Stereoselective reduction of flavanones by marine-derived fungi

Article abstract of DOI:10.1016/j.mcat.2021.111734

Biotransformation is an alternative with great potential to modify the structures of natural and synthetic flavonoids. Therefore, the bioreduction of synthetic halogenated flavanones employing marine-derived fungi was described, aiming the synthesis of flavan-4-ols 3a-g with high enantiomeric excesses (ee) of both cis- and trans-diastereoisomers (up to >99% ee). Ten strains were screened for reduction of flavanone 2a in liquid medium and in phosphate buffer solution. The most selective strains Cladosporium sp. CBMAI 1237 and Acremonium sp. CBMAI1676 were employed for reduction of flavanones 2a-g. The fungus Cladosporium sp. CBMAI 1237 presented yields of 72–87% with 0–64% ee cis and 0–30% ee trans with diastereoisomeric ratio (dr) from 52:48 to 64:36 (cis:trans). Whereas Acremonium sp. CBMAI 1676 resulted in 31% yield with 77–99% ee of the cis and 95–99% ee of the trans-diastereoisomers 3a-g with a dr from 54:46 to 96:4 (cis:trans). To our knowledge, this is the first report of the brominated flavon-4-ols 3e and 3f. The use of fungi, with emphasis for these marine-derived strains, is an interesting approach for enantioselective reduction of halogenated flavanones. Therefore, this strategy can be explored to obtain enantioenriched compounds with biological activities.

Full text of DOI:10.1016/j.mcat.2021.111734

Molecular Catalysis 513 (2021) 111734
Contents lists available at ScienceDirect
Molecular Catalysis
journal homepage: www.journals.elsevier.com/molecular-catalysis
Stereoselective reduction of flavanones by marine-derived fungi
,
b
c
d
Iara L. de Matosa, Willian G. Birollia , Darlisson de A. Santos , Marcia Nitschke , Andre Luiz
´
,
*
M. Portoa
a
˜
˜
˜
Laboratory of Biocatalysis and Organic Chemistry, Sao Carlos Institute of Chemistry, University of Sao Paulo, Av. Joao Dagnone, 1100, Ed. Química Ambiental, J.
˜
Santa Angelina, 13563-120, Sao Carlos, SP, Brazil
b
˜
˜
Chemistry Department, Center for Exact Sciences and Technology, Federal University of Sao Carlos, Via Washington Luiz, km 235, 13565-905, Sao Carlos, SP, Brazil
c
´
´
´
Institute of Exact Sciences, Federal University of Southern and Southeastern Para, Folha 17, Quadra 04, Nova Maraba, 68505080, Maraba, PA, Brazil
d
˜
˜
˜
Laboratory of Microbial Biotechnology, Sao Carlos Institute of Chemistry, University of Sao Paulo, Av. Joao Dagnone, 1100, Ed. Química Ambiental, J. Santa Angelina,
˜
13563-120, Sao Carlos, SP, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
Biotransformation is an alternative with great potential to modify the structures of natural and synthetic fla-
vonoids. Therefore, the bioreduction of synthetic halogenated flavanones employing marine-derived fungi was
described, aiming the synthesis of flavan-4-ols 3a-g with high enantiomeric excesses (ee) of both cis- and trans-
diastereoisomers (up to >99% ee). Ten strains were screened for reduction of flavanone 2a in liquid medium and
in phosphate buffer solution. The most selective strains Cladosporium sp. CBMAI 1237 and Acremonium sp.
CBMAI1676 were employed for reduction of flavanones 2a-g. The fungus Cladosporium sp. CBMAI 1237 pre-
sented yields of 72–87% with 0–64% ee cis and 0–30% ee trans with diastereoisomeric ratio (dr) from 52:48 to
64:36 (cis:trans). Whereas Acremonium sp. CBMAI 1676 resulted in 31% yield with 77–99% ee of the cis and
95–99% ee of the trans-diastereoisomers 3a-g with a dr from 54:46 to 96:4 (cis:trans). To our knowledge, this is
the first report of the brominated flavon-4-ols 3e and 3f. The use of fungi, with emphasis for these marine-
derived strains, is an interesting approach for enantioselective reduction of halogenated flavanones. There-
fore, this strategy can be explored to obtain enantioenriched compounds with biological activities.
Flavanol
Flavonoid
Marine fungi
Enantioselective
Biotransformation
1. Introduction
presented better anticancer activity than the natural quercetin, pro-
moting interest for the obtention of these compounds [9]. Also, bromi-
Flavonoids possess a basic C6-C3-C6 flavan skeleton that presents
two aromatic rings linked by a heterocyclic pyran ring [1]. Based on
their chemical structure, degree of oxidation, linking chain, and unsa-
turation, flavonoids can be further classified into 6 major groups: iso-
nated and chlorinated flavanones, flavones and catechins showed
antifungal activities against potential pathogens for humans [10], and
halogenated derivatives of 5,7-dihydroxyflavanone presented increased
activity against bacteria and fungi [11].
flavonoids,
flavanones,
flavanols,
flavonols,
flavones
and
Flavanones present a heterocyclic ring C with no conjugation be-
tween both rings A and B (Fig. 1C), and different strategies have been
employed to obtain new derivatives [12]. Biocatalysis has had an impact
in the conversion of starting materials and preparation of different
compounds [13]. Therefore, biotransformation is an alternative with
great potential to modify the structures of natural and synthetic flavo-
noids [14]. Different biocatalytic approaches were already employed,
such as microbial biotransformation, enzymatic engineering, metabolic
engineering, and the use of plant cells [15–19].
anthocyanidins [2].
Among these types of flavonoids, flavone and isoflavone derivatives
deserve to be highlighted due to occurrence in nature and stablished
biological activities [3]. However, flavanones also deserve attention due
to antioxidant, antiviral, anticancer, anti-inflammatory, and anti-
estrogenic activities [4,5]. Moreover, these compounds are present at
high concentrations in different fruits, such as oranges, grapefruit,
lemons, limes, and some aromatic herbs like oregano, rosemary, and
peppermint [6].
Biotransformation reactions were carried out by different bio-
catalysts aiming to overcome the cell toxicity of flavonoid aglycones and
producing novel bioactive molecules with interesting activities [20]. For
Synthetic flavonoids have also been studied for their biological ac-
tivities [7,8]. In this context, halogenated chalcones and flavanols
* Corresponding author.
E-mail address: almporto@iqsc.usp.br (A.L.M. Porto).
https://doi.org/10.1016/j.mcat.2021.111734
Received 8 March 2021; Received in revised form 5 June 2021; Accepted 28 June 2021
Available online 9 August 2021
2468-8231/© 2021 Elsevier B.V. All rights reserved.

I.L. de Matos et al.
Molecular Catalysis 513 (2021) 111734
example, dihydrochalcones and an alcohol product of 4′-methyl-
chalcones biotransformation presented interesting bactericidal activity
[21].
(Brazil). The solvents ethyl acetate (PA) and hexane (PA) were pur-
chased from Synth. Chromatographic grade solvents (hexane, methanol
and 2-propanol) were obtained from Panreac. All reagents were used
without prior purification.
Engineered microorganisms have also been employed for biotrans-
formation of flavanones. Quercetin was converted to quercetin 3-O-
gentiobioside by Escherichia coli expressing two uridine diphosphate
dependent glycosyltransferases and two nucleotide sugar biosynthetic
genes [22], and the cytochrome P450 CYP52G3 from Aspergillus oryzae
expressed in E. coli converted flavanone into 4′-hydroxyflavanone and
6-hydroxyflavanone [23]. Moreover, some native bacterial strains were
employed for flavanones biotransformation [24].
The flash silica gel with pore diameter of 6 nm (0.035–0.070 nm)
used in the purifications of the compounds by column chromatography
was obtained from Acros Organics. The salts used in the preparation of
synthetic seawater were obtained from Synth. Agar and malt extract
were purchased from Kasvi. The deuterated solvents, DMSO‑d6 (99.9%)
and CDCl3 (99.9%) were purchased from Cambridge Isotope Labora-
tories. L-proline and anhydrous Na2SO4 were obtained from Synth. The
sterile and disposable Petri plates were obtained from Olen.
For reduction reactions, promising results have been presented with
the screening of fungi from distinct environments with substrates like
flavanone [25], 7-hydroxyflavanone [26], 6-hydroxyflavanone [27],
chalcones [28], 4-chromanol, 4-flavanol, xanthydrol [29], and glabra-
nin [30]. Moreover, it is important to emphasize the successful use of
marine-derived fungi for biotransformation of flavonoids like chalcones
[31] and 2′-hydroxychalcones [32].
2.2. Synthesis of chalcones 1a-g
The chalcones 1a-g were synthesized in a 125 mL round-bottom flask
with 50 mL of ethanol, 2-hydroxyacetophenone (0.01 mol) and benz-
aldehyde derivatives a-g (0.01 mol). Then, 5 mL of NaOH (5 M) was
added dropwise, and the reaction mixture was stirred for 12–24 h. The
reactions were monitored by Thin Layer Chromatography (TLC)
(Macherey-Nagel, 20 × 20 cm, silica gel 60, G / UV 254 nm). After that
period, the reaction mixture was transferred to a beaker flask (500 ml),
and 5 mL of HCl (10%) was added slowly. The precipitate was filtered
and washed 4 times with cold distilled water [31,32]. The yields were
determined after purification by column chromatography using hexane
and ethyl acetate, ratio of 9.5:0.5 (Fig. 1A).
In this work, the biotransformation of synthetic halogenated flava-
nones employing marine-derived fungi aiming to obtain flavan-4-ols
with high enantiomeric excesses (ee) of both cis- and trans-di-
astereoisomers was reported for the first time.
2. Material and methods
2.1. Reagents, solvents and culture media
The reagents 2-hydroxyacetophenone (99%), benzaldehyde (99%),
3-bromobenzaldehyde (97%), 4-bromobenzaldehyde (99%), 3-chloro-
benzaldehyde (97%), 3-fluorobenzaldehyde (97%), 4-fluorobenzalde-
hyde (98%) and NaBH4 were obtained from Sigma-Aldrich. 4-
Methoxybenzaldehyde (98%) was purchased from Vetec. Sodium hy-
droxide (97%) and hydrochloric acid (37%) were acquired from Quemis
2.3. Synthesis of (±)-flavanones 2a-g
In a 25 mL flask, 2′-hydroxychalcones 1a-g (0.45 mmol), KOH (8 M,
1 mL), L-proline (0.01 g) and distilled water (5.0 mL) were added. The
suspension was kept under magnetic stirring at room temperature for
1–24 h. Then, the formed precipitate was collected by filtration, washed
Fig. 1. (A) Synthesis of 2′-hydroxychalcones derivatives 1a-g by Claisen-Schmidt aldolic condensation; (B) Synthesis of flavanones 2a-g from 2′-hydroxychalcones in
basic medium; (C) Synthesis of flavan-4-ols 3a-g from the reduction of (±)-flavanones with NaBH4.
2

I.L. de Matos et al.
Molecular Catalysis 513 (2021) 111734
with distilled water and dried at room temperature [33]. When neces-
sary, purification was performed by column chromatography using
hexane and ethyl acetate (9.5:0.5). Products 2a-g were characterized by
IV, MS, NMR (1H and 13C) and their yields determined (Fig. 1B).
Condition A: reaction by 2.5 g of wet cells of fungi in 50 mL of malt
broth, and Condition B: reaction by 2.5 g of wet cells of fungi in 50 mL of
phosphate buffer solution (Na2HPO4/KH2PO4 0.1 mol Lꢀ 1, pH 7.0). Both
were carried out in sterilized 125 mL Erlenmeyer flasks employing
cotton plugs with the addition of 25 mg of 2a-g dissolved in 400 µL of
DMSO. The screening reactions were performed for 7 days, but different
periods were tested further.
2.4. Synthesis of (±)-flavan-4-ols 3a-g
Control experiments employing each of the fungus strains were
performed since the production of compounds with similar structure can
occur as natural products. Moreover, experiments in abiotic conditions
were carried out to evaluate the occurrence of spontaneous reactions
with the employed substrates in the experimental conditions.
In a 25 mL flask, 1 mmol of the flavanone 2a-g and 10 mL of ethanol
were added. The mixture was placed in an ice bath under magnetic
stirring for 5 min. Then NaBH4 was added with an excess of 0.5 mmol in
relation to (±)-flavanone. The reaction was monitored by TLC until the
complete conversion of the reagents. After the reaction, methanol was
removed in a rotoevaporator and a solid was obtained. Then, 10 mL of
distilled water were added, and a liquid-liquid extraction was performed
with ethyl acetate (3 × 10 mL). Anhydrous sodium sulfate was added to
the organic phase and the solvent was filtered and evaporated under
reduced pressure. Subsequently, the sample was purified by column
chromatography, dried and its yield was determined (Fig. 1C). The
(±)-flavan-4-ols 3a-b and 3d-g were characterized by IR, MS and NMR
(1H and 13C). The employed methodological procedure was not suitable
for the synthesis of flavan-4-ol 3c, so, it was not obtained by chemical
synthesis. However, 3c was obtained by biotransformation and
characterized.
2.8. Extraction of the biotransformation reactions
The samples were extracted by the addition of ethyl acetate in the
proportion of 1:1 in relation to the amount of reaction medium. Then,
the flasks were stirred magnetically for 30 min, and the mixture was
centrifuged (10,000 rpm, 20 min) in a Hitachi CR22GIII. The liquid
phase (organic phase and aqueous phase) was then transferred to a
separating funnel. A liquid-liquid extraction was performed with two
following steps by the addition of 25 mL ethyl acetate. After that, the
organic phase was collected and anhydrous sodium sulfate was added,
then filtered, and the solvent was evaporated under reduced pressure.
2.5. Marine-derived fungi
2.9. Chromatographic analyses
The strains of marine-derived fungi employed for the biotransfor-
mation of flavanones 2a-g were already available at the Group of
Organic Chemistry and Biocatalysis (colony images shown in Supporting
Material (SM), Item 1). The fungi were isolated from marine sponges and
provided by Prof. Roberto G. S. Berlinck (IQSC / USP) and Prof. Mirna H.
R. Seleghim (DBE / UFSCar) in previous studies [34–36]. These strains
were selected for reduction of flavanones based on good results pre-
sented in the biotransformation and biodegradation of xenobiotics
described in the literature [37,38].
The Gas Chromatography-Mass Spectrometry analyzes were per-
formed in a Shimadzu GC2010plus coupled to a selective mass detector
(Shimadzu MS2010plus) in Electronic Impact mode (E.I., 70 eV) with a
DB-5MS column (30 m x 0.25 mm x 0.25 µm, Agilent J & W Advanced)
as already described in the literature [32].
High-Performance Liquid Chromatography (HPLC-UV) was per-
formed in a Shimadzu 2010 chromatograph equipped with a CBM-20A
controller, LC-20AT pump, DGU-20A5 degasser, SIL-20AHT sampler,
CTO-20A oven and SPD-M20A UV–VIS detector. The oven temperature
was 40 ◦C. The analysis for determination of diastereoisomeric ratios
(dr) of the flavan-4-ols 3a-g were performed on isocratic reverse mode
with a Luna C18 column (0.46 × 25 cm; 5 µm) equipped with a pre-
column. The measurements of racemic and/or enantiomerically
enriched flavanones 2a-g and flavon-4-ols 3a-g were performed using a
Chiralcel OD-H column (0.46 × 25 cm; 5 µm) in normal phase and
isocratic mode. All methods employed for the analysis of the compounds
in C18 column and in the chiral OD-H column are described in SM Item
2.
These strains are deposited in the Brazilian Collection of Microor-
ganisms from the Environment and Industry (CBMAI - http://webdrm.
cpqba.unicamp.br/cbmai/, WDCM823), deposited as Fusarium sp.
CBMAI 1830, Acremonium sp. CBMAI 1676, Aspergillus sp. CBMAI 1829,
Aspergillus sydowii CBMAI 935, Penicillium oxalicum CBMAI 1996, Peni-
cillium citrinum CBMAI 1186, Penicillium raistrickii CBMAI 931, Clado-
sporium sp. CBMAI 1237, Mucor racemosus CBMAI 847 and Westerdykella
sp. CBMAI 1679.
2.6. Culture medium
Marine-derived fungi were cultivated in 2% malt agar prepared with
20 g of malt extract and 15 g of Agar added to 1 L of synthetic seawater
composed of: CaCl2•2H2O (1.36 g), MgCl2•6H2O (9.68 g), KCl (0.61 g),
NaCl (30 g), Na2HPO4 (0.014 mg), Na2SO4 (3.47 g), NaHCO3 (0.17 g),
KBr (100 mg), SrCl2•6H2O (40 mg), and H3BO3 (30 mg). The pH was
adjusted to 7.0 with KOH 0.5 mol Lꢀ 1. Then, the medium was sterilized
in autoclave for 20 min (121 ◦C, 1 atm) and poured into disposable Petri
plates under aseptic conditions [39].
2.10. Product characterization
The determination of the melting point of the solid compounds was
performed using closed capillaries in a Fisatom device (model 431). The
absorption spectra in the infrared region were obtained using a Shi-
madzu IRAffinity-1 spectrometer operating with Fourier Transform. The
analyzes were performed using KBr tablets at wavelengths in the region
of 400 to 4000 cmꢀ 1
.
The 1H and 13C Nuclear Magnetic Resonance (NMR) analyzes were
performed on an Agilent Technologies 500/54 Premium Shielded or on
an Agilent Technologies 400/54 Premium Shielded spectrometer.
Deuterated chloroform (CDCl3) or dimethylsulfoxide (DMSO‑d6) were
used to solubilize the samples. Moreover, tetramethylsilane (TMS) was
used as reference signal. The chemical shifts were expressed in parts per
million (ppm) and referenced to the internal standard TMS and the
deuterated solvent used, DMSO‑d6 (δ 2.50, δ 39.52) and CDCl3 (δ
2.7. Biotransformation reactions by marine fungi
The screening of biocatalysts for biotransformation of 2a was per-
formed with 10 different strains of marine-derived fungi. Each strain
was cultivated in five 250 mL flasks with 100 mL of 2% malt broth
covered with cotton plugs. All sets were sterilized in autoclave (121 ◦C,
15 min, 1 atm) and inoculated with seven slices of microbial inoculum
previously prepared in malt agar during 7 days at 32 ◦C. After growth
(32 ◦C, 130 rpm, 7 days), the cells were harvested by filtration using a
Buchner apparatus and employed for biotransformation in two different
conditions, A and B.
H
C
H
7.26, δC 77.16). The coupling constants (J) values were reported in Hz.
Optical rotation measurements were performed using a Jasco
polarimeter model P-2000 equipped with a sodium lamp (λ = 589 nm).
The measurements were performed at 23 ◦C using a 1.0 dm long cell.
3

I.L. de Matos et al.
Molecular Catalysis 513 (2021) 111734
Samples were diluted in spectroscopic grade chloroform with concen-
trations expressed in g/100 mL.
Flavanone 2a possess a stereogenic center, and consequently, a pair
of (S)ꢀ 2a and (R)ꢀ 2a enantiomers. Moreover, this compound also
presents a pro-stereogenic ketone in its structure, being able to provide a
The description of compounds characterization is presented in SM
Item 3, whereas an additional discussion was presented in SM Item 4 and
spectra at SM Item 5. The obtained data was in accordance with the
literature [40–48]. To our knowledge, this is the first report of com-
pounds 3e and 3f.
new chirality center in the molecule generating
a pair of di-
astereoisomers and two pairs of enantiomers. The produced flavan-4-ol
3a can present the (2R,4R)-cis-3a and (2S,4S)-cis-3a and/or (2R,4S)-
trans-3a and (2S, 4R)-trans-3a stereoisomers, where the ratio of the
stereoisomeric products will be determined by the selectivity of the
enzymes involved in the reduction process, Fig. 2B.
3. Results and discussion
The screened strains can be classified into three groups according to
the products from the biotransformation of flavanone 2a. In Group I,
which resulted in the production of the dihydrochalcone 4a as unique
product, is the fungus P. raistrickii CBMAI 931. In Group II were Fusarium
sp. CBMAI 1830 and A. sydowii CBMAI 935 that produced the dihy-
drochalcone 4a and dihydroxy-dihydrochalcone 5a. Most of strains,
Westerdykella sp. CBMAI 1679, Acremonium sp. CBMAI 1676, Cladospo-
rium sp. CBMAI 1237, Aspergillus sp. CBMAI 1829, P. oxalicum CBMAI
1996, P. citrinum CBMAI 1186 and M. racemosus CBMAI 847 were at
Group III, in which flavan-4-ol 3a was the main product (Table 1).
Chromatograms in SM Item 6 Fig. 6.1.
3.1. Screening of marine-derived fungi for biotransformation of flavanone
2a
Flavanones possess different biotransformation sites for obtaining
interesting products, such as alcohols and hydroxylated compounds
[14]. In this sense, a screening was carried out with ten fungi from
marine environment for biotransformation employing the substrate 2a
as model. For each strain, the biotransformation was performed in malt
medium (Condition A) and phosphate buffer solution 0.1 M pH 7.0
(Condition B).
The analyses of the samples by HPLC-UV and GC–MS revealed that
different products were obtained depending on the microorganism
employed as biocatalyst, resulting in reactions of reduction, hydroxyl-
ation, and cleavage of the heterocyclic ring (Fig. 2a). An abiotic control
for spontaneous reactions assessment was prepared in both conditions,
and no transformation product was observed after a period of 7 days.
Moreover, in the biotic controls, in which the fungi biocatalyst without
substrate addition was used, no natural flavonoids were identified by the
employed methods. Chromatograms were presented in SM Item 6.
The obtention of dihydrochalcone 4a by marine-derived fungi was
explored in a previous study [32], and hydroxylation reactions for
synthesis of dihydroxy-dihydrochalcone 5a will be further investigated.
In this manuscript, the enantioselective reduction of flavanone 2a to
flavan-4-ol 3a was the main objective.
P. raistrickii CBMAI 931 (Group I) promoted the opening of ring C of
flavanone 3a producing dihydrochalcone 4a in both conditions A and B.
In general, the opening of ring C of flavanones leads to the formation of
chalcones [49], however, the formation of chalcone 1a was not
observed, probably because of a reduction reaction catalyzed by fungal
ene-reductases. Dihydrochalcone 4a was isolated with 30% yield in
condition A.
It is also observed a greater selectivity for the consumption of (S)ꢀ 2a
for the formation of dihydrochalcone 4a. After 7 days of reaction, (R)ꢀ
2a was obtained with an enantiomeric excess of 57% and 56% ee in
conditions A and B, respectively. Chiral chromatograms are in SM Item 6
Fig. 6.2. In the literature, the production of dihydrochalcone from
flavanone was reported with 13% yield by the bacteria Gordonia sp. DSM
44456 [50] The biotransformation reactions by Fusarium sp. CBMAI
Fig. 2. A) Biotransformation of flavanone 2a to obtain flavan-4-ol 3a, dihydrochalcone 4a, and/or dihydroxy-dihydrochalcone 5a by fungi from marine environ-
ment. B) Structures of the stereoisomers of flavanone 2a and flavan-4-ol 3a.
4

I.L. de Matos et al.
Molecular Catalysis 513 (2021) 111734
Table 1
Biotransformation of (±)-flavanone 2a by marine derived fungi (32 ◦C, 130 rpm, 7 days).
Strain
Condition
Remaining 2a
3a (%)
(cis:trans)ꢀ 3a dr
ee (%) of 3a
cis for (2S,4S)
Isolated Yield of 3a
4a (%)
5a (%)
(%)
ee (%)
trans
Group I
P. raistrickii CBMAI 931
A
B
45
45
57 (R)
56 (R)
–
–
–
–
–
–
–
–
28
30
–
–
Group II
Fusarium sp. CBMAI 1830
A
B
A
B
57
7
23 (R)
12 (R)
22 (R)
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
6
5
3
6
34
64
11
30
A. sydowii CBMAI 935
10
7
Group III
Westerdykella sp. CBMAI 1679
A
B
A
B
A
B
A
B
A
B
A
B
A
B
14
39
28
62
13
11
75
71
68
75
72
76
62
62
0
57
36
11
22
25
35
8
82:18
72:28
85:15
91:9
13
29
96
97
54
49
–
49
43
98
88
24
24
–
63%
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
12 (R)
28 (R)
27 (R)
91 (R)
38 (R)
–
–
Acremonium sp. CBMAI 1676
Cladosporium sp. CBMAI 1237
Aspergillus sp. CBMAI 1829
P. oxalicum CBMAI 1996
P. citrinum CBMAI 1186
–
31%
42:58
44:56
78:22
68:32
47:53
30:70
26:74
33:67
22:78
29:71
–
83%
–
–
–
–
–
–
–
–
–
<2
<2
<2
<2
<2
<2
6
–
–
2 (R)
1 (R)
1 (R)
3 (R)
1 (R)
7 (R)
–
–
–
–
–
33
ꢀ 19
23
3
–
M. racemosus CBMAI 847
–
–
Condition A: biotransformation in malt 2% medium; Condition B: biotransformation in phosphate buffer (Na2HPO4/KH2PO4 0.1 mol Lꢀ 1, pH 7.0).
1830 and A. sydowii CBMAI 935 led to the formation of two main
products, dihydrochalcone 4a and 2′,4-dihydroxy-dihydrochalcone 5a.
For Fusarium sp. CBMAI 1830, (S)ꢀ 2a consumption was favored in both
experimental conditions, leading to the opening of ring C of flavanone
2a and hydroxylation of ring B to obtain 4a and 5a, respectively.
Enantiomeric excesses of 23 and 12% were obtained for unreacted (R)ꢀ
2a in conditions A and B, respectively. Chiral chromatograms are in SM
Item 6 Fig. 6.3.
3a from flavanone (±)ꢀ 2a in a mixture of diastereoisomers. The dia-
stereoisomeric ratio for flavan-4-ol 3a in condition A was 82:18 (cis:
trans) and in condition B was 72:28 (cis:trans). The enantiomeric ex-
cesses in conditions A (13% ee for peak 2, 42.4 min, cis-3a; 49% ee for
peak 1, 26.2 min, trans-3a) and B (29% ee for peak 2, 42.4 min, and later
defined as (2S,4S)-cis-3a; 43% ee for peak 1, 26.2 min, trans-3a) were
similar. It is important to emphasize that the absolute configuration of
the trans-3a enantiomers was not assigned.
Westerdykella sp. CBMAI 1679 promoted the formation of flavan-4-ol
In condition A, Westerdykella sp. CBMAI 1679 presented a higher
Fig. 3. Chromatograms obtained by HPLC-UV with chiral column for biotransformation by Westerdykella sp. CBMAI 1679 (32 ◦C, 130 rpm, 7 days). Analysis
conditions: HPLC-UV with chiral column Chiralcel OD-H (0.46 cm x 25 cm; 5 µm), isocratic elution with hexane and isopropanol (95:5) at a flow of 0.5 mL minꢀ 1
,
total analysis time of 60 min. Chromatograms obtained at wavelength of 215 nm. Retention times: (S)ꢀ 2a (RT = 16.5 min), (R)ꢀ 2a (RT = 19.8 min), trans-3a (RT =
26.2 min and 31.8 min), cis-3a (RT = 35.1 min for 2R,4R-3a and 42.4 min for 2S,4S-3a). Solv. = solvent residue of ethyl acetate (RT = 7.0 min) and isopropanol (RT
= 3.8 min). NI = not identified.
5

I.L. de Matos et al.
Molecular Catalysis 513 (2021) 111734
conversion of 2a (c = 83%) than condition B (c = 59%). Possibly, the
malt broth provided a higher concentration of enzymes and more
effectiveness of the natural regeneration system of cofactors due to the
increased availability of nutrients. The flavan-4-ol 3a formed in the
reaction of 2a was isolated under condition A with a yield of 63% and
49% ee for trans-3a and 13% ee for cis-3a. However, it was not possible to
isolate the cis-3a and trans-3a diastereoisomers separately by column
chromatography because the bands were too close. Therefore, only a
mixture of the diastereoisomers was obtained. Chiral chromatograms
were presented in Fig. 3.
42.4 min, 2S,4S) and for the trans-3a enantiomers was 24% ee (peak 1,
26.2 min)
Despite having lower diastereoselectivity, condition B showed a
conversion of 90%, which was slightly higher when compared to con-
dition A with 87% conversion. Flavan-4-ol 3a was then isolated from
condition B as a mixture of diastereoisomers with 83% yield. Chiral
chromatograms are presented in SM Item 6 Fig. 6.5.
In biotransformation reactions using the fungi Acremonium sp.
CBMAI 1676, Westerdykella sp. CBMAI 1679 and Cladosporium sp.
CBMAI 1237, the formation of the cis-3a product was predominant. The
same pattern was observed in the reactions of synthesis of flavan-4-ols
using NaBH4 and methanol, where the product cis-(±)ꢀ 3a was ob-
tained in 95:5 in relation to the product trans-3a.
Although reactions with Acremonium sp. CBMAI 1676 presented
decreased yield of 3a, this biocatalyst was more selective than West-
erdykella sp. CBMAI 1679 and Cladosporium sp. CBMAI 1237. The dia-
stereoselectivity of flavan-4-ol 3a formed in condition A was 85:15 (cis:
trans), while for condition B was 91:9 (cis:trans). For the diastereoisomer
cis-3a, 96% ee and 97% ee (peak 2, 42.4 mim, later defined as 2S,4S)
were obtained in conditions A and B, respectively. The enantiomeric
excesses for the trans-3a diastereoisomer pair in conditions A and B were
98% ee and 88% ee (peak 1, 26.2 min), respectively. Flavan-4-ol 3a was
isolated from the reaction at condition A with 31% yield and an enan-
tiomeric excess of 98% for trans-3a and 96% ee for cis-3a. See chiral
chromatograms in SM Item 6 Fig. 6.4.
The major formation of the cis-3a product suggests that the hydrogen
approached by the face with reduced steric impediment. Therefore, the
attack must be carried out from the opposite face of the bulkier
substituting group (ring B) located in the stereogenic center of flavanone
2a. Thus, the nucleophilic attack on the carbonyl group of the R enan-
tiomer of flavanone occurs through the Si face, while the nucleophilic
attack on the S enantiomer occurs through the Re face of the pro-
stereogenic ketone (Fig. 4).
The reactions with Fusarium sp. CBMAI 1830, Aspergillus sp. CBMAI
1829, A. sydowii CBMAI 934, P. citrinum CBMAI 1186, M. racemosus
CBMAI 847 and A. sclerotiorum CBMAI 849 showed formation of flavan-
4-ol 3a with low conversion. Despite the reduced production of 3a, the
reactions with P. citrinum CBMAI 1186 and M. racemosus CBMAI 847
presented an interesting characteristic, the formation of the trans
configuration of flavan-4-ol 3a was favored in relation to the cis
configuration. The diastereoisomeric ratio obtained with M. racemosus
CBMAI 847 were 22:78 and 29:71 (cis:trans) in conditions A and B,
respectively.
The unreacted flavanone 2a was isolated with an enantiomeric
23,
excess for (R)ꢀ 2a which had [
α]
589 + 4.2◦ (c 1.00, CHCl3, 28% ee) and
23,
was designated by comparison with the literature [
α]
+ 54.8◦ (c
589
0.80, CHCl3, 85% ee) [51]. The measurement of optical rotation for
flavan-4-ol 3a isolated from the reaction with the fungus Acremonium sp.
23,
CBMAI 1676 was determined as [
α
]
589 + 8.1◦ (c 1.00, CHCl3, 97% ee)
23,
and compared with the rotation presented in the literature [
α]
+
589
59.8 (c 1.20, CHCl3, 95% ee) for the cis-(2S,4S)ꢀ 3a [51]. In view of the
optical rotation values, the diastereoisomeric ratio, the enantiomeric
excess, the preferential consumption of (S)ꢀ 2a and the fact that this
stereogenic center was not the target of the transformation, it can be
suggested that the cis-(2S,4S)ꢀ 3a was the major product from the
biotransformation of 2a by the fungus Acremonium sp. CBMAI 1676.
The biotransformation of 2a by Cladosporium sp. CBMAI 1237
resulted in the formation of cis and trans flavan-4-ol 3a with a discrete
selectivity for the trans-3a diastereoisomer. The dr of 3a was 42:58 (cis:
trans) in condition A and 44:56 (cis:trans) in condition B. In condition A
with Cladosporium sp. CBMAI 1237 the enantioselectivity for the cis-3a
enantiomer pair was 54% ee (peak 2, 42.4 min, 2S,4S) and for the trans-
3a diastereoisomer was 24% ee (peak 1, 26.2 min). In condition B, the
enantioselectivity for the cis-3a enantiomer pair was 49% ee (peak 2,
However, the ee values of the trans-3a diastereoisomer were low for
both conditions A and B with 27% and 3% (peak 1, 26.2 min), respec-
tively. The determination of cis-3a ee was not possible because of the low
2a conversion to this diastereoisomer (8–10%). For reaction with
P. citrinum CBMAI 1186, the diastereoisomeric ratios were 26:74 (cis:
trans) in condition A and 33:67 (cis:trans) in condition B, presenting 33%
ee (peak 1, 26.2 min) in condition A and 19% ee (peak 2, 31.8 min) in
condition B for the trans-3a diastereoisomer.
The biotransformation of flavanone 2a has been reported in the
literature describing the bioreduction by yeasts of the genera Rhodo-
torula, Candida and Saccharomyces to form flavan-4-ol 3a [51]. The
authors observed that the bioreduction of the R enantiomer of flavanone
Fig. 4. Representation of the nucleophilic attack of the hydride ion by the Re and Si faces of the pro-stereogenic ketone of flavanone 2a to form the flavan-4-ol 3a.
6

I.L. de Matos et al.
Molecular Catalysis 513 (2021) 111734
was favored in most of cases. However, Rhodotorula yeasts were more
selective for the formation of trans-3a enantiomers with high enantio-
meric excesses (> 99% ee) for trans-(2R,4S)ꢀ 3a, while cis-3a enantio-
mers showed selectivity for cis-(2S,4S)-flavan-4-ol 3a.
Table 2
Bioreduction of flavanone 2a by Acremonium sp. CBMAI 1676 over time (32 ◦C,
130 rpm, 1–14 days).
In the literature, the reactions promoted by fungi of the Aspergillus
genus in general were selective for the formation of the cis enantiomers
of flavan-4-ols. The terrestrial fungus Aspergillus niger KB reduced the
carbonyl group of racemic 7-methoxyflavone and led to the formation of
(±)ꢀ 2,4-cis-7-methoxyflavane-4-ol [26]. A. niger KB also promoted the
biotransformation of flavanone and 6-hydroxyflavanone to obtain
(±)-cis-flavan-4-ol and (±)ꢀ 2,4-cis-6-hydroxiflavan-4-ol, respectively
[25]. Also, the derivatives 6-acetoxy-, 6-propionoxy and 6-butyryloxy-
flavanones were hydrolyzed and enantioselective hydroxylated to pro-
duce (S)ꢀ 6,4-dihydroxyflavanone [27].
Time (days)
2a
3a
ee (%) de 3a
cis (2S,4S)
c (%) remaining
ee (%)
dr (cis:trans)
trans
1
86
80
70
64
53
4 (R)
95:5
95:5
95:5
94:5
85:15
>99
99
–
3
13 (R)
17 (R)
26 (R)
27 (R)
>99
>99
>99
93
5
99
In another example, the natural flavanone Bavachinin, that can be
found in species of the genus Psoralea (P. corylifolia, P. corylifoliaos) and
be useful for treatment of asthma and diarrhea, was biotransformed by
the terrestrial fungus P. raistrickii ATCC 10490 resulting in a high se-
lective reduction for the production of (2S,4R)ꢀ 2-(4-hydroxyphenyl)ꢀ
7‑methoxy-6-(3-methylbut-2-en-1-yl)chromen-4-ol with 73% yield
[52].
7
>99
96
14
c = remaining 3a concentration determined by analytical curve using HPLC-UV
employing C18 column.
ee = enantiomeric excess determined by chiral HPLC-UV analysis.
dr = diastereoisomeric ratio determined by HPLC-UV analysis employing C18
column.
Alcohol dehydrogenases (ADH) are the enzymes responsible for the
reduction of aldehydes and ketones to obtain enantiomerically pure or
enriched alcohols. ADH enzymes are dependent on cofactors, such as
NAD+ or NADP+, which in their respective reduced form (NADH/
NADPH) possess hydride ions that can be transferred to another mole-
cule [53,54]. However, these coenzymes are expensive and therefore
must be accompanied of a recycling system using an easily accessible
co-substrate, for example, 2-pronanol. Moreover, promiscuous reactions
are also a possibility [55].
3.3. Biotransformation of flavanones 2b-g by Acremonium sp. CBMAI
1676 and Cladosporium sp. CBMAI 1237
Racemic flavanones 2b-g were submitted to biotransformation with
the fungi Acremonium sp. CBMAI 1676 and Cladosporium sp. CBMAI
1237 for the formation of the respective flavan-4-ols 3b-g(Table 3).
These products were obtained in reactions with the fungus Cladosporium
sp. CBMAI 1237 with good isolated yields (67–87%), and those obtained
from reactions with Acremonium sp. CBMAI 1676 presented lower iso-
lated yields (15–46%) but higher enantioselectivities (up to 99% ee).
Chromatograms are shown in the SM Item 7 Fig. 7.1.
In the use of whole cells of microorganisms as in this work, the
addition of cofactors and/or co-substrates was not necessary, since fungi
produce the cofactors and the metabolic pathways for their regenera-
tion, maintaining the enzymatic catalysis that can accept natural and
synthetic substrates. Multiple dehydrogenase enzymes can be produced
by a living organism, each of them can have different specificities for the
substrate enantiomers [56]. Thus, in the biotransformation reactions of
2a that resulted in both cis-3a and trans-3a isomers, it was not possible
to establish that a single oxidoreductase was responsible for the trans-
formation of the substrate.
The diastereoisomeric ratios were low in reactions with the fungus
Cladosporium sp. CBMAI 1237, but the compounds with smaller sub-
stituents 2a (-H), 2c (F) and 2d (-F) and 2 g (-Cl) showed some pre-
dominance for the formation of trans stereoisomers, while flavanones
with bulkier substituents 2b (–OCH3), 2e (Br) and 2f (-Br) favored the
formation of the cis stereoisomers. In the literature, a study with whole
cells of yeast presented the cis preference in substrates with small sub-
stituents, but the opposite diastereoselectivity was observed when bulky
substituents were tested [57].
3.2. Biotransformation of 2a over time by Acremonium sp. CBMAI 1676
Among the flavan-4-ols obtained in the biotransformation reactions
promoted by Cladosporium sp. CBMAI 1237, flavan-4-ol 3c showed the
highest diastereoselectivity and enantioselectivity with a 35:65 dr (cis:
trans) with 64% ee in the cis-3c and 30% ee in the trans-3c. The reactions
with flavanone 2c using Acremonium sp. CBMAI 1676 presented an
opposite diastereopreference in flavan-4-ol 3c 66:34 (cis: trans) when
compared to that obtained with the fungus Cladosporium sp. CBMAI
1237, showing 77% ee in the cis-3c and >99% ee in the trans-3c. Chiral
chromatograms for product 3c are presented in SM Item 7 Fig. 7.2.
The biotransformation reaction of flavanone 2e employing Acre-
monium sp. CBMAI 1676 showed greater selectivity than by Cladospo-
rium sp. CBMAI 1237. Flavan-4-ol 3e was isolated from the reaction with
Acremonium sp. CBMAI 1676 as a mixture of diastereoisomers with 15%
yield and diastereoisomeric ratio of 81:19 (cis:trans). Chiral chromato-
grams for product 3e are presented in SM Item 3 Fig. 7.3.
The 2a biotransformation reaction by the fungus Acremonium sp.
CBMAI 1676 was monitored using the condition A for periods of 1, 3, 5,
7 and 14 days for evaluation of the reaction course and product for-
mation. The consumption of flavanone 2a and formation of flavan-4-ol
3a was determined (Table 2).
The biotransformation of 2a by Acremonium sp. CBMAI 1676 pro-
moted the formation of 3a in the first 24 h with a diastereoisomeric ratio
of 95:5 (cis:trans) and 99% ee for the cis and trans enantiomers. Over
time, there was an increase from 14% to 36% of conversion for 7 days of
reaction and the diastereoselectivity remained high during this period.
After 14 days, the conversion continued to increase to 47%, but there
was a decrease in the diastereoisomeric ratio to 85:15 (cis:trans), as well
as in the ee of the cis (96% ee) and trans (93% ee) enantiomers of flavan-
4-ol 3a.
The trans-3e and cis-3e diastereoisomers were produced with 97%
and 95% ee, respectively, highlighting that the enzymes of the fungus
Acremonium sp. CBMAI 1676 showed selectivity for the transfer of the
hydride ion over the Si face of flavanone 2e. Flavan-4-ol 3e was obtained
by the reaction with Acremonium sp. CBMAI 1676 with 67% yield and
52:48 diastereoisomeric ratio (cis:trans), but with low enantiomeric ex-
cesses (<4% ee). To our knowledge, this is the first report of compound
3e.
Despite the progressive increase in the conversion of flavanone 2a in
reactions with the fungus Acremonium sp. CBMAI 1676, selectivity loss
was observed over time. Thus, it was decided to carry out the
biotransformation of flavanones 2b-g by the fungus Acremonium sp.
CBMAI 1676 in the condition A with 7 days of incubation. The fungus
Cladosporium sp. CBMAI 1237 was also used in the biotransformation of
flavanones 2b-g because of the high yields determined in the formation
of cis-3a and trans-3a isomers (Section 3.1).
7

I.L. de Matos et al.
Molecular Catalysis 513 (2021) 111734
Table 3
Bioreduction of flavanones 2b-g promoted by the fungi Cladosporium sp. CBMAI 1237 and Acremonium sp. CBMAI 1676 (32 ◦C, 130 rpm, 7 days).
Product
Isolated yield (%)
dr (cis:trans)
ee (%)
cis*
trans**
Cladosporium sp. CBMAI 1237a
3a
83
87
65
82
67
77
72
44:56
61:39
35:65
42:58
52:48
64:36
45:55
49 (peak 2)
24 (peak 1)
3b
–
–
3c
64 (peak 2)
30 (peak 2)
3d
–
4
–
3
3e
3f
22 (peak 2)
4
29 (peak 2)
2
3g
Acremonium sp. CBMAI 1676b
3a
3b
3c
3d
3e
3f
31
46
44
29
15
19
25
85:15
96:4
96 (peak 2)
94 (peak 2)
77 (peak 2)
–
97 (peak 2)
95 (peak 2)
93 (peak 2)
>99 (peak 1)
>99 (peak 2)
>99 (peak 2)
–
95 (peak 2)
>99 (peak 2)
97 (peak 2)
66:34
76:24
81:19
54:46
75:25
3g
Reaction conditions:.
a
Fungal fungal cells (5 g) and flavanones 3a-g (50 mg) in phosphate buffer (Na2HPO4 / KH2PO4 0.07 M, 100 mL, pH 7) incubated in an orbital shaker (32 ◦C, 130
rpm, 7 days);.
b
Wet fungal cells (5 g) and flavanones 3a-g (50 mg) in culture broth (100 mL) incubated on orbital shaker (7 days, 130 rpm, 32 ◦C).
ee = enantiomeric excess determined by chiral HPLC-UV analysis.
dr = diastereoisomeric ratio determined by HPLC-UV analysis.
4a: cis* = Peak 2 (42.4 min) and trans** = peak-1 (26.2 min); 4c: cis* = peak 2 (46.8 min) and trans** = peak 2 (28.8 min); 4e: cis* = peak 2 (55.6 min) and trans**
= peak 2 (32.0 min); 4f: cis* = peak 2 (52.4 min) and trans** = peak 2 (36.4 min); 4 g: cis* = peak 2 (45.3 min) and trans** = peak 2 (28.7 min).
The bioreduction of flavanone 2f by Cladosporium sp. CBMAI 1237
followed the same behavior observed in the previous biotransformation
reactions promoted by this fungus. Flavan-4-ol 3f was obtained as a
mixture of diastereoisomers with 77% yield, 64:36 dr (cis:trans) and 22%
ee (peak 2) of cis-3f and 29% ee of trans-3f diastereoisomer.
This feature is one of the disadvantages of using whole cells of mi-
croorganisms, since more than one enzyme can catalyze the same type of
transformation. Experiments with prebiotics and lactic bacteria
employing hesperetin-7-O-rutinoside, naringenin-7-O-rutinoside, hes-
peretin, and naringenin showed that different classes of enzymes can act
on the substrate, promoting ring fission and consumption of the sub-
strate as energy source [58,59].
A lower selectivity in the formation of diastereoisomers was
observed for reactions with Acremonium sp. CBMAI 1676 when
compared to other flavan-4-ols, obtaining a dr of 54:46 (cis:trans) with
19% isolated yield. However, enantiomeric selectivity was not affected,
as flavan-4-ol 3f was obtained with > 99% ee in the cis-(2S,4S)ꢀ 3f and,
95% ee in the trans-3f diastereoisomer. Chiral chromatograms for
product 3f are presented in SM Item 7 Fig. 7.4. To our knowledge, this is
the first report of compound 3f.
For products 3c-g, Acremonium sp. CBMAI 1676 presented low dia-
stereoselectivity, differently from the sample of 3a and 3b. Therefore,
high diastereoselective or diastereopure products of 3c-g were not ob-
tained, and it was not possible to separate these diastereoisomers by
chromatographic column. Consequently, the absolute configuration of
the obtained compounds was not assigned. Although optical rotation
measurements were presented in SM Item 8.
Flavan-4-ols obtained with Cladosporium sp. CBMAI 1237 were iso-
lated as a mixture of the two diastereoisomers cis-3g and trans-3g in 72%
yield. However, flavan-4-ol 3g showed low diastereoselectivities 45:55
(cis:trans) and enantioselectivities (2 and 4% ee).
In summary, different strains of marine-derived fungi bio-
transformed flavanone 2a to dihydrochalcone 4a, 2′,4-dihydroxy-dihy-
drochalcone 5a and/or flavan-4-ol 3a with enantioselectivity and
diastereoselectivity, showing the potential of these biocatalysts for
reduction of this class of compounds. Acremonium sp. CBMAI 1676 and
Cladosporium sp. CBMAI 1237 reduced flavanones 2a-g to the respective
flavan-4-ols 3a-g with good yields, enantioselectivity and diaster-
eoselectivty, which were defined by the action of the alcohol de-
hydrogenases produced by each fungus strain. Indicating that this
bioreduction process can be further explored employing enzyme isola-
tion, characterization and genetic engineering.
The 2g biotransformation reaction by Acremonium sp. CBMAI 1676
showed a lower isolated yield for 3g (25%) when compared to that
obtained with the fungus Cladosporium sp. CBMAI 1237. However, the
reaction was more selective in relation to both diastereoselectivity and
enantioselectivity. Flavan-4-ol 3g obtained from reactions with Acre-
monium sp. CBMAI 1676 was isolated as a mixture of cis and trans di-
astereoisomers (75:25 dr) with high ees in the trans-3 g (97% ee) and cis-
3g (93% ee). Chiral chromatograms for product 3g are presented in SM
Item 7 Fig. 3.5.
The fungus Cladosporium sp. CBMAI 1237 can be an important source
of alcohol dehydrogenases. In addition, Acremonium sp. CBMAI 1676
was the most enantioselective biocatalyst of this study presenting values
of 99% ee for both diastereoisomers of flavanones. Also, the presence of
substituents on the substrates did not inhibited the enantioselectivity of
the alcohol dehydrogenases present in these biocatalysts. Moreover, the
obtained products can be assessed for biological activities in future
studies.
In general, the biotransformation reactions of flavanones 2a-g by the
marine-derived fungus Cladosporium sp. CBMAI 1237 resulted in good
conversions of the substrates in their respective flavan-4-ols 3a-g with
different diastereoselectivities and enantioselectivities. The low selec-
tivity may be due to the presence of different enzymes capable of car-
rying out the same reaction, thus, flavan-4-ols 3a-g were not necessarily
produced by the action of a single alcohol dehydrogenase.
8

I.L. de Matos et al.
Molecular Catalysis 513 (2021) 111734
[4] D. Barreca, G. Gattuso, E. Bellocco, A. Calderaro, D. Trombetta, A. Smeriglio,
4. Conclusion
`
G. Lagana, M. Daglia, S. Meneghini, S.M. Nabavi, Flavanones: citrus phytochemical
with health-promoting properties, BioFactors 43 (2017) 495–506, https://doi.org/
10.1002/biof.1363.
Fungi derived from the marine environment presented potential for
asymmetric reduction of flavanones with a pro-stereogenic center. The
employed strains reduced flavanones 2a-g with Cladosporium sp. CBMAI
1237 presenting high yields (about 80%), and Acremonium sp. CBMAI
1676 resulting in up to 99% ee in the cis and trans diastereoisomers. The
use of fungi, with emphasis for these marine-derived strains, was an
interesting approach for enantioselective reduction of halogenated fla-
vanones. To our knowledge, this was the first report of brominated
flavan-4-ols. Therefore, this strategy can be important for obtaining
enantioenriched compounds with biological activities.
[5] A.N. Panche, A.D. Diwan, S.R. Chandra, Flavonoids: an overview, J. Nutr. Sci. 5
(2016) 1–15, https://doi.org/10.1017/jns.2016.41.
´
ˇ
´
ˇ
[6] I. Najmanova, M. Voprsalova, L. Saso, P. Mladenka, The pharmacokinetics of
flavanones, Crit. Rev. Food Sci. Nutr. 60 (2020) 3155–3171, https://doi.org/
10.1080/10408398.2019.1679085.
[7] L.G. Sarbu, L.G. Bahrin, C. Babii, M. Stefan, M.L. Birsa, Synthetic flavonoids with
antimicrobial activity: a review, J. Appl. Microbiol. 127 (2019) 1282–1290,
https://doi.org/10.1111/jam.14271.
[8] J. Chen, C.-.F. Liu, G.-.W. Rao, Progress in the synthesis, angiogenesis activity and
mechanism of chalcone derivatives, Mini. Rev. Org. Chem. 17 (2019) 814–827,
https://doi.org/10.2174/1570193x17666191223161941.
[9] T.A. Dias, C.L. Duarte, C.F. Lima, M.F. Proença, C. Pereira-Wilson, Superior
anticancer activity of halogenated chalcones and flavonols over the natural
flavonol quercetin, Eur. J. Med. Chem. 65 (2013) 500–510, https://doi.org/
10.1016/j.ejmech.2013.04.064.
Funding
[10] R. Bernini, M. Pasqualetti, G. Provenzano, S. Tempesta, Ecofriendly synthesis of
halogenated flavonoids and evaluation of their antifungal activity, New J. Chem.
39 (2015) 2980–2987, https://doi.org/10.1039/c5nj00258c.
The authors thank all the support of the Chemical Analysis Center
˜
˜
(Sao Carlos Institute of Chemistry, University of Sao Paulo) for the NMR
[11] X. Zhang, O. Khalidi, S.Y. Kim, R. Wang, V. Schultz, B.F. Cress, R.A. Gross, M.A.
G. Koffas, R.J. Linhardt, Synthesis and biological evaluation of 5,7-dihydroxyfla-
vanone derivatives as antimicrobial agents, Bioorg. Med. Chem. Lett. 26 (2016)
3089–3092, https://doi.org/10.1016/j.bmcl.2016.05.003.
˜
analysis. A.L.M. Porto thanks to Sao Paulo Research Foundation
(FAPESP, 2016/20155-7 and 2014/18257-0), Brazilian National Coun-
cil for Scientific and Technological Development (CNPq, 302528/2017-
2) and Coordination for the Improvement of Higher Education Personnel
(CAPES, Finance Code 001) for funding.
[12] F.L.A. Shah, A.B. Ramzi, S.N. Baharum, N.M. Noor, H.H. Goh, T.C. Leow, S.
N. Oslan, S. Sabri, Recent advancement of engineering microbial hosts for the
biotechnological production of flavonoids, Mol. Biol. Rep. 46 (2019) 6647–6659,
https://doi.org/10.1007/s11033-019-05066-1.
[13] R. Wohlgemuth, Biocatalysis – Key enabling tools from biocatalytic one-step and
multi-step reactions to biocatalytic total synthesis, N. Biotechnol. 60 (2021)
113–123, https://doi.org/10.1016/j.nbt.2020.08.006.
CRediT authorship contribution statement
[14] J.F. Wang, S.S. Liu, Z.Q. Song, T.C. Xu, C.S. Liu, Y.G. Hou, R. Huang, S.H. Wu,
Naturally occurring flavonoids and isoflavonoids and their microbial
transformation: a review, Molecules 25 (2020), https://doi.org/10.3390/
molecules25215112.
Iara L. de Matos: Conceptualization, Methodology, Validation,
Software, Formal analysis, Investigation, Resources, Data curation,
Writing – original draft, Writing – review & editing, Visualization.
Willian G. Birolli: Conceptualization, Methodology, Writing – original
draft, Writing – review & editing, Visualization. Darlisson de A. San-
tos: Methodology, Writing – review & editing. Marcia Nitschke:
Conceptualization, Resources, Writing – review & editing, Visualization,
[15] H. Cao, X. Chen, A.R. Jassbi, J. Xiao, Microbial biotransformation of bioactive
flavonoids, Biotechnol. Adv. 33 (2015) 214–223, https://doi.org/10.1016/j.
biotechadv.2014.10.012.
[16] L.L. Chu, R.P. Pandey, N. Jung, H.J. Jung, E.H. Kim, J.K. Sohng, Hydroxylation of
diverse flavonoids by CYP450 BM3 variants: biosynthesis of eriodictyol from
naringenin in whole cells and its biological activities, Microb. Cell Fact. 15 (2016)
135, https://doi.org/10.1186/s12934-016-0533-4.
´
Supervision, Project administration, Funding acquisition. Andre Luiz
M. Porto: Conceptualization, Resources, Writing – review & editing,
[17] A. Zerva, E. Koutroufini, I. Kostopoulou, A. Detsi, E. Topakas, A novel thermophilic
laccase-like multicopper oxidase from Thermothelomyces thermophila and its
application in the oxidative cyclization of 2′,3,4-trihydroxychalcone,
N. Biotechnol. 49 (2019) 10–18, https://doi.org/10.1016/j.nbt.2018.12.001.
[18] H. Harwoko, R. Hartmann, G. Daletos, E. Ancheeva, M. Frank, Z. Liu, P. Proksch,
Biotransformation of host plant flavonoids by the fungal endophyte epicoccum
nigrum, ChemistrySelect 4 (2019) 13054–13057, https://doi.org/10.1002/
slct.201903168.
Visualization, Supervision, Project administration, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the study reported in this paper.
[19] J. Seo, S. Il Kang, M. Kim, J. Han, H.G. Hur, Flavonoids biotransformation by
bacterial non-heme dioxygenases, biphenyl and naphthalene dioxygenase, Appl.
Microbiol. Biotechnol. 91 (2011) 219–228, https://doi.org/10.1007/s00253-011-
3334-z.
[20] Z.T.A. Shakour, N.M. Fayek, M.A. Farag, How do biocatalysis and
biotransformation affect Citrus dietary flavonoids chemistry and bioactivity? A
review, Crit. Rev. Biotechnol. 40 (2020) 689–714, https://doi.org/10.1080/
07388551.2020.1753648.
Acknowledgments
I. L. de Matos thanks to the Coordination for the Improvement of
Higher Education Personnel (CAPES) for the provision of her doctorate
scholarship (CAPES, process n◦ 1313652) and W. G. Birolli thanks to the
Brazilian National Council for Scientific and Technological Develop-
ment (CNPq, 141656/2014-0) for his doctorate’s degree scholarship. W.
[21] J. Kozłowska, B. Potaniec, B. Zarowska, M. Anioł, Microbial transformations of 4′-
methylchalcones as an efficient method of obtaining novel alcohol and
dihydrochalcone derivatives with antimicrobial activity, RSC Adv 8 (2018)
30379–30386, https://doi.org/10.1039/c8ra04669g.
[22] A.R. Cho, D.G. An, Y. Lee, J.H. Ahn, Biotransformation of quercetin to quercetin 3-
O-gentiobioside using engineered Escherichia coli, Appl. Biol. Chem. 59 (2016)
689–693, https://doi.org/10.1007/s13765-016-0212-5.
˜
G. Birolli also thanks to Sao Paulo Research Foundation (FAPESP, 2017/
19721-0) for his post-doctoral scholarship.
[23] T. Uno, T. Yanase, H. Imaishi, Functional characterization of CYP52G3 from
Aspergillus oryzae and its application for bioconversion and synthesis of hydroxyl
flavanone and steroids, Biotechnol. Appl. Biochem. 64 (2017) 385–391, https://
doi.org/10.1002/bab.1496.
Supplementary materials
[24] E. Kostrzewa-Suslow, M. Dymarska, U. Guzik, D. Wojcieszynska, T. Janeczko,
Stenotrophomonas maltophilia: a gram-negative bacterium useful for
transformations of flavanone and chalcone, Molecules (2017) 22, https://doi.org/
10.3390/molecules22111830.
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.mcat.2021.111734.
´
[25] E. Kostrzewa-Susłow, J. Dmochowska-Gładysz, A. Białonska, Z. Ciunik,
W. Rymowicz, Microbial transformations of flavanone and 6-hydroxyflavanone by
Aspergillus niger strains, J. Mol. Catal. B 39 (2006) 18–23, https://doi.org/
10.1016/j.molcatb.2006.01.020.
References
[1] D.M. Kopustinskiene, V. Jakstas, A. Savickas, J. Bernatoniene, Flavonoids as
anticancer agents, Nutrients 12 (2020), https://doi.org/10.3390/nu12020457.
[2] I.A. Parshikov, J.B. Sutherland, Biotransformation of steroids and flavonoids by
cultures of aspergillus niger, Appl. Biochem. Biotechnol. 176 (2015) 903–923,
https://doi.org/10.1007/s12010-015-1619-x.
[26] E. Kostrzewa-Suslow, T. Janeczko, Microbial transformations of 7-
Methoxyflavanone, Molecules 17 (2012) 14810–14820, https://doi.org/10.3390/
molecules171214810.
´
[27] E. Kostrzewa-Susłow, M. Dymarska, A. Białonska, T. Janeczko, Enantioselective
conversion of certain derivatives of 6-hydroxyflavanone, J. Mol. Catal. B 102
˜
[3] R. Santos, D. Pinto, C. Magalhaes, A. Silva, Halogenated flavones and isoflavones: a
(2014) 59–65, https://doi.org/10.1016/j.molcatb.2014.01.013.
state-of-art on their synthesis, Curr. Org. Synth. 17 (2020) 415–425, https://doi.
org/10.2174/1570179417666200530213737.
9

I.L. de Matos et al.
Molecular Catalysis 513 (2021) 111734
´
[28] J. Alarcon, J. Alderete, C. Escobar, R. Araya, C.L. Cespedes, Aspergillus niger
relationship study, Bioorganic Med. Chem. Lett. 27 (2017) 1205–1209, https://doi.
org/10.1016/j.bmcl.2017.01.061.
catalyzes the synthesis of flavonoids from chalcones, Biocatal. Biotransform. 31
(2013) 160–167, https://doi.org/10.3109/10242422.2013.813489.
[29] M. Nagaki, Y. Kikuchi, S. Maekawa, T. Kidachi, K. Honda, Y. Chounan,
Biotransformation of 4-chromanol, 4-flavanol, xanthydrol, and their analogs by
tissue cultured cells, Nat. Prod. Commun. 12 (2017), https://doi.org/10.1177/
1934578X1701201120, 1934578X1701201.
[43] E.V. Stoyanov, Y. Champavier, A. Simon, J.P. Basly, Efficient liquid-phase synthesis
of 2′-hydroxychalcones, Bioorganic Med. Chem. Lett. 12 (2002) 2685–2687,
https://doi.org/10.1016/S0960-894X(02)00553-X.
[44] P. Tang, T. Furuya, T. Ritter, Silver-catalyzed late-stage fluorination, J. Am. Chem.
Soc. 132 (2010) 12150–12154, https://doi.org/10.1021/ja105834t.
[45] Y. Yong, S. Ahn, D. Hwang, H. Yoon, G. Jo, Y.H. Kim, S.H. Kim, D. Koh, Y. Lim, 1H
and 13C NMR spectral assignments of 2′-hydroxychalcones, Magn. Reson. Chem.
51 (2013) 364–370, https://doi.org/10.1002/mrc.3949.
[30] A. Cano-Flores, M. Espinoza, G. Delgado, Bio- and chemo- transformations of
glabranin and 7-O-methylglabranin and cytotoxic evaluations of the transformed
products, Nat. Prod. Res. (2020), https://doi.org/10.1080/
14786419.2020.1862835.
[46] Y.L. Zhang, Y.Q. Wang, Enantioselective biomimetic cyclization of 2′-
hydroxychalcones to flavanones, Tetrahedron Lett 55 (2014) 3255–3258, https://
doi.org/10.1016/j.tetlet.2014.04.032.
[31] I. Maciel Ferreira, L. Coutinho Rocha, S. Akinobo Yoshioka, M. Nitschke,
A. Haroldo Jeller, L. Pizzuti, M.H.R. Seleghim, A.L.M. Porto, Chemoselective
reduction of chalcones by whole hyphae of marine fungus Penicillium citrinum
CBMAI 1186, free and immobilized on biopolymers, Biocatal. Agric. Biotechnol. 3
(2014) 358–364, https://doi.org/10.1016/j.bcab.2014.04.001.
[47] D. Zhao, B. Beiring, F. Glorius, Ruthenium-NHC-catalyzed asymmetric
hydrogenation of flavones and chromones: general access to enantiomerically
enriched flavanones, flavanols, chromanones, and chromanols, Angew. Chemie Int.
Ed. 52 (2013) 8454–8458, https://doi.org/10.1002/anie.201302573.
[32] I.L. de Matos, M. Nitschke, A.L.M. Porto, Hydrogenation of halogenated 2′-
hydroxychalcones by mycelia of marine-derived fungus Penicillium raistrickii,
Mar. Biotechnol. 21 (2019) 430–439, https://doi.org/10.1007/s10126-019-09893-
y.
´
[48] E. Kostrzewa-Susłow, J. Dmochowska-Gładysz, A. Białonska, Z. Ciunik, Microbial
transformations of flavanone by Aspergillus niger and Penicillium chermesinum
cultures, J. Mol. Catal. B 52–53 (2008) 34–39, https://doi.org/10.1016/j.
molcatb.2008.01.002.
[33] K. Tanaka, T. Sugino, Efficient conversion of 2′-hydroxychalcones into flavanones
and flavanols in a water suspension medium, Green Chem. R. Soc. Chem. (2001)
133–134, https://doi.org/10.1039/b101826b.
[49] L.M.B. Ferreira, M. Kobelnik, L.O. Regasini, L.A. Dutra, V. da Silva Bolzani, C.
A. Ribeiro, Synthesis and evaluation of the thermal behavior of flavonoids: thermal
decomposition of flavanone and 6-hydroxyflavanone, J. Therm. Anal. Calorim. 127
(2017) 1605–1610, https://doi.org/10.1007/s10973-016-5896-6.
[50] M. Stompor, B. Potaniec, A. Szumny, P. Zielinski, A.K. Zonierczyk, M. Anioł,
Microbial synthesis of dihydrochalcones using Rhodococcus and Gordonia species,
J. Mol. Catal. B 97 (2013) 283–288, https://doi.org/10.1016/j.
molcatb.2013.09.009.
[34] L.C. Rocha, H.V. Ferreira, R.F. Luiz, L.D. Sette, A.L.M. Porto, Stereoselective
bioreduction of 1-(4-methoxyphenyl)ethanone by whole cells of marine-derived
fungi, Mar. Biotechnol. 14 (2012) 358–362, https://doi.org/10.1007/s10126-011-
9419-y.
[35] C.M. Mizuno, Chemical and Microbiological Studies of Microorganisms Associated
With the Marine Sponge Dragmacidon reticulatum, Aiming At the Isolation of
˜
´
´
Bioactive Secondary Metabolites, Universidade Federal de Sao Carlos, 2010.
[51] T. Janeczko, M. Dymarska, M. Siepka, R. Gniłka, A. Lesniak, J. Popłonski,
E. Kostrzewa-Susłow, Enantioselective reduction of flavanone and oxidation of cis-
and trans-flavan-4-ol by selected yeast cultures, J. Mol. Catal. B 109 (2014) 47–52,
https://doi.org/10.1016/j.molcatb.2014.08.006.
[36] S.N. Ortega, M. Nitschke, A.M. Mouad, M.D. Landgraf, M.O.O. Rezende, M.H.
R. Seleghim, L.D. Sette, A.L.M. Porto, Isolation of Brazilian marine fungi capable of
growing on DDD pesticide, Biodegradation 22 (2011) 43–50, https://doi.org/
10.1007/s10532-010-9374-8.
[52] J. Luo, Q. Liang, Y. Shen, X. Chen, Z. Yin, M. Wang, Biotransformation of
bavachinin by three fungal cell cultures, J. Biosci. Bioeng. 117 (2014) 191–196,
https://doi.org/10.1016/j.jbiosc.2013.08.001.
[37] W.G. Birolli, I.M. Ferreira, N. Alvarenga, D.D.A. Santos, I.L. de Matos, J.
V. Comasseto, A.L.M. Porto, Biocatalysis and biotransformation in Brazil: an
overview, Biotechnol. Adv. (2015) 33, https://doi.org/10.1016/j.
biotechadv.2015.02.001.
[53] F. Hollmann, D.J. Opperman, C.E. Paul, Biocatalytic reduction reactions from a
chemist’s Perspective, Angew. Chem. Int. Ed. 60 (2020) 5644–5665, https://doi.
org/10.1002/anie.202001876.
[38] W.G. Birolli, L.I. Souza, A.L.M. Porto, E. Rodrigues-Filho, Biodegradation and
Bioremediation of Pyrethroids, a recent update and Experiments in Soil, in:
J. Ruijten (Ed.), Pyrethroids Expo. Appl. Resist., Nova Science Publishers,
Hauppauge, NY, USA, 2020, pp. 1–89.
[54] J. Zhou, G. Xu, Y. Ni, Stereochemistry in asymmetric reduction of bulky–bulky
ketones by alcohol dehydrogenases, ACS Catal. 10 (2020) 10954–10966, https://
doi.org/10.1021/acscatal.0c02646.
[39] W.G. Birolli, L.L. Zanin, D.E.Q. Jimenez, A.L.M. Porto, Synthesis of Knoevenagel
adducts under microwave irradiation and biocatalytic ene-eeduction by the
marine-derived fungus Cladosporium sp. CBMAI 1237 for the production of 2-
cyano-3-phenylpropanamide derivatives, Mar. Biotechnol. 22 (2020) 317–330,
https://doi.org/10.1007/s10126-020-09953-8.
[55] W.G. Birolli, A.L.M. Porto, L.P. Fonseca, Miniemulsion in biocatalysis, a new
approach employing a solid reagent and an easy protocol for product isolation
applied to the aldol reaction by Rhizopus niveus lipase, Bioresour. Technol. 297
(2020), https://doi.org/10.1016/j.biortech.2019.122441.
[56] L. Di, A. Balesano, S. Jordan, S.M. Shi, The role of alcohol dehydrogenase in drug
metabolism: beyond ethanol oxidation, AAPS J. 23 (2021), https://doi.org/
10.1208/s12248-020-00536-y.
[40] M. Flammang, D. Frederic, C.-.G. Wermuth, ChemInform Abstract: effects of the
nature and the position of substituents in a novel synthesis method of β-flavanols
and flavanones, Chem. Informationsd. 9 (1978), https://doi.org/10.1002/
chin.197845199.
[57] K. Faber, Biotransformations in Organic Chemistry, Springer International
Publishing, 2018, https://doi.org/10.1007/978-3-319-61590-5.
[41] N. Ahmed, N.K. Konduru, A. Praveen, Kamaluddin Kumar, Silica supported-double
metal cyanides (DMCs): a green and highly efficient catalytic protocol for
isomerisation of 2′-hydroxychalcones to flavanones, J. Mol. Catal. A 373 (2013)
135–141, https://doi.org/10.1016/j.molcata.2013.03.009.
[58] X. Guo, A. Guo, E. Li, Biotransformation of two citrus flavanones by lactic acid
bacteria in chemical defined medium, Bioprocess Biosyst. Eng. 44 (2020) 235–246,
https://doi.org/10.1007/s00449-020-02437-y.
´
´
[59] G. Pereira-Caro, B. Fernandez-Quiros, I.A. Ludwig, I. Pradas, A. Crozier, J.
M. Moreno-Rojas, Catabolism of citrus flavanones by the probiotics
Bifidobacterium longum and Lactobacillus rhamnosus, Eur. J. Nutr. 57 (2018)
231–242, https://doi.org/10.1007/s00394-016-1312-z.
[42] G. Bist, N.T. Pun, T.B.T. Magar, A. Shrestha, H.J. Oh, A. Khakurel, P.H. Park, E.
S. Lee, Inhibition of LPS-stimulated ROS production by fluorinated and
hydroxylated chalcones in RAW 264.7 macrophages with structure-activity
10