine and terrestrial fungi
On the basis of the work by Jimenez et al,18 a new path
of reaction has been proposed here to yield the compound
3a (Scheme 4). In this system, the production of
enantiomerically enriched α‐cyanoamide 3a can be
obtained from E‐2‐cyano‐3‐(furan‐2‐yl) acrylamide 2a
using microorganisms to reduce the carbon‐carbon dou-
ble bond (Scheme 4). The compound 2a was synthetized
using furfuraldehyde 1a and cyanoacetamide 1b under
microwave irradiation.
The adduct 2a illustrated in Scheme 1 was prepared
via Knoevenagel condensation using furfural 1a and
cyanoacetamide 1b in the presence of NaOH under
microwave irradiation with 95% yield. The reaction
mechanism is based on the removal of acidic hydrogen
of cyanoacetamide 1b by NaOH affording a carbanion
as intermediate. The formation of Knoevenagel adduct
2a occurs by the nucleophilic attack of the carbanion into
the carbonyl group of furfural generating an intermediate
that suffers a dehydration reaction. The marine‐derived
fungi (P. citrinum CBMAI 1186, Trichoderma sp. CBMAI
932, and A. sydowii CBMAI 935) and terrestrial fungi
(Aspergillus sp. FPZSP 146 and Aspergillus sp. 152) were
Isolated
Yield(%) 3a Configuration 3a
Enantiomeric ratio (%) /Absolute
Fungi
Penicillium
citrinum
CBMAI
1186
98
92
99/S
Aspergillus
sydowii
CBMAI
935
97/R
Aspergillus
sp. FPZSP
152
86
90
91/R
39/R
98/R
Aspergillus
sp. FPZSP
146
Trichoderma 94
sp. 932
SCHEME 3 Bioreduction and
biotransformation of 2‐(furan‐2‐
ylmethylene)malononitrile (adapted from
Jimenez et al18)

6
JIMENEZ ET AL.
The enantioselectivity of reduced product 3a was
product was confirmed by the analysis of racemic 3a, pre-
viously synthetized and purified.
The LC results showed that the fungi A. sydowii CBMAI
935, Aspergillus sp. FPZSP 152, Trichoderma sp. 932 pre-
sented enantiomeric ratio up to 95% to R‐configuration,
and only the fungi P. citrinum CBMAI 1186 with high e.
determined by enantioselective LC analysis. It can be
observed by LC chromatograms that the bioreduction in
the presence of all fungi yielded the compound 2‐cyano‐
3‐(furan‐2‐yl)propanamide 3a with good enantiomeric
ratio (Table 1 and Figure 1). The presence of bioreduced
FIGURE 1 LC—chiral chromatograms of compound 3a obtained by different fungi
FIGURE 2 Chromatograms of ( )‐2‐cyano‐3‐(furan‐2‐yl) propanamide 3a at different times of analyses (15, 30, 45, 60, 75 and 90 minutes).
Chromatographic conditions: CHIRALPAK AD‐H chiral column (0.46 × 25 cm), mobile phase: Hexane/ethanol (60:40), flow rate:
0.5 mL min−1, λ =215 nm). R‐enantiomer 3a: tR~7.7 minutes and S‐enantiomer 3a: tR ~8.3 minutes

JIMENEZ ET AL.
7
e. up to 95% to S‐configuration. Finally, the fungi Aspergil-
lus sp. FPZSP 146 showed low value of e.e. (39%) to R‐
configuration.
It was observed that for ( )‐2‐cyano‐3‐(furan‐2‐yl)
acrylamide 3a loss of chromatographic resolution for the
racemic compound occurred after successive analysis
onto LC system (Figure 2).
A tautomeric equilibrium was proposed for the ( )‐2‐
cyano‐3‐(furan‐2‐yl) propanamide 3a in the presence of
polar solvents (MeOH, EtOH). The formation of a
ketenimine derivative could justify the loss of chromato-
graphic resolution since it bears no stereogenic center.
Basically, this equilibrium occurs between the nitrile
and prototropic ketenimine forms (Scheme 5), and it is
common in compounds containing cyano groups with
one or more acidic hydrogens on the α‐C position.
In the equilibrium, the cyano forms generally arises as
the major compound.34 The ketenimine tautomer has
been previously reported by Kasturi et al35 in the pres-
ence of polar solvents such as water and methanol at
240 nm (malononitrile) and around 350 nm (extensive
conjugation) bands. In apolar solvents, eg, hexane, the
formation of ketenimine tautomer was not detectable by
Ultraviolet (UV) absorption.
In our study, biotransformation with fungi (Figure 1)
did not showed the equilibrium of ketenimine because
extraction reactions were performed with ethyl acetate.
Thus, it was possible under these conditions to obtain
the compound 3a in the chiral form with their respective
enantiomeric ratio.
FIGURE 3 Comparison of the observed UV and electronic
circular dichroism (ECD) spectra of ( )‐3a with the calculated
[CAM‐B3LYP/TZVP//B3LYP/6‐31G(d)] UV and ECD spectra of the
Boltzmann average of the lowest energy conformers identified for
(R)‐3a (Supporting Information for lowest energy conformers)
However, when the samples of compound 3a obtained
of biotransformation reactions were placed in the pres-
ence of methanol, the equilibrium to ketenimine form
was observed by chiral LC analysis.
It is also worth observed by LC analysis that both the
samples obtained by the fungi and the racemic synthesis,
after the removal of methanol, the chiral racemic form
prevailed.
The formation of the ketenimine tautomer was con-
firmed by 1H NMR and 13C NMR analyses in CDCl3
(aprotic solvent). The 2‐cyano‐3‐(furan‐2‐yl) propanamide
(3a) showed a signal at 3.70 ppm (dd, J = 8.0 and 5.4 Hz,
1H), which corresponds to a double doublet from a
hydrogen adjacent to the stereogenic center. Also,
between 3.40 and 3.23 ppm (m, 2H), a multiplet assigned
to two methylene protons adjacently attached to the
furan ring was observed (Supporting Information). From
the 13C NMR spectra analysis, it was observed a signal
at 165.7 ppm, which corresponds to the carbonyl amide
group, as well as a methine carbon at 37.5 ppm (stereo-
genic center) and a methylene carbon at 28.4 ppm signal.
These data were in accordance with those in the litera-
ture18 (Supporting Information).
1
From the H NMR analyses for the compound 3a in
CD3OD (protic solvent) it was not observed the signal at
3.70 ppm (dd, J = 8.0 and 5.4 Hz, 1H); however, a signal
at 3.36 ppm (s, 2H) was observed, which corresponds to
two methylene protons adjacent to the furan ring
(Supporting Information). Signals at 165.7 and 28.4 ppm
was observed for the 13C NMR spectra analysis; however,
the signal at 37.5 ppm corresponding to the stereogenic
methine carbon was not observed (Supporting Information).
These findings supported the formation of the
ketenimine tautomer (Scheme 5) in the presence of polar
solvents (eg, MeOH, EtOH). The signals referent to the
1
stereogenic center were presented in both H and 13C
SCHEME
5
Tautomerization
of
2‐cyano‐3‐(furan‐2‐yl)
NMR spectra recorded in aprotic deuterated solvents
propanamide 3a in protic solvent

8
JIMENEZ ET AL.
(eg, CDCl3) but was not observed when polar protic deu-
terated solvents (eg, CD3OD) (Supporting Information).
In order to assess the selectivity in the biotransforma-
tion of 2a by marine and terrestrial fungi, the absolute
configuration of the enantiomers of the product 3a was
assigned by ECD spectroscopy and TD‐DFT calculations.
The racemic synthetic 3a was used, and each peak eluting
from the chiral column was trapped into the ECD
detector.
The experimental ECD spectra of both enantiomers
were then compared with those calculated at the CAM‐
B3LYP/TZVP and wB97XD/aug‐cc‐pVDZ (Supporting
Information) levels of theory. The very good agreement
between observed and theoretical data at both levels of the-
ory (Figure 3 and Supporting Information) allowed the
unambiguous determination of the absolute configuration
of the first eluting peak as R‐enantiomer 3a and, conse-
quently, that of the last eluted peak as S‐enantiomer 3a.
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de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES) ‐ Finance Code 001 for the financial support
provide to the grants for DEQJ and JCB. Also, thanks to
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Tecnológico (CNPq) and Fundação de Amparo à Pesquisa
do Estado de São Paulo (FAPESP ‐ 18257‐0/2014, 25222‐
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ORCID
Juliana C. Barreiro https://orcid.org/0000-0002-3657-2620
João M. Batista Jr https://orcid.org/0000-0002-0267-2631
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SUPPORTING INFORMATION
Additional supporting information may be found online
in the Supporting Information section at the end of the
article.
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How to cite this article: Jimenez DEQ, Barreiro
JC, dos Santos Jr FM, de Vasconcellos SP, Porto
ALM, Batista Jr JM. Enantioselective ene‐reduction
of E‐2‐cyano‐3‐(furan‐2‐yl) acrylamide by marine
and terrestrial fungi and absolute configuration of
(R)‐2‐cyano‐3‐(furan‐2‐yl) propanamide determined
by calculations of electronic circular dichroism
(ECD) spectra. Chirality. 2019;1–9. https://doi.org/
10.1002/chir.23078
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