Stereoselective reduction of 2-azido-1-phenylethanone derivatives by whole cells of marine-derived fungi applied to synthesis of enantioenriched β-hydroxy-1,2,3-triazoles

Article abstract of DOI:10.1080/10242422.2017.1352585

Several marine-derived fungi were evaluated by the bioreduction of 2-azido-1-phenylethanone 1, and the strains A. sydowii CBMAI 935 and M. racemosus CBMAI 847 were selected for the reduction of 2-azido-1-phenylethanone derivatives 2–4. Whole cells of A. s

Full text of DOI:10.1080/10242422.2017.1352585

Biocatalysis and Biotransformation
ISSN: 1024-2422 (Print) 1029-2446 (Online) Journal homepage: http://www.tandfonline.com/loi/ibab20
Stereoselective reduction of 2-azido-1-
phenylethanone derivatives by whole cells of
marine-derived fungi applied to synthesis of
enantioenriched β-hydroxy-1,2,3-triazoles
Natália Alvarenga & André L. M. Porto
To cite this article: Natália Alvarenga & André L. M. Porto (2017): Stereoselective reduction
of 2-azido-1-phenylethanone derivatives by whole cells of marine-derived fungi applied to
synthesis of enantioenriched β-hydroxy-1,2,3-triazoles, Biocatalysis and Biotransformation, DOI:
10.1080/10242422.2017.1352585
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Date: 17 August 2017, At: 10:20

BIOCATALYSIS AND BIOTRANSFORMATION, 2017
https://doi.org/10.1080/10242422.2017.1352585
RESEARCH ARTICLE
Stereoselective reduction of 2-azido-1-phenylethanone derivatives by whole
cells of marine-derived fungi applied to synthesis of enantioenriched
b-hydroxy-1,2,3-triazoles
ꢀ
ꢀ
Natalia Alvarenga and Andre L. M. Porto
ꢀ
~
~
~
Instituto de Quımica de Sao Carlos, Universidade de Sao Paulo, Sao Carlos, Brazil
ABSTRACT
ARTICLE HISTORY
Received 25 February 2017
Accepted 5 July 2017
Several marine-derived fungi were evaluated by the bioreduction of 2-azido-1-phenylethanone 1,
and the strains A. sydowii CBMAI 935 and M. racemosus CBMAI 847 were selected for the reduc-
tion of 2-azido-1-phenylethanone derivatives 2–4. Whole cells of A. sydowii CBMAI 935 promoted
the reduction of 2-azido-1-phenylethanones 1–4 with high selectivities to yield the (S)-2-azido-1-
phenylethanols 1a–4a. Bioreduction of compounds 1–4 by M. racemosus CBMAI 847 led to (R)-2-
azido-1-phenylethanols for 1, 2 and 4 and (S)-2-azido-1-phenylethanol 3. Enantiomerically
enriched 2-azido-1-phenylethanols 1a–4a and phenylacetylene 5 were applied in the synthesis
of b-hydroxy-1,2,3-triazoles using CuSO4 and sodium ascorbate leading to regioselective forma-
tion of enantioenriched 1,4-disubstituted 1,2,3-triazole compounds 1b–4b.
KEYWORDS
Arylazides; 1,2,3-triazoles;
biocatalysis; microorgan-
isms; click reaction
Introduction
enzymes to be applied in biocatalytic reactions, since
they present unique properties such as hyperthermost-
ability, salt tolerance, stability at high and low temper-
atures and in a range of pH (Trincone 2010).
Aryl-azide derivatives are an important class of com-
pounds and due to its high stability can be applied
both to biological and industrial purposes, as in the
synthesis of polymers and as key intermediate in
organic synthesis. In this study, aryl-azide derivatives
were applied in the synthesis of 1,2,3-triazoles, but
azides can also be intermediate at the synthesis of tet-
razoles, aziridines, indazoles, benzofuroxans and vari-
ous classes of compounds obtained by various
One of major advantages of biocatalysis as a tool in
organic synthesis is the production of enantiomerically
pure or enriched compounds. In many cases, the bio-
logical activity of pharmaceuticals and agrochemicals
depends on the optical purity of the compound (Lima
1997; Faber 2011). Enantiomerically pure or enriched
alcohols obtained via selective reduction of ketones
has become an important tool for the synthesis of sim-
ple molecules, which have been applied as important
building blocks for the synthesis of optically active
molecules (Rocha et al. 2010).
The bioreduction of ketones is promoted by
dehydrogenase enzymes. The biotransformation of a
keto group by isolated enzymes and whole cells has
been widely studied, however, the use of whole cells
is convenient since microorganisms may present
multiple dehydrogenases which can accept unusual
substrates and also present the cofactor needed for
the hydride transfer (Faber 2011).
€
reaction pathways (Brase et al. 2005).
The 1,2,3-triazoles are an important class of hetero-
cyclic compounds which are only obtained by
synthetic methodologies. They have attracted consid-
erable attention due to the wide range of applications
such as organocatalysis, optical brighteners, in ionic
liquids and corrosion retarding agents (Silva et al.
2015; Yadav et al. 2001; Freitas et al. 2011). 1,2,3-
Triazoles have been extensively studied as potential
targets for drug discovery since they possess a broad
range of biological properties, including antimicrobial,
antiviral, antiepileptic, anti-HIV and activities against
Bioreduction of different class of compounds by
whole cells of marine-derived fungi have been suc-
cessfully applied by our research group (Rocha et al.
2009, 2010, 2012; Mouad et al. 2011, 2015). Marine-
derived microorganisms are an important source of
ꢀ
ꢀ
~ ~ ~
Instituto de Quımica de Sao Carlos, Universidade de Sao Paulo, Av. Joao Dagnone, 1100,
CONTACT Andre L. M. Porto
almporto@iqsc.usp.br
ꢀ
~
~
Quımica Ambiental, J. Santa Angelina, Sao Carlos, Sao Paulo 13563-120, Brazil
Supplemental data for this article can be accessed here.
ß 2017 Informa UK Limited, trading as Taylor & Francis Group

2
N. ALVARENGA & A. L. M. PORTO
several neglected diseases (Silva et al. 2015; Yadav
et al. 2001).
prepared as thin films on KBr disks (solid samples) or
liquid film (liquid samples) in the 4000–400 cmꢁ1 region.
The copper-catalyzed Huisgen 1,3-dipolar cycloadd-
ition between azides and terminal alkynes to give 1,4-
disubstituted 1,2,3-triazoles can be performed under
mild conditions, using water as solvent, with high
yields and regioselectivity. This reaction has become a
major example of click reaction or click chemistry (Lutz
2007; Agalave et al. 2011). The click chemistry concept
is applied to describe simple, stereospecific and effi-
cient reactions used as strategy to obtain compound
libraries that could be applied for drug discovery,
material science, bioconjugation reaction, among
others (Agalave et al. 2011; Freitas et al. 2011).
In this study, we report the use of the multienzy-
matic system of marine-derived fungi in the bioreduc-
tion of 2-azido-1-phenylethanone derivatives 1–4 for
further application in the synthesis of enantiomerically
enriched b-hydroxy-1,2,3-triazoles 1b–4b (Scheme 1).
Chemicals reagents
2-Bromo-1-phenylethanone 98%, 2-bromo-1-(4-metho-
xyphenyl)ethanone 97%, 2-bromo-1-(4-chlorophenyl)e-
thanone 98%, 2-bromo-1-(4-bromophenyl)ethanone
98%, phenylacetylene 98% and (þ)-sodium L-ascorbate
~
(98%) were purchased from Sigma-Aldrich (Sao Paulo,
Brazil). Sodium borohydride (97%) was purchased from
Vetec (Duque de Caxias, Brazil). Sodium azide was pur-
chased from Merck (Sao Paulo, Brazil). The solvents ethyl
~
acetate, hexane and acetone were purchased from
Synth (Diadema, Brazil). Hexane and 2-propanol HPLC-
grade were purchased from Panreac (Barcelona, Spain).
Deuterated chloroform and deuterated dimethylsulfox-
ide purchased from Cambridge Isotope Laboratories
(Tewksbury, MA). All reagents and solvents were used
without further purification. Salts used in the prepar-
ation of artificial seawater and CuSO4 were purchased
from Synth, Merck or Vetec (Brazil). Malt extract was pur-
chased from Acumedia (Indaiatuba, Brazil) and Agar was
purchased from Himedia (Mumbai, India).
Methods
General methods
All manipulations involving marine-derived fungi were
carried out under sterile conditions in a Veco laminar
flow cabinet. Fungi were grown in a Technal TE-421 or
Superohm G-25 orbital shaker. High performance liquid
chromatography (HPLC) analysis were performed in a
Preparation of 2-azido-1-phenylethanone
derivatives (1–4)
The 2-azido-1-phenylethanones 1–4 were obtained by
the reaction of 2-bromo-1-phenylethanone derivatives
(5.0 mmol) and sodium azide (10.0 mmol, 0.65 g) in a
round-bottomed flask (100 mL) containing acetone
(30 mL). The reactions were stirred at room tempera-
ture for 4–6 h and monitored by TLC. After the reac-
tion went to completion, the mixture was extracted
with EtOAc (3 ꢀ 20 mL), dried over anhydrous Na2SO4,
filtered and evaporated under reduced pressure. The
products 1–4 were purified by column chromatog-
raphy (CC) on silica gel eluted with mixtures of hexane
and EtOAc (7:3). The 2-azido-1-phenylethanones 1–4
were obtained in good yields (Scheme 2), and the
R
Shimadzu LC-20ATV model equipped with detector
diode array SPD-M20A, degasser DGU-20A3, control cen-
ter CBM-20AV, automatic injector Sil-20A and oven CTO
20-A in a Chiralpack AS-H (0.46 cm ꢀ25 cm) chiral col-
umn (Supplementary data). 1H NMR and 13C nuclear
magnetic resonance (NMR) spectra were recorded on an
Agilent Technologies 500/54 Premium Shielded or
Agilent Technologies 400/54 Premium Shielded spec-
trometer (1H NMR and 13C at 400 and 100 Hz, respect-
ively), with CDCl3 or DMSO-d6 as solvent and TMS as the
internal standard. The chemical shifts were given in
ppm and coupling constants (J) in Hz. Fourier transform
infrared spectroscopy (FTIR) spectra were recorded on a
Shimadzu IRAffinity-1 spectrometer samples were
1
compounds were identified using H and 13C NMR and
FTIR (Supplementary content).
Scheme 1. Chemoenzymatic synthesis of b-hydroxy-1,2,3-triazoles enantiomerically enriched.

BIOCATALYSIS AND BIOTRANSFORMATION
3
Scheme 2. Synthesis of reference compounds.
Preparation of racemic 2-azido-1-phenylethanol
Reduction of b-keto-1,2,3-triazoles derivatives by
derivatives (1a–4a)
sodium borohydride
The 2-azido-1-phenylethanols 1a–4a were obtained by
the addition of the 2-azido-1-phenylethanones 1–4
(50 mg) in a round-bottomed flask (100 mL) containing
methanol (20 mL) and stirred in ice bath. Sodium boro-
hydride (1.5 equivalent) was added and the reaction
was stirred for 1.5 h. The reaction was monitored by
TLC and after the reaction went to completion the
solvent was evaporated under pressure and the resi-
due was extracted with EtOAc (3 ꢀ 10 mL), dried over
anhydrous Na2SO4, filtered and evaporated under
reduced pressure. The products 1a–4a were purified
by CC on silica gel eluted with mixtures of hexane and
EtOAc (95:5). The 2-azido-1-phenylethanols 1a–4a
were obtained in good yields (Scheme 2), and the
The b-hydroxy-1,2,3-triazoles 1b–4b were obtained by
the addition of the b-keto-1,2,3-triazoles (50 mg) in a
round-bottomed flask (50 mL) containing methanol
(20 mL) and stirred in ice bath. Sodium borohydride
(1.5 equivalent) was added and the reaction was
stirred for 1.5 h. The reaction was monitored by TLC
and after the reaction went to completion the solvent
was evaporated under pressure and the residue was
extracted with EtOAc (3 ꢀ 10 mL), dried over anhyd-
rous Na2SO4, filtered and evaporated under reduced
pressure. The products 1b–4b were purified by CC on
silica gel eluted with mixtures of Dichloromethane,
hexane and methanol (85:15:5). The b-hydroxy-1,2,3-tri-
azoles 1b–4b were obtained in good yields (Scheme
1
compounds were identified using H and 13C NMR and
1
2), and the compounds were identified using H and
13C NMR and FTIR (Supplementary data).
FTIR (Supplementary content).
Preparation of racemic b-hydroxy-1,2,3-triazole
derivatives 1b–4b
Marine-derived fungi strains
The marine-derived fungi strain Aspergillus sydowii
CBMAI 935 was isolated from sponge Chelonaplysilla
erecta, Mucor racemosus CBMAI 847 was isolated from
cnidarian Mussismilia hispida and Penicillium miczynskii
CBMAI 930 was isolated from sponge Geodia corticosty-
lifera. The sponges and cnidarian were collected by
the group of Laboratory of Organic Chemistry of
Natural Products (IQSC/USP) by Prof. R. G. S. Berlinck.
The marine-derived fungi Botryosphaeria sp. CBMAI
1197 and Hydropisphaera sp. CBMAI 1194 were iso-
lated from alga Bostrychia radicans, collected by the
group of Laboratory of Organic Chemistry from Marine
Environment (FCFRP/USP) by Prof. H. M. Debonsi. The
fungi strains were identified using both conventional
and molecular methods at the Chemical, Biological
Preparation of b-keto-1,2,3-triazole derivatives
The b-keto-1,2,3-triazoles were obtained by the reac-
tion of 2-azido-1-phenylethanones 1–4 (1.24 mmol),
phenylacetylene (1.86 mmol, 190 mg) using CuSO4
(1% mol) and (þ)-sodium L-ascorbate (10% mol) as
catalysts in a round-bottomed flask (50 mL) contain-
ing 20 mL of distilled water. The reactions were
stirred at room temperature for 4 h and monitored
by TLC. After the reaction went to completion, the
mixture was extracted with EtOAc (3 ꢀ 20 mL), dried
over anhydrous Na2SO4, filtered and evaporated
under reduced pressure. The products were purified
by CC on silica gel eluted with mixtures of hexane
and EtOAC (2:1).

4
N. ALVARENGA & A. L. M. PORTO
and Agricultural Pluridisciplinary Research Center
(CPQBA) at the University of Campinas, SP, Brazil. All
the strains were deposited in the Brazilian Collection
of Environmental and Industrial Microorganisms
(CBMAI – http://webdrm.cpqba.unicamp.br/cbmai/).
eluted with mixtures of hexane and EtOAc (95:5) and
analyzed by HPLC. Marine-derived fungi A. sydowii
CBMAI 935 and M. racemosus CBMAI 847 were studied
for the bioreduction of 2-azido-1-phenylethanone
derivatives 2–4 (50 mg each compound) according to
the same procedures described for the 2-azido-1-phe-
nylethanone 1.
Preparation of culture medium and artificial
seawater
Preparation of enantioenriched b-hydroxy-1,2,3-
triazoles 1b-4b from 2-azido-1-phenylethanols
1a–4a
Composition of artificial sea water: CaCl2ꢂ2H2O (1.36 g),
MgCl2 ꢂ6H2O (9.68 g), KCl (0.61 g), NaCl (30.0 g),
Na2HPO4 (0.014 mg), Na2SO4 (3.47 g), NaHCO3 (0.17 g),
KBr (100.0 mg), SrCl2 6H2O (40.0 mg), H3BO3 (30.0 mg)
in 1 L of distilled water. Composition of solid culture
medium: 20 g Lꢁ1 of malt extract and 20 g Lꢁ1 of Agar
in artificial seawater at pH 7. Composition of liquid cul-
ture medium: 20 g Lꢁ1 of malt extract in artificial sea-
water at pH 7. The solid and liquid culture medium
were sterilized in autoclave (121 ꢃC, 1.5 kPa, 20 min).
The enantioenriched b-hydroxy-1,2,3-triazoles 1b–4b
were synthetized by the reaction of 2-azido-1-phenyle-
thanols 1a–4a (0.24 mmol) obtained from bioreduction
by A. sydowii CBMAI 935 and M. racemosus CBMAI 847
and phenylacetylene (0.37 mmol, 38 mg) using CuSO4
(1% mol) and (þ)-sodium L-ascorbate (10% mol) as cat-
alysts in a round-bottomed flask (50 mL) containing
20 mL of distilled water. The reactions were stirred at
room temperature for 24 h, monitored by TLC and
after the reaction went to completion the mixture was
extracted with EtOAc (3 ꢀ 10 mL), dried over anhyd-
rous Na2SO4, filtered and evaporated under reduced
pressure. The products 1b–4b were purified by CC on
silica gel eluted with mixtures of dichloromethane,
hexane and methanol (85:15:5). The enantioenriched
b-hydroxy-1,2,3-triazoles were compared with the
racemic standards by HPLC analysis.
Bioredution of 2-azido-1-phenylethanones (1–4)
The biotransformation experiments were initially con-
ducted with 2-azido-1-phenylethanone 1 and the mar-
ine-derived fungi strains (A. sydowii CBMAI 935, M.
racemosus CBMAI 847, P. miczynskii CBMAI 930,
Hydropisphaera sp. CBMAI 1194 and Botryosphaeria sp.
CBMAI 1197). Ten small disks (0.5 cm diameter) bearing
the mycelia of fungi strains were cut from a solid cul-
ture previously grown for 7 d at 32 ꢃC and inoculated
in 100 mL of a liquid culture medium in Erlenmeyer
flasks (250 mL). After incubation in orbital shaker for 7
d at 32 ꢃC, the mycelia were harvested by filtration on
Absolute configuration
The optical rotations of the purified 2-azido-1-phenyle-
thanol derivatives 1a–4a were measured in
€
a Buchner funnel under sterile conditions and 5 g of
a
wet cells were suspended in 100 mL of phosphate buf-
fer (Na2HPO4/KH2PO4, pH 7, 0.1 M). After that, the 2-
Schmidt þ Haensch Polartronic H polarimeter with a
1 dm cuvette and referenced to the sodium D-line. The
absolute configurations were determined by compari-
son of the signals of the measured specific rotations of
the 2-azido-1-phenylethanols with those reported in
the literature (Fardelone et al. 2006) and it is pre-
sented at Table 1.
azido-1-phenylethanone
1 (50 mg, 0.31 mmol) was
solubilized in DMSO (300 lL), added to the medium
and incubated in orbital shaker (130 rpm) for 6 d at
32 ꢃC. The aliquot samples (3 mL) were taken from the
whole reaction flask in 2, 4 and 6 d and transferred to
Falcon tubes (15 mL), added 7 mL of EtOAc, shaking in
Vortex and centrifugated (10 min, 6000 rpm). The
organic phase was dried over anhydrous Na2SO4, fil-
tered and analyzed in HPLC. After 6 d, the whole reac-
tion was filtered on a Buchner funnel and the wet
cells were suspended in 40 mL of distilled water and
EtOAc (1:1) and stirred for 30 min at room tempera-
ture. The mixture obtained by cell lysis were filtered
together with the medium obtained in the previous fil-
tration and extracted with EtOAc (3 ꢀ 25 mL), dried
over anhydrous Na2SO4, filtered, evaporated under
reduced pressure and purified by CC on silica gel
Results
Bioreduction of 2-azido-1-phenylethanones 1–4 by
marine-derived fungi
Five marine-derived fungi strains (A. sydowii CBMAI
935, M. racemosus CBMAI 847, P. miczynskii CBMAI 930,
Hydropisphaera sp. CBMAI 1194 and Botryosphaeria sp.
CBMAI 1197) were screened by the bioreduction of 2-
azido-1-phenylethanone 1 over time. Samples of the
reaction were analyzed in 2, 4 and 6 d (Table 2).

BIOCATALYSIS AND BIOTRANSFORMATION
5
Table 1. Optical rotations of (S)-2-azido-1-phenylethanols 1a–4a obtained by bioreduction with
A. sydowii CBMAI 935 and literature data.
23
25
2-Azido-1-phenylethanols
Experimental data [a]D
Literature data [a]D
(S)-1a
(S)-2a
(S)-3a
(S)-4a
þ68.6 (c 0.5, CHCl3, 80% ee)
þ60.5 (c 1.0, CHCl3, 85% ee)
þ74.1 (c 1.0, CHCl3, 96% ee)
þ76.1 (c 1.0, CHCl3, 95% ee)
þ34.6 (c 1.0, CHCl3, 40% ee)
þ39.1 (c 1.0, CHCl3, >99% ee)
þ79.3 (c 1.0, CHCl3, >99% ee)
þ38.7 (c 1.0, CHCl3, >99% ee)
Table 3. Bioreduction of 2-azido-1-phenylethanone 1 by mar-
Table 2. Bioreduction of 2-azido-1-phenylethanone 1 by mar-
ine-derived fungi in 6 days.
ine-derived fungi over time.
Fungi strain
Yield (%)a
ee (%)
ac
Time (days)
c (%)
ee (%)
ac
A. sydowii CBMAI 935
M. racemosus CBMAI 847
P. miczynskii CBMAI 930
Hydropisphaera sp. CBMAI 1194
Botryosphaeria sp. CBMAI 1197
78
72
48
60
20
80
81
14
42
12
S
R
R
R
S
Aspergillus sydowii CBMAI 935
2
4
98
99
83
78
77
S
S
S
6
100
Mucor racemosus CBMAI 847
2
4
6
93
95
95
88
90
88
R
R
R
Reaction conditions: 5 g of wet cells from marine derived fungi, 2-azido-1-
phenylethanone
1 (50 mg) in DMSO (300 lL) in phosphate buffer
(Na2HPO4/KH2PO4, pH 7, 0. 1 M) incubated for 6 days (32 ꢃC, 130 rpm).
Penicillium miczynskii CBMAI 930
aIsolated yield.
2
4
6
83
88
94
24
33
31
R
R
R
ac: absolute configuration; ee: enantiomeric excess determined by
HPLC-UV.
Hydropisphaera sp. CBMAI 1194
2
4
6
94
100
100
42
45
34
R
R
R
bioreduction of 1 by marine-derived fungi are sum-
marized in Table 3.
Bioreduction of 1 by A. sydowii CBMAI 935 and M.
racemosus CBMAI 847 reached high isolated yields
(78% and 72%, respectively) and enantiomeric
excesses for (S)- and (R)-1a (80% and 81%, respect-
ively). Total extraction of bioreduction of 1 by P. mic-
zynskii CBMAI 930 led to 48% isolated yield and 14%
ee for the (R)-1a.
Botryosphaeria sp. CBMAI 1197
2
4
6
2
3
7
78
79
78
R
R
R
Reaction conditions: 5 g of wet cells from marine derived fungi, 2-azido-1-
phenylethanone
1 (50 mg) in DMSO (300 lL) in phosphate buffer
(Na2HPO4/KH2PO4, pH 7, 0.1 M) incubated at 32 ꢃC (130 rpm).
ac: absolute configuration; c: conversion determined by HPLC-UV; ee:
enantiomeric excess determined by HPLC-UV.
Reaction with Hydropisphaera sp. CBMAI 1194 pre-
sented 60% yield and 42% ee for the experiment with
extraction of whole reaction, unlike the samples
obtained by aliquot, which presented a complete dis-
appearance of the substrate 1. Surprisingly, after
whole extraction of reaction with Botryosphaeria sp.
CBMAI 119, 1a was obtained as the (S)-alcohol (12%
ee), unlike the product obtained from the samples by
aliquot, (R)-1a in 78% ee.
The strains of M. racemosus CBMAI 847, P. miczynskii
CBMAI 930 and Hydropisphaera sp. CBMAI 1194 pre-
sented selectivity towards the formation of the (R)-1a,
which corresponds to hydride transfers from Si-face of
1, in accordance with the Prelog rule. In the reaction
with Botryosphaeria sp. CBMAI 1197, the anti-Prelog
(S)-1a was formed, even with low selectivity. The
reduction of 1 by A. sydowii CBMAI 935 yielded the
anti-Prelog product (S)-1a.
Variations in the ee of 2-azido-1-phenylethanol 1a
in the analyses over time were observed in presence
of all strains (Table 2). Almost all the 2-azido-1-phenyl-
ethanone was converted already in 2 days (98%) to
the 2-azido-1-phenylethanol 1a by A. sydowii CBMAI
935 with 83-77% ee between
2 and 6 days.
Biotransformation of 2-azido-1-phenylethanone 1 by
M. racemosus CBMAI 847 yield the (R)-1a in 93% con-
version and 88% ee already in 2 days. Reactions with
P. miczynskii CBMAI 930 presented good conversion
values over time (83–94%), however, with low selectiv-
ity for the formation of (R)-1a (24–33% ee). High con-
versions (94–100%) and low selectivity (34-45% ee) for
the (R)-1a were achieved in bioreduction of 1 by
Hydropisphaera sp. CBMAI 1194. Bioreduction by
Botryosphaeria sp. CBMAI 1197 led to 7% conversion
with 78% ee in 6 days of reaction.
After 6 days, the extraction of the whole reaction
was performed promoting the cellular lysis through
magnetic stirring of filtered mycelia with EtOAc (rt,
30 min) and the 2-azido-1-phenylethanol 1a obtained
in the presence of each strain was isolated for deter-
mination of yields and absolute configuration. The
results of isolated yields and enantiomeric excesses of
Bioreduction of 2-azido-1-phenylethanones 2–4 by
A. sydowii CBMAI 935 and M. racemosus CBMAI
847
The search for enantiocomplementary enzymes, as the
enzymes from A. sydowii CBMAI 935 and M. racemosus

6
N. ALVARENGA & A. L. M. PORTO
Table 4. Bioreduction of 2-azido-1-phenylethanones 1–4 by A. sydowii CBMAI 935 and M. racemosus CBMAI 847.
2-Azido-1-phenylethanols
Yield (%)a
ee (%)
ac
A. sydowii CBMAI 935
1a
2a
3a
78
66
73
76
80
85
96
95
S
S
S
S
4a
M. racemosus CBMAI 847
1a
2a
3a
4a
72
48
59
57
81
59
7
R
R
S
R
13
Reaction conditions: 5 g of wet cells from marine derived fungi, 2-azido-1-phenylethanone 1–4 (50 mg) in DMSO (300 lL) in phosphate buffer (Na2HPO4/
KH2PO4, pH 7, 0.1 M) incubated for 6 d (32 ꢃC, 130 rpm).
aIsolated yield.
ac: absolute configuration; ee: enantiomeric excess determined by HPLC-UV.
CBMAI 847, is important for the production of both
enantiomers for a specific compound. The potential of
these strains in the bioreduction of 2-azido-1-phenyle-
thanones 2–4 to produce enantiocomplementary com-
pounds was evaluated in 6 d of reaction (Table 4).
High ee were obtained for the compounds 2a–4a in
biotransformation by A. sydowii CBMAI 935 with select-
ivity for the formation of anti-Prelog products, the (S)-
alcohols. The reduction of the 2-azido-1-(4-methoxy-
phenyl)ethanone 2 by A. sydowii CBMAI 935 reached
66% of isolated yield with 85% ee. Bioreduction of 2-
azido-1-(4-chlorophenyl)ethanone 3 and 2-azido-1-(4-
bromophenyl)ethanone 4 by A. sydowii CBMAI 935
proceeded with high selectivity (96% and 95% ee,
respectively) and yield (73% and 76%, respectively).
Low enantiomeric excesses (7–59%) were obtained
for the 2-azido-1-phenylethanols 2a–4a in the reaction
with M. racemosus CBMAI 847. The bioreduction of
para-substituted –Cl and –Br were specially detrimen-
tal to M. racemosus CBMAI 847 enzymes, since poor
selectivity was obtained for this compounds (7% and
13% ee, respectively) and even a shift in the selectivity
pattern occurred. For the compounds 1, 2 and 4 the
Prelog products, the (R)-alcohols, were obtained, how-
ever, in the case of the 2-azido-1-(4-chlorophenyl)etha-
none 3, it was obtained (S)-3a in low ee.
(R)-2-azido-1-phenylethanols 1a–2a from reaction with
M. racemosus CBMAI 847 were used to produce b-(R)-
hydroxy-1,2,3-triazoles since 3a and 4a presented very
low ee. The results of 1,3-dipolar cycloaddition
between 1a–4a and the terminal alkyne, phenylacety-
lene 5 are summarized in Table 5.
The b-hydroxy-1,2,3-triazoles 1b–4b were obtained
with good isolated yields and since the chiral center of
1a–4a was not the reaction center, the b-hydroxy-
1,2,3-triazoles were obtained with same configuration
as the starting material.
Discussion
The bioreduction of 2-azido-1-phenylethanone 1 were
monitored over time by samples extractions of the
reaction in presence of various marine-derived strains.
The results of the aliquots analysis obtained over time
presented a variation in the enantiomeric excesses for
each reaction. The presence of endogenous enzymes
at the whole cell catalyst may present complementary
selectivity or even substrate competition, which may
cause reduced yields, low selectivity and lead to com-
plex mixture of compounds (Hollmann et al. 2011).
Since the dehydrogenases are usually intracellular
enzymes (Van Loon and Young 1986; Smidt et al.
2008; Bhattacharyya et al. 2010), after 6 days, the
whole reaction was extracted to promote the cellular
lysis of mycelia and proceed to isolation of the 2-
azido-1-phenylethanol 1a obtained from reaction with
each strain. The cellular lysis is an important step in
the extraction of reactions employing whole cell as
catalysts, since allows the releasing of intracellular con-
tent. In this specific bioreduction reaction, the cellular
Synthesis of enantiomerically enriched b-hydroxy-
1,2,3-triazoles 1b–4b
The (S)-2-azido-1-phenylethanols 1a–4a obtained in
bioreduction with A. sydowii CBMAI 935 were
employed at the synthesis of enantiomerically
enriched b-(S)-hydroxy-1,2,3-triazoles 1b–4b. Only

BIOCATALYSIS AND BIOTRANSFORMATION
7
Table 5. Synthesis of enantioenriched b-hydroxy-1,2,3-triazoles 1b–4b with 2-azido-1-phenylethanols 1a–4a obtained by biore-
duction with A. sydowii CBMAI 935 and M. racemosus CBMAI 847.
2-Azido-1-phenylethanols
Yield (%)a
ee (%)
ac
A. sydowii CBMAI 935
1a
2a
3a
75
79
63
69
80
85
96
95
S
S
S
S
4a
M. racemosus CBMAI 847
1a
2a
68
66
81
59
R
R
Reaction conditions: 2-azido-1-phenylethanol 1a–4a (40 mg), phenylacetylene (38 mg), CuSO4 (1% mol) and sodium ascorbate (10% mol) in distilled water
stirred for 24 h (rt).
aIsolated yield.
ac: absolute configuration; ee: enantiomeric excess determined by HPLC-UV.
OH
O
OH
cells +
liquid medium
N
N
N
3
3
3 liquid medium
aliquot
total extraction
(S)-alcohol (12% ee)
(R)-alcohol (78% ee)
Scheme 3. Formation of (R)- and (S)-2-azido-1-phenylethanol 1a by bioreduction with Botryosphaeria sp. CBMAI 1197 in liquid
medium and extract of whole cells.
lysis affects not only the concentration of 1, which
needs to be absorbed by cell membrane to proceed
to biotransformation, but also 1a which may or may
not be released to reaction medium after been formed
(Smidt et al. 2008).
the bioreduction with this particular strain. The differ-
ences obtained by analysis of reactions by
Botryosphaeria sp. CBMAI 1197 illustrate a particular
drawback concerning the use of wild type whole cells
in bioreduction reactions.
By the results of the screening with the five marine-
derived fungi strains, A. sydowii CBMAI 935 and M.
racemosus CBMAI 847 were selected as enantiocomple-
mentary source of oxidoreductases for the bioreduc-
tion of 2-azido-1-phenylethanones 1–4.
Bioreduction studies with para-substituted aceto-
phenone derivatives usually show that the rate of
hydride transfer mediated by DHs from NADH or
NADPH is decreased by electron donating groups, as
–OCH3. However, the presence of electronegative
groups attached to the a-carbon, as –N3, make the car-
bonyl group more active towards the hydride transfer
from the cofactor. Indeed, in spite of the decreasing
effect in hydride transfer by 4-methoxyphenyl group,
good yields were obtained for bioreduction of 2-azido-
1-(4-methoxyphenyl)ethanone 2 by both strains.
The bioreduction of para-substituted –Cl and –Br 3
and 4 presented better results than 1 and 2 in pres-
ence of A. sydowii CBMAI 935, indicating also an acti-
vation of the carbonyl group towards the bioreduction
with aromatic ring para-substituted with electro
Furthermore, variations were also observed
between enantiomeric excess in the analysis of ali-
quots samples and isolated compounds obtained by
whole reaction extraction. In the case of reactions by
Botryosphaeria sp. CBMAI 1197 it was obtained even
the products in different absolute configurations
(Scheme 3). The analysis by aliquots from reaction
evaluates mainly the compounds present at liquid
medium, unlike the whole extraction, which evaluates
both liquid medium and cells extract.
Through the cellular lysis and extraction of intracel-
lular content, the majority enantiomer obtained in low
selectivity was different than the one obtained by ali-
quot samples. Since dehydrogenases are endogenous
in living organisms, various dehydrogenases with com-
plementary selectivity may be active at the same time
causing the variation of selectivity by this strain
(Hollmann et al. 2011). The fungi Botryosphaeria sp.
CBMAI 1197 was also evaluated by the deracemization
of 1a, but no changes in the ee were observed for the
whole extraction over time (data not showed) and fur-
ther experiments must be conducted for evaluation of

8
N. ALVARENGA & A. L. M. PORTO
withdrawing groups for this strain. However, the biore-
duction of 2-azido-1-phenylethanone derivatives 2–4
by M. racemosus CBMAI 847 showed that the para-sub-
stitution of the phenyl group was harmful to hydride
transfer, since reduced yields and poor selectivity were
obtained when compared to 2-azido-1-phenyletha-
none 1. Reactions where the substituent may affect
the activity and enantioselectivity in bioreduction of
aryl ketones were already reported by Zhu et al.
(2005).
The asymmetric reduction of 2-azido-1-phenyletha-
nones 1–4 by oxidoreductases enzymes can be pre-
dicted according the enantiospecificity postulated by
the empiric model of Prelog. The enzyme-mediated
hydride transfer from the cofactor to the carbonyl car-
bon occurs by the Si face from prochiral azidoketones
with the formation of (R)-azidoalcohols since the bulk
substituent, the phenyl group, is bigger than the
CH2N3 group (Fardelone et al. 2006).
whole cells of the marine-derived fungi M. racemosus
CBMAI 847 and A. sydowii CBMAI 935, respectively.
Conclusions
Bioreduction of 2-azido-1-phenylethanones by Aspergillus
sydowii CBMAI 935 afforded the enantioenriched 2-
azido-1-phenylethanols 1a–4a with high selectivity.
However, Mucor racemosus CBMAI 847 was able to pro-
mote the bioreduction of only 2-azido-1-phenylethanone
1 with high selectivity. This study presents the applica-
tion of two important tools in organic chemistry, the
biocatalysis to the production of enantiomerically com-
plementary and enriched compounds and click chemis-
try to the synthesis of molecules in high yields avoiding
the waste of reagents and solvents.
Disclosure statement
The authors report no declarations of interest. The authors
alone are responsible for the content and writing of the
paper.
The strain M. racemosus CBMAI 847 presented
selectivity mainly for the production of Prelog prod-
ucts, the (R)-alcohols 1a, 2a and 4a. A. sydowii CBMAI
935 produced preferentially the (S)-alcohols in the bio-
reduction of 2-azido-1-phenylethanones 1–4, demon-
strating to be a promising source of dehydrogenase
with anti-Prelog selectivity. Enzymes with anti-Prelog
selectivity are described in literature, however, they
are more rare than the enzymes with Prelog selectivity
and only few of them are commercially available
(Faber 2011).
Funding
N. Alvarenga thanks the Conselho Nacional de
ꢀ
ꢀ
Desenvolvimento Cientıfico e Tecnologico (CNPq) for scholar-
ship (141844/2013-2). A.L.M. Porto thanks the CNPq (400202/
~
ꢁ
2014-0 and 301987/2013-0) and the Fundac¸ao de Amparo a
~
Pesquisa do Estado de Sao Paulo (2014/18257-0) for financial
support.
The 2-azido-1-phenylethanols 1a–4a obtained in
good ee were employed at the synthesis of enantio-
merically enriched b-(S)-hydroxy-1,2,3-triazoles 1b–4b.
In this specific reaction, the azide acts as nucleophile
and the use of Copper as catalyst increases the select-
ivity and reactivity of the reaction with phenylacety-
lene 5. This reaction used to be developed with
metallic Copper, however, due to its toxicity, the pro-
tocols involving the metal was a limiting factor for bio-
logical purposes. This problem was circumvented by
using a salt of Cu(II) and a reducing agent (CuSO4 and
sodium ascorbate, respectively in this study) to the for-
mation of the desired form of catalyst, Cu(I), in situ
(Cintas et al. 2010).
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