Aspergillus niger catalyzes the synthesis of flavonoids from chalcones

Article abstract of DOI:10.3109/10242422.2013.813489

Flavonoids, which have many biological activities and have been widely used in nature, can be artificially synthesized. However, regioselective cyclization of chalcones is difficult by chemical methods. In this study, we demonstrated that Aspergillus nige

Full text of DOI:10.3109/10242422.2013.813489

Biocatalysis and Biotransformation, 2013; 31(4): 160–167
ORIGINAL ARTICLE
Aspergillus niger catalyzes the synthesis of flavonoids from chalcones
JULIO ALARCÓN1, JOEL ALDERETE2, CARLOS ESCOBAR3, RAMIRO ARAYA4 &
CARLOS L. CESPEDES1
1Laboratorio de Síntesis y Biotransformación de Productos Naturales, Departamento de Ciencias Básicas,
Universidad del Bío-Bío, Chillán, Chile, 2Departamento Química Orgánica, Universidad de Concepción,
Concepción, Chile, 3Departamento Química Orgánica, Facultad de Ciencias, Universidad de Chile, Santiago, Chile,
4
and Universidad Nacional Andrés Bello, Santiago, Chile
Abstract
Flavonoids, which have many biological activities and have been widely used in nature, can be artificially synthesized.
However, regioselective cyclization of chalcones is difficult by chemical methods. In this study, we demonstrated
that Aspergillus niger is capable of cyclizing chalcones to flavanones, affording a mimic of plant biosynthetic processes.
Chalcones 1–6 were biotransformated to the modified chalcones 8–14 and to the flavanones 15–27. The biotransforma-
tion showed that enzymatic cyclization and demethylation occurred during the first days of biotransformation; in
contrast, hydroxylation is a later process. With a longer culturing time, it is possible to obtain more hydroxylated
flavanones with excellent yields.
Keywords: A. niger, chalcones biotransformation, flavonoids
Introduction
are able to catalyze different reactions. The use of
Flavonoids are a major class of oxygen-containing
heterocyclic natural product that are widespread in
green plants.This group of natural products plays an
important role in plant growth,development,defence
against microorganisms and pests, and in human
health. In addition to physiological roles in plants,
flavonoids are important components in the human
diet as antioxidants that can scavenge free radicals.
The biological effects of flavonoids have been inten-
sively studied due to their beneficial effects on
human health (Bandgar et al. 2010).The interaction
of flavonoids with numerous enzymes has been
reported and a picture emerged that each flavonoid
is likely to affect a spectrum of cellular target pro-
teins. Biosynthetically, flavonoids are derived from
chalcone precursors which in turn are derived from
the condensation of p-coumaroyl CoA and three
malonyl CoA’s by chalcone synthases (CHS) (Dixon
& Steele 1999).
enzymes or microorganisms as biocatalysts for the
building of new molecules has been well documented
(Faber 2000; Alarcon et al. 2007). Biotransforma-
tions of bioactive natural and synthetic compounds
by microorganisms including Aspergillus species have
been investigated (Das & Rosazza 2006; Alarcon
et al. 2008).Thus, regioselective O-demethylation of
flavones to their 4′-O-demethylated metabolites was
performed using an A. niger strain (Buisson et al.
2007); reduction of the carbonyl group and
dehydrogenation at C-2 and C-3 positions; reduc-
tion of the carbonyl group with hydroxylation at C-7;
and dehydrogenation at C-2 and C-3 and hydroxyla-
tion at C-3 producing a flavanol have all been
reported by biotransformation of flavanone with
A. niger (Kostrzewa-Suslow et al., 2006, 2008).
Hydroxylation at C-6 and C-4′ were obtained as a
result of biotransformation of flavanone by a UV-
induced mutant strain of A. niger. Other studies with
Aspergillus strains show the biotransformation of
cyclic ketones (Keppler et al. 2005). However, the
Biocatalysis is a valuable tool for organic chemi-
stry. Microorganisms, whole cells or their enzymes
Correspondence: Julio Alarcón, Laboratorio de Síntesis y Biotransformación de Productos Naturales, Departamento de Ciencias Básicas, Universidad del
Bío-Bío, Chillán, Chile. Tel: ϩ56-42-253156. Fax: ϩ56-42-253046. E-mail: jualarcon@ubiobio.cl
(Received 15 January 2013; accepted 6 June 2013)
ISSN 1024-2422 print/ISSN 1029-2446 online © 2013 Informa UK, Ltd.
DOI: 10.3109/10242422.2013.813489

A. niger catalyzes the synthesis of flavonoids 161
literature contains few reports on biotransformation
of chalcones (Corrêa et al. 2011; Sanchez-Gonzalez
& Rosazza, 2004), although there is experimental
evidence to show that Aspergillus spp are able to
catalyze cyclization of chalcones to flavones. This
cyclization according to our results requires the pres-
ence of a hydroxyl group at position 2.
et al. 2010). The residue was purified by column
chromatography (silica gel with 10% ethyl acetate in
hexane) affording pure chalcones 1–6 (Table I). Note
that a further chalcone 7 was prepared but reference
to it has been deleted.The physical and spectral data
of chalcones are given below.
Due to the ability of Aspergillus spp to promote
these biotransformations, we decided to study the
biotransformation of different type of chalcone with
the fungus A. niger, and identify its potential use as
a tool for synthesis of flavonoids.
Spectroscopic data of precursors
2″,4″-dimethoxy-2’-hydroxychalcone (1) was obtained
as yellow crystals (59%) with mp.102–103°C. H
1
NMR (CDCl3, 300 MHz) δ/ppm 12.9 (1H, s, OH),
8.18(1H,d,Jϭ15Hz,H-3),7.90(1H,dd,Jϭ7.9/1.3
Hz, H-6′), 7.75 (1H,d, Jϭ15.5 Hz, H-2), 7.50 (1H,
dd, Jϭ7.8/1.65 Hz, H-5′), 7.48 (1H, t, Jϭ8/1.3 Hz,
H-4′), 7.16 (1H,dd, Jϭ 8/1.4 Hz, H-6″), 6.92
(1H, d, Jϭ7.8 Hz, H-3′), 3.83 (3H, s, OCH3), 3.79
(3H, s, OCH3).
Materials and methods
General experimental procedures
NMR spectra (250 MHz 1H and 65 MHz 13C) were
measured in CDCl3 (which also provided the lock
signal) with a Bruker spectrometer (AC-250). The
13C chemical shift was assigned with distortion-free
enhancement of polarization transfer (DEPT) using
a flip angle of 135°. IR spectra were recorder on a
Shimadzu FTIR-8400. High-performance liquid
chromatography (HPLC) was performed with a
Merck-Hitachi L-7100 with a Merck-Hitachi L-7455
Photodiode Array Detector. Separations were car-
ried out in a Kromasil C-18 (250 mmϫ4.6 mm i.d.,
10 μm particle size). The mobile phase consisted of
two solvent systems: CH3CN/H2O (10:90) contain-
ing 5% of HCOOH (A) and CH3CN/H2O (90:10)
containing 5% of HCOOH (B). The gradient was
0%–100% B over 65 min. at a flow rate of 1 ml/min.
UV absorbances of eluting peaks were recorded at
328 nm.
2″,3″-dimethoxy-2´-hydroxychalcone (2):was obtai-
ned as yellow crystals (58%) with mp.107–109°C. 1H
NMR (CDCl3, 300 MHz) δ/ppm 11.9 (1H, s, OH),
8.20 (1H, d, Jϭ15.5 Hz, H-3), 8.10 (1H, dd, Jϭ8/1.4
Hz, H-6´), 7.90 (1H, d, Jϭ15.5 Hz, H-2), 7.52 (1H,
dt, Jϭ8/1.4 Hz, H-4´), 7.35 (1H, dd, Jϭ8/1.4 Hz,
H-6″), 7.02 (1H, t, Jϭ8 Hz, H-5″), 6.98 (1H, dt,
Jϭ8/1.4 Hz, H-5´), 6.97 (1H,dd, Jϭ8/1.4 Hz,
H-36″), 6.96 (1H, dd, Jϭ8/1.4 Hz, H-4″), 6.93
(1H, d, Jϭ7.6 Hz, H-3′), 3.91 (3H, s, 3″-OCH3),
3.83 (3H, s, 2″-OCH3).
3″,4″-dimethoxy-2′-hydroxy-chalcone (3) was
obtained as purple-brown crystals (41%). Mp 112–
1
115°C. H NMR (CDCl3, 300 MHz) δ/ppm 12.9
(s, 1H, OH), 7.94 (dd, 1H, Jϭ7.8/0.7 Hz, H-6′),
7.89 (d, 1H, Jϭ15.4 Hz, H-2), 7.53 (d, 1H, Jϭ15.4
Hz, H-3), 7.50 (ddd, 1H, Jϭ7.8/1.65 Hz, H-5′),
7.28 (dd, 1H, Jϭ8.24/2.02 Hz, H-6″), 7.18 (d, 1H,
Jϭ2.0 Hz, H-2″), 7.03 (dd, 1H, Jϭ8.2 Hz, H-5″),
6.95 (ddd, 1H, Jϭ7.8/0.7 Hz, H-4′), 6.92 (d, 1H,
Jϭ7.8 Hz, H-3′), 3.98 (3H, s, OCH3), 3.95 (3H, s,
OCH3).
Synthesis of chalcones
A mixture of substituted 1-phenyl-ethanone (1)
(1 mmol) and substituted benzaldehyde (2) (1 mmol)
was dissolved in 10 mL ethanol. To this mixture,
potassium hydroxide (40%, 1 ml) was added at
0–5°C. The reaction mixture was stirred at room
temperature for 12 h. Then it was poured over
crushed ice and acidified with 2M HCl. The light
yellow or orange solid thus obtained was filtered,
washed with water, and dried (Scheme 1) (Hwang
3″, 5″-dimethoxy-2′-hydroxy-chalcone (4) was
obtained as yellow crystals (52%). Mp 125–127°C.
1H NMR (CDCl3, 300 MHz) δ/ppm 12.4 (s, IH,
OH), 7.91 (dd, 1H, Jϭ8.4, 1.0 Hz, H-6′), 7.82
(d, 1H, Jϭ15.6 Hz, H-2), 7.59 (d, 1H, Jϭ15.6 Hz,
H-3), 7.50 (td, 1H, Jϭ8.4, 1.0 Hz, H-4′), 7.03
(d, 1H, Jϭ8.4 Hz, H-3′), 6.95 (t, 1H, Jϭ8.4 Hz,
O
O
O
1) EtOH, KOH
12hrs
H
R
+
2) 2M HCl
pH = 3–4
R
R
R
2
1
3
Scheme 1. Chalcone synthesis by the Claisen–Schmidt procedure.

162 J. Alarcón et al.
Table I. Chemical structures of synthetized chalcones.
125 ml of medium after 5 days of preculture. After
incubation for a further 4 days in a shaker at room
temperature, the cultures were filtered, the cells were
washed thoroughly with ethyl acetate, and the fil-
trates pooled and extracted with ethyl acetate. The
combined extracts were dried over anhydrous
Na2SO4 and evaporated under reduced pressure to
give a mixture of compounds. Pure compounds were
obtained by preparative TLC on silica gel using an
n-hexane:ethyl acetate mixture. The pure metabo-
lites were identified by 1H NMR and 13C NMR.
O
R1
R3
R4
R5
1
3
3'
2''
5''
2
6'
R2
R6
Chalcone
R1
R2
R3
R4
R5
R6
1
2
3
4
5
6
OH
OH
OH
OH
OH
OH
H
OMe
OMe
H
H
OMe
OMe
H
OMe
H
OMe
H
H
OMe
H
H
H
OMe
H
H
H
H
OMe
OMe
OMe
OMe
OMe
OMe
H
H
Time-course experiment
Spores of A. niger (about 6ϫ105 spores) were trans-
ferred to 250 ml Erlenmeyer flasks containing
125 ml Hagen medium, and cultured with continu-
ous shaking for 5 days at room temperature. Com-
pound 1 (100 mg) was added to the suspension
cultures and incubated at room temperature on a
rotatory shaker (100 rpm). At regular 12-h time
intervals over 144 h, three Erlenmeyer flasks of the
incubation mixture were removed and extracted with
ethyl acetate. This fraction was analyzed by HPLC
using a Merck-Hitachi L-7100 quaternary pumping
system connected to a Merck-Hitachi diode array
detector L-7455. Separations and quantifications
were performed as previously described.
H-5′), 6.79 (d, 2H, Jϭ2.2 Hz, H-2″, H-6″), 6.55
(t, 1H, Jϭ2.2 Hz, H-4″), 3.85 (3H, s, OCH3).
2″,3″,4′-trimethoxy-2′-hydroxy-chalcone (5) was
obtained as orange crystals (58%), Mp. 129–130°C.
1H NMR (CDCl3, 300 MHz) δ/ppm 12.8 (s, 1H,
OH), 8.21 (d, 1H, Jϭ15.5 Hz, H-3), 8.1 (d, 1H,
Jϭ8.5 Hz, H-6′), 7.90 (d, 1H, Jϭ15.5 Hz, H-2),
7.52 (dt, 1H, Jϭ8, 1.4 Hz, H-4″), 6.98 (dt, 1H,
Jϭ8, 1.3 Hz, H-5″), 6.49 (m, 1H, H-5′), 6.47 (m,
1H, H-3′), 3.92 (s, OCH3), 3.91 (3H, s, OCH3),
3.88 (3H, s, OCH3).
2″,4″,4′-trimethoxy-2′-hydroxy-chalcone (6):was
obtained as orange crystals (46%).1H NMR (CDCl3,
300 MHz) δ/ppm 12.1 (s, 1H, OH), 8.18 (d, 1H,
Jϭ15.6 Hz, H-3), 7.92 (d, 1H, Jϭ8.6 Hz, H-6′),
7.68 (d, 1H, Jϭ15.6 Hz, H-2), 7.15 (dd, 1H,
Jϭ8/1.3 Hz, H-6″), 6.49 (m, 1H, H-5′), 6.47 (m,
1H, H-3′), 3.93 (3H, s, OCH3), 3.88 (3H, s, OCH3),
3.83 (3H, s, OCH3).
Results and discussion
Biotransformation results
The chalcones 1–6 (Table I), prepared by Claisen–
Schmidt condensation were biotransformed with
A. niger LSPN001 over 5 days of reaction. After that
time the media was extracted with ethyl acetate, and
19 biotransformation products (Compounds 8–27)
were separated and purified by preparative TLC
Medium and culture conditions
A. niger LSPN001 was obtained from Colección de
Cultivos, Laboratorio de Síntesis y Biotrans-
formación de Productos Naturales, Facultad de
Ciencias, Universidad del Bio-Bio, Chillán, Chile.
It was cultured in Hagen medium containing
CaCl2 (0.005%), KH2PO4 (0.0025%), (NH4)2HPO4
(0.025%),MgSO4.7H2O(0.015%),FeCl3(0.0012%),
malt extract (0.3%) and glucose (1%) in H2O at pH
6.5.Two set of Erlenmeyer flasks (250 ml) containing
125 ml of medium were inoculated with LSPN001
and incubated with shaking (100 rpm) at 25°C for
different periods of preculture, after which a solution
of substrate in ethanol was added.
1
Products were identified by H and 13C NMR and
by comparison with authentic samples.
Identification of biotransformation compounds
The biotransformation produced two types of prod-
ucts: modified chalcones 8–14, and flavanones 15–27
(Scheme 2 and Table II). 1H NMR of Compounds
8–14 contained two sets of doublets at δ 8.20–
7.50 ppm (Jϭ15 Hz), signals characteristic of chal-
1
cone olefinic protons. For the H NMR spectra of
products 15–24 the olefinic proton H-2 and H-3 of
the precursors were absent. These signal were
replaced by a doublet of doublets at δ 5.80 (Jϭ17,
13.8 Hz, H-3ax) and 2.8 (Jϭ17, 2.8 Hz, H-3eq)
indicating that a flavanone ring had been formed.
Incubation of chalcones 1–6 with A. niger strain
Chalcone 1–6 (300 mg) were dissolved in 3 ml
ethanol and the solution added in equal portions to
three Erlenmeyer flask culture, each containing

A. niger catalyzes the synthesis of flavonoids 163
R1
R3
O
R4
R5
8–14
R2
R6
R4
R1
O
R3
R6
R4
R5
+
A. niger
R2
R3
R5
R6
chalcones 1–6
R2
O
15–27
O
Scheme 2. Metabolites obtained from biotransformation of chalcones 1–6 with A. niger.
The modified chalcones according to their spec-
tral data were identified as:
(C-2), 119.6 (C-5″), 118.9 (C-5′), 114.3 (C-1′),
114.1 (C-4″), 112.1 (C-3′), 55.8 (2″-OMe).
2″-Methoxy-2′,5″-dihydroxy-chalcone (8): 1H
NMR (CDCl3, 300 MHz) δ/ppm 12.6 (s, 1H, OH),
8.18 (d, Jϭ15 Hz, 1H, H-3), 7.90 (dd, Jϭ7.9, 1.3
Hz, 1H, H-6′), 7.75 (d, Jϭ15.5 Hz, 1H, H-2), 7.50
(dd, Jϭ7.8, 1.65 Hz, 1H, H-5′), 7.48 (t, Jϭ8, 1.3
Hz, 1H, H-4′), 7.16 (dd, Jϭ8, 1.4 Hz, 1H, H-6″),
6.92 (d, Jϭ7.8 Hz, 1H, H-3′), 6.64 (d, Jϭ2.4
Hz,1H, H-3″), 6.58 (d, Jϭ8.8, 2.4 Hz, 1H, H-5″).
13C NMR (CDCl3, 100 MHz) δ/ppm 193.2 (C-1),
163.4 (C-4″), 160.3 (C-2″), 158.3 (C-2′), 144.5
(C-3), 133.1 (C-4′), 130.5 (C-6′), 129.1 (C_6″),
121.4 (CϪ2), 120.9 (C-5′), 116.5 (C-1″), 114.3
(C-1′), 111.8 (C-3′), 105.6 (C-5″), 98.3 (C-3″), 55.5
(2″-OMe).
2′,3″,4″-trihydroxy-chalcone (11): 1H NMR
(CDCl3, 300 MHz) δ/ppm 8.08 (dd, Jϭ7.9, 1.7,
1H, H-6′), 7.81 (d, Jϭ15.3 Hz, 1H, H-2), 7.65 (d,
Jϭ15.3 Hz, 1H, H-3), 7.49 (dt, Jϭ7.4, 1.7 Hz, 1H,
H-4′), 7.22 (d, Jϭ2.3 Hz, 1H, H-2″), 7.15 (dd,
Jϭ8.3, 2.3 Hz, 1H, H-6″), 6.94 (d, Jϭ8.3, 1.4 Hz,
1H, H-5′), 6.92 (d, Jϭ8.3 Hz, 1H, H-3′), 6.83 (d,
Jϭ8.3 Hz, H-5″). 13C NMR (CDCl3, 100 MHz) δ/
ppm 193.1 (C-1), 158.1 (C-2′), 151.0 (C-3″), 148.8
(C-4″), 143.8 (C-3), 132.8 (C-4′), 130.9 (C-1″),
129.9 (C-6′), 122.7 (C-6″), 121.2 (C-2), 119.6
(C-5′), 114.6 (C-1′), 110.9 (C-5″), 110.5 (C-3′),
109.9 (C-2″),
5″-methoxy-2′,4″-dihydroxy-chalcone (12): 1H
NMR(CDCl3,300MHz)δ/ppm8.21(d,Jϭ15.5Hz,
1H, H-3), 8.1 (d, Jϭ8.6 Hz, 1H, H-6′), 7.90 (d,
Jϭ15.5 Hz, 1H, H-2), 7.11 (d, Jϭ8.1 Hz, 1H,
H-6″), 6.87 (d, Jϭ7.7 Hz, 1H, H-4″), 6.68 (dd,
Jϭ8.1, 7.2 Hz, 1H, H-5″), 6.49 (m, 1H, H-5′), 6.47
(m, 1H, H-3′). 13C NMR (CDCl3, 100 MHz) δ/ppm
191.9 (C-1), 161.4 (C-3″), 161.2 (C-5″), 157.6
(C-2′), 143.7 (C-3), 137.2 (C-1″), 131.4 (C-6′),
130.2 (C-4′), 120.6 (C-2), 120.2 (C-5′), 113.9
(C-1′), 111.8 (C-3′), 106. 7 (C-6″), 106.5 (C-2″),
102.6 (C-4″), 55.6 (5″-OMe).
4′,2″-dimethoxy-2′,3″-dihydroxy-chalcone (13):
1H NMR (CDCl3, 300 MHz) δ 8.08 (d, Jϭ15.2 Hz,
1H, H-2), 7.88 (d, Jϭ8.8 Hz, 1H, H-6″), 7.69 (d,
Jϭ15.2 Hz, 1H, H-3), 7.50 (d, Jϭ8.6 Hz, 1H,
H-6′), 6.39 (dd, Jϭ8.8, 2.4 Hz, 1H, H-5′), 6.36 (dd,
Jϭ8.6, 2.5 Hz, 1H, H-5″), 6.34 (d, Jϭ2.4 Hz, 1H,
H-3″), 6.27 (d, Jϭ2.4 Hz, 1H, H-3′). 13C NMR
(CDCl3, 100 MHz) δ/ppm 192.4 (C-1), 166.8 (C-4′),
166.5 (C-2′), 153.2 (C-2″), 148.8 (C-3″), 144.1
(C-3), 131.5 (C-6′), 129.2 (C-1″), 123.4 (C-6″),
121.8 (C-2), 119.6 (C-5″), 114.4 (C-1′), 114.1
(C-4″), 108.0 (C-5′), 101.2 (C-3′), 54.8 (4′-OMe).
1
2″-methoxy-2′,3′,5″-trihydroxychalcone (9): H
NMR (CDCl3,300 MHz) δ/ppm 12.9 (s,1H,OH-2′),
8.15 (d, Jϭ15.5 Hz, 1H, H-3), 7.80 (d, Jϭ15.5 Hz,
1H, H-2), 7.32 (dd, Jϭ8,1.4 Hz, 1H, H-6′), 7.16
(dd, Jϭ8, 1.4 Hz, 1H, H-6″), 7.08 (dd, Jϭ8,1.4 Hz,
1H, H-4′), 6.78 (t, Jϭ8 Hz, 1H, H-5′), 6.64 (d,
Jϭ2.4 Hz, 1H, H-3″), 6.59 (dd, Jϭ8.8, 2.4 Hz, 1H,
H-5″), 5.98 (s, 1H, OH-5″). 13C NMR (CDCl3,
100 MHz) δ/ppm 192.1 (C-1), 163.2 (C-4″), 159.9
(C-2″), 156.7 (C-2′), 148.1 (C-3′), 144.3 (C-3),
129.3 (C-6″), 122.1 (C-2), 121.5 (C-4′), 121.2
(C-6′), 121.0 (C-1′), 119.4 (C-5′), 116.3 (C-1″),
104.3 (C-5″), 99.8 (C-3″), 56.4 (2″-OMe).
2″-Methoxy-2′,3′-dihydroxy-chalcone (10): 1H
NMR(CDCl3,300MHz)δ/ppm8.20(d,Jϭ15.5Hz,
1H, H-3), 8.10 (dd, Jϭ8, 1.4 Hz, 1H, H-6′), 7.90
(d, Jϭ15.5 Hz, 1H, H-2), 7.52 (t, Jϭ8, 1.4 Hz, 1H,
H-4′), 7.02 (t, Jϭ8, 1H, H-5″), 6.98 (dt, Jϭ8, 1.4,
1H, H-5′), 6.96 (dd, Jϭ8, 1.4 Hz, 1H, H-4″), 6.93
(d, Jϭ7.6 Hz, 1H, H-3′). 13C NMR (CDCl3,
100 MHz) δ/ppm 192.6 (C-1), 157.1 (C-2′), 153.2
(C-2″), 148.8 (C-3″), 144.9 (C-3), 133.2 (C-4′),
130.6 (C-6′), 129.4 (C-1″), 123.4 (C-6″), 120.6

164 J. Alarcón et al.
Table II. Substitution patterns of chalcones and flavanones obtained by biotransformation of chalcones 1–6.
R1
R4
O
3
2'
2''
R5
R2
3'
1
3''
2
R34'
6'
4''
R6
5''
5'
R7
Precursor
Products
R1
R2
R3
R4
R5
R6
R7
Yield (%)
1
8
9
OH
OH
OH
OH
OH
OH
OH
H
OH
H
H
H
H
H
H
H
H
OMe
OH
OMe
OMe
OMe
H
H
OMe
OH
H
H
OH
OH
H
OH
H
H
H
H
H
OMe
H
H
25
22
18
21
16
19
23
2
3
4
5
6
10
11
12
13
14
OH
OH
OH
OH
H
H
H
H
OH
R4
R3
R5
4'
R1
1'
R2
O
R6
2
3
4
6
5
O
Precursor
Products
R1
R2
R3
R4
R5
R6
Yield (%)
1
15
16
17
18
19
20
21
22
23
24
25
26
27
H
H
O
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
H
H
H
OMe
OMe
OMe
OH
H
H
H
OMe
OH
OH
H
H
H
H
H
H
H
H
OMe
OMe
H
H
H
27
16
14
12
11
16
14
18
16
17
10
20
18
2
3
4
5
6
OMe
OH
OMe
OH
OMe
OH
OMe
OH
H
H
OMe
OH
H
H
H
H
OMe
OMe
H
H
156.6 (C-2′),136.5 (C-7),131.7 (C-6′),129.8(C-1′),
126.0 (C-5), 122.3 (C-6), 121.2 (C-10), 119,6
(C-8), 106,7 (C-5′), 102.9 (C-3′),76.0 (C-2), 55.8
(O-CH3) 55.4 (O-CH3), 44.8 (C-3).
2′,4′,2″,4″-tetrahydroxy-chalcone (14): 7.90 (dt,
Jϭ8.1, 8 Hz, 1H, H-5), 7.56 (dt, Jϭ8, 1.6 Hz, 1H,
H-7), 7.10 (dt, Jϭ8, 1.6 Hz, 1H, H-6), 7.06 (dd,
Jϭ8,1.7 Hz, 1H, H-8), 5.81 (dd, Jϭ13.8, 2.8 Hz,
1H, H-2), 3.14 (dd, Jϭ17, 13.8 Hz, 1H, H-3ax),
2.76 (dd, Jϭ17, 2.8 Hz, 1H, H-3eq). 13C NMR
(CDCl3, 100 MHz) δ/ppm 193.2 (C-1), 167.2
(C-4′), 166.2 (C-2′), 162.8 (C-2″), 160.3 (C-4″),
144.6 (C-3), 131.5 (C-6′), 130.8 (C-6″), 119.8
(C-2), 117.3 (C-1″), 114.3 (C-1′), 108.7 (C-54′),
105.3 (C-5″), 101.3 (C-3′), 98.4 (C-3″).
1
2´-methoxy-4′-hydroxyflavanone (16): H NMR
(CDCl3, 300 MHz) δ/ppm 7.91 (dt, Jϭ8, 1.8 H-5),
7.56 (dt, Jϭ8, 1.6 Hz, H-7), 7.11 (dt, Jϭ8, 1.6 Hz,
H-6), 7.08 (dd, Jϭ8, 1.7 Hz, H-8), 6.93 (d, Jϭ8.4,
H-6′), 6.87 (d, Jϭ7.1, H-5′), 5.80 (dd, Jϭ13.8,
2.8 Hz, H-2), 3.16 (dd, Jϭ17, 3.8 Hz, H-3ax), 2.72
(dd, Jϭ17, 2.8 Hz, H-3eq). 13C NMR (CDCl3,
100 MHz) δ/ppm 194 (C-4), 163 (C-9), 158.6
(C-4′), 156.6 (C-2′), 137.5 (C-7), 131.7 (C-6′), 130
(C-1′), 126.0 (C-5), 121.3 (C-6), 121.2 (C-10), 119
(C-8), 106 (C-5′), 102.7 (C-3′), 76 (C-2), 56.5
(O-CH3), 44.9 (C-3).
The flavanones according their spectral data were
identified as:
2′,4′-dimethoxyflavanone(15):1HNMR(CDCl3,
300 MHz) δ/ppm 7.90 (dt, Jϭ8, 1.8 H-5), 7.56 (dt,
Jϭ8, 1.6 Hz, H-7), 7.10 (dt, Jϭ8, 1.6 Hz, H-6),
7.06 (dd, Jϭ8, 1.7 Hz, H-8), 6.93 (d, Jϭ8.4, H-6′),
6.87 (d, Jϭ7.1, H-5′), 5.81 (dd, Jϭ13.8, 2.8 Hz,
H-2), 3.14 (dd, Jϭ17, 3.8 Hz, H-3ax), 2.76 (dd,
Jϭ17, 2.8 Hz, H-3eq). 13C NMR (CDCl3, 100
MHz) δ/ppm 194.3 (C-4),163.0 (C-9),158.8 (C-4′),
4′,8-dihydroxy-2′-methoxyflavanone (17): 1H
NMR (CDCl3, 300 MHz) δ/ppm 7.49 (dd,
Jϭ8,1.5 Hz, 1H, H-5), 7.16 (dd, Jϭ8, 1.4 Hz, 1H,
H-7), 6.97 (t, Jϭ8 Hz, 1H, H-6), 6.92 (d, Jϭ8.4,
H-6′), 6.63 (d, Jϭ2.4 Hz, 1H, H-3′), 6.58 (dd,

A. niger catalyzes the synthesis of flavonoids 165
126.7 (C-5), 121.5 (C-10), 122.1 (C-6), 119.0
(C-8), 118.1 (C-6′), 115.3 (C-5′), 114.4 (C-2′), 78.8
(C-2), 42.2 (C-3).
Jϭ8.8, 2.4 Hz, 1H, H-5´), 5.59 (s, 1H, 8-OH), 5.53
(dd, Jϭ13, 3 Hz, 1H, H-2), 3.87 (s, 3H, OCH3),
3.16 (dd, Jϭ13, 17 Hz, 1H, H-3ax), 2.89 (dd, Jϭ17,
3 Hz, 1H, H-3eq). 13C NMR (CDCl3, 100 MHz) δ/
ppm 195.8 (C-4), 158.6 (C-9), 157.7 (C.4′), 154.2
(C-2′), 129 (C-6′), 128.1 (C-5), 121.3 (C-10), 117.2
(C-1′), 111.3 (C-6), 106.2 (C-5′)105.2 (C-3′), (C-7),
(C-8), 76.3 (C-2), 53.2 (OCH3), 41.8 (C-3).
3′,5′-dimethoxyflavanone(22):1HNMR(CDCl3,
300 MHz) δ/ppm 7.89 (1H, dd, Jϭ8,1.7 Hz, H-5),
7.60 (1H, dt, Jϭ8,1.7 Hz, H-7), 7.07 (1H, dt, Jϭ8,
1.7 Hz, H-6), 7.03 (1H, dt, Jϭ8, 1.7 Hz, H-8), 5.30
(dd, Jϭ12,3.5 Hz, H-2), 3.04 (dd, Jϭ17, 12 Hz,
H-3ax), 2.75 (dd, Jϭ17, 3.5 Hz, H-3eq). 13C NMR
(CDCl3, 100 MHz) δ/ppm 195.2 (C-4), 163.3 (C-9),
157.3 (C-5′), 156.8 (C-3′), 136.7 (C-7), 130.6
(C-1′), 126.8 (C-5), 122.2 (C-6), 122.0 (C-10),
119.1 (C-8), 118.8 (C-4′), 114.6 (C-2′), 112.8
(C-6′), 77.8 (C-2), 56.8 (-OCH3), 56.4 (-OCH3),
44.7 (C-3).
2′,3′-dimethoxyflavanone(18):1HNMR(CDCl3,
300 MHz) δ/ppm 7.88 (dd, Jϭ8, 1.7, H-5), 7.54
(dt, Jϭ8.0, 1.7 Hz, H-7), 7.05 (dd, Jϭ8, 1.7 Hz,
H-8), 7.07 (dt, Jϭ8, 1.7 Hz, H-6), 7.05 (dd,
Jϭ8,1.6 Hz, H-6′), 6.99 (t, Jϭ8 Hz, H-5′), 6.87
(dd, Jϭ8, 1.6, H-4′), 5.77 (dd, Jϭ13.8, 2.8 Hz,
H-2), 3.13 (dd, Jϭ17, 13.8 Hz, H-3ax), 2.79 (dd,
Jϭ17,2.8Hz,H-3eq).13CNMR(CDCl3,100MHz)
δ/ppm 194.0 (C-4), 164.0 (C-9), 152.0 (C-3′), 146.0
(C-2′), 137.0 (C-7), 133.0 (C-1′), 127.0 (C-5),
125.3 (C-5′), 122.3 (C-6), 121.3 (C-10), 119.5
(C-8), 117.8 (C-6′), 117.5 (C-4′), 76.0 (C-2), 61.4
(-OCH3 ϫ2), 44.5 (C-3).
3′-hydroxy-5′-methoxy-flavanone (23): 1H NMR
(CDCl3, 300 MHz) δ/ppm 7.88 (1H, dd, Jϭ8,1.7
Hz, H-5), 7.61 (1H, dt, Jϭ8,1.7 Hz, H-7), 7.08
(1H, dt, Jϭ8, 1.7 Hz, H-6), 7.03 (1H, dt, Jϭ8, 1.7
Hz, H-8), 13C NMR (CDCl3, 100 MHz) δ 197.1
(C-4), 161.6 (C-9), 157.3 (C-5′), 156.9 (C-3′),
136.7 (C-7), 130.2 (C-1′), 127,1 (C-5), 122.0
(C-10) 121.8 (C-6), 120.2 (C-8), 118.9 (C-4′),
114.5 (C-2′), 1121.5 (C-6´), 80.2 (C-2), 56.4
(-OCH3), 44.7 (C-3).
1
3′-hydroxy-2′-methoxyflavanone (19): H NMR
(CDCl3, 300 MHz) δ/ppm 7.88 (dd, Jϭ8, 1.7 Hz,
H-5), 7.54 (dt, Jϭ8, 1.7 Hz, H-7), 7.07 (dt, Jϭ8,
1.7 Hz, H-6), 7.05 (dd, Jϭ8, 1.7 Hz, H-8, H-6′),
6.99 (t, Jϭ8 Hz, H-5′), 6.87 (dd, Jϭ8, 1.6 Hz,
H-4′), 5.77 (dd, Jϭ13.8, 2.8 Hz, H-2), 3.13 (dd,
Jϭ17, 13.8 Hz, H-3ax), 2.79 (dd, Jϭ17, 2.8 Hz,
H-3eq). 13C NMR (CDCl3, 100 MHz) δ/ppm 194.5
(C-4), 151.8 (C-9), 151.2 (C-3′), 146.3 (C-2′),
137.0 (C-7),133.0 (C-1′),127.0 (C-5),125.3 (C-5′),
122.5 (C-10), 122.3 (C-6), 119.6 (C-8), 118.7
(C-6′), 117.9 (C-4′), 75.0 (C-2), 61.4 (-OCH3),
44.3 (C-3).
7,2′,3′-trimethoxyflavanone (24): 1H NMR
(CDCl3, 300 MHz) δ/ppm 7.83 (d, Jϭ8.9 Hz, H-5),
7.32 (dd, Jϭ7.8, 1.2 Hz, H-6′), 7.17 (t, Jϭ7.8 Hz,
H-5′), 7.06 (dd, Jϭ7.8, 1.2 Hz, H-4′), 6.72 (dd,
Jϭ8.9, 2.4 Hz, H-6), 6.56 (d, Jϭ2.4 Hz, H-8). 13
C
NMR (CDCl3, 100 MHz) δ/ppm 196.8 (C-4), 157.1
(C-7), 154.5 (C-9), 153.1 (C-3′), 146.2 (C-2′),
132.8 (C-1´), 128.1 (C-5), 125.4 (C-5′), 120.5
(C-10), 116.9 (C-6′), 116.8 (C-4′), 116.0 (C-6),
103.2 (C-8), 79.1 (C-2), 55.2 (-OCH3 ϫ2), 54.2
(-OCH3), 45.7 (C-3).
3′,4′-dimethoxy-flavanone(20):1HNMR(CDCl3,
300 MHz) δ/ppm 7.91 (dd, Jϭ8,1.8 Hz, H-5), 7.58
(dt, Jϭ8,1.7 Hz, H-7), 7.14 (dt, Jϭ8.0, 1.6 Hz,
H-6), 7.04 (dd, Jϭ8, 1.7 Hz, H-8), 7.0 (dd, Jϭ8.5,
1.7 Hz, H-6′), 6.99 (d, Jϭ1.7 Hz, H-2′), 6.90
(d, Jϭ8.5 Hz, H-5′), 5.34 (dd, Jϭ13.3, 2.6 Hz,
H-2), 3.0 (dd, Jϭ16.7, 13.3 Hz, H-3ax), 2.77 (dd,
Jϭ16.7, 2.6 Hz, H-3eq). 13C NMR (CDCl3,
100 MHz) δ/ppm 189.4 (C-4), 161.6 (C-9), 149.6
(C-4′), 149.4 (C-3′), 136.2 (C-7), 131.2 (C-1′),
127.1 (C-5), 121.6 (C-6), 121.0 (C-10), 119.9
(C-6′), 118.2 (C-8), 111.4 (C-5′), 109.6 (C-2′), 79.2
(C-2), 56.14 (-OCH3), 56.05 (-OCH3), 45.6 (C-3).
3′,4′-dihydroxyflavanone (21): 1H NMR (CDCl3,
300 MHz) δ/ppm 7.89 (dd, Jϭ8, 1.7 Hz, H-5), 7.55
(dt, Jϭ7.9,1.7 Hz, H-7), 7.10 (dt, Jϭ8,1.6 Hz,
H-6), 7.02 (dd, Jϭ8,1.7 Hz, H-8), 6.87 (s, H-2′),
6.74 (s, H-5′), 6.74 (s, H-6′), 5.42 (dd, Jϭ12.5,
3.0 Hz, H-2), 3.28 (dd, Jϭ17,12 Hz, H-3ax), 2.73
(dd, Jϭ17.1,3 Hz, H-3eq). 13C NMR (CDCl3,
100 MHz) δ/ppm 197.3 (C-4), 162.0 (C-9), 145.8
(C-4′), 145.2 (C-3′), 137.0 (C-7), 129.1 (C-1′),
1
3′-hydroxy, 7,2′-dimethoxyflavanone (25): H
NMR (CDCl3, 300 MHz) δ/ppm 7.81 (d, Jϭ8.8
Hz, H-5), 7.12 (dd, Jϭ8,1.6 Hz, H-6′), 7.02 (t,
Jϭ8 Hz, H-5′), 6.89 (d, Jϭ8 Hz, H-4′), 6.58 (dd,
Jϭ8.5, 2.4 HZ, H-6), 6.42 (d, Jϭ2.4 Hz, H-8). 13
C
NMR (CDCl3, 100 MHz) δ/ppm 197.2 (C-4), 156.3
(C-7), 155.4 (C-9), 154.1 (C-3′), 146.4 (C-2′),
120
1
8
14
16
17
9
100
80
60
40
20
0
0
50
100
Time (h)
150
200
Figure 1.Time-course of chalcone 1 metabolism and metabolites
8, 9, 14, 16, and 17 formation after incubation with A. niger.

166 J. Alarcón et al.
OMe
O
O
OMe
O
OMe
OH
a
O
O
15
OH
OH
OMe
OMe
OMe
OH
b
O
OMe
Chalcone 1
O
O
16
OMe
O
OH
OH
17
8
OH
OH
9
OH
Scheme 3. Biocatalytic pathway for the conversion of chalcone 1 to flavanone and altered chalcone by A. niger.
the amount of metabolites 8 and 15 subsequently
gradually decreased, with a proportionate increase
in metabolites 9, 16, and 17. As flavanone 15 is the
cyclization product of 1, flavanone 16 could be
formed through two routes; one involving demeth-
ylation of metabolite 15 and the second, cyclization
of metabolite 8. Chalcone 9 is the hydroxylation
product of 8, which is then converted to flavanone
17, which appears after formation chalcone 9
(Scheme 3).
The progress of the biotransformation shows that
cyclization and demethylation are early steps in the
process, with hydroxylation occurring later. So with
a longer incubation time it was possible to obtain
more hydroxylated flavanones. The chemical oxida-
tion of chalcones has been described by Kurosawa
and Higuchi (1972).
132.6 (C-1′), 128.3 (C-5), 125.2 (C-5′), 120.6
(C-10), 116.5 (C-4′), 115.9 (C-6′), 108.8 (C-6),
103.0 (C-8), 78.7 (C-2), 55.1 (-OCH3), 54.3
(-OCH3), 46.1 (C-3).
7,2′,4′-trimethoxy-flavanone (26): 1H NMR
(CDCl3, 300 MHz) δ/ppm 8.21 (d, Jϭ8.8 Hz, H-5),
7.21 (d, Jϭ8.1 Hz, H-6′), 6.98 (dd, Jϭ8.8, 2.3 Hz,
H-6), 6.85 (d, Jϭ2.3 Hz, H-8), 6.42 (d, Jϭ2.1 Hz,
H-3′), 6.30 (dd, Jϭ9, 2.1 Hz, H-5′). 13C NMR
(CDCl3, 100 MHz) δ/ppm 196.8 (C-4), 160.2 (C-9),
158.6 (C-7), 158.3 (C-2′), 157.2 (C-4′), 128.1
(C-6′), 127.7 (C-5), 122.4 (C-10), 116.8 (C-1′),
111.1 (C-6), 107.6 (C-5′), 104.5 (C-3′), 102.9
(C-8), 78.1 (C-2), 55.1 (-OCH3), 42.3 (C-3).
2′,4′-dihydroxy-7-methoxyflavanone (27): 1H
NMR (CDCl3,300 MHz) δ/ppm 8.19 (d,Jϭ8.8 Hz,
H-5), 7.09 (d, Jϭ8.5 Hz, H-6′), 6.89 (dd, Jϭ8.8,
2.3 Hz, H-6), 6.83 (d, Jϭ2.4 Hz, H-8), 6.45(d,
Jϭ2 Hz, H-3′), 6.42 (dd, Jϭ8.5,2 Hz, H-5′), 2.86
(dd, Jϭ17, 2.5 Hz, H-3eq). 13C NMR (CDCl3,
100 MHz) δ/ppm 196.7 (C-4), 160.1 (C-7), 159.8
(C-9), 157.1 (C-4′), 155.4 (C-2′), 128.0 (C-6′),
127.5 (C-5), 122.4 (C-10), 116.6 (C-1′), 111.1
(C-6), 107.8 (C-5′), 104.3 (C-3′), 102.6 (C-8), 77.5
(C-2), 55.2 (-OCH3), 41.9 (C-3).
Acknowledgements
The authors wish to thank to internal grant from
Dirección de Investigación, DIUBB N°083009-2R,
Universidad del Bio Bio, Chillán, Chile.
Declaration of interest: The authors report no
conflicts of interest. The authors alone are respon-
sible for the content and writing of the paper.
Biotransformation pathway
Having identified the chemical structure of the
metabolites, the biotransformation kinetics were
evaluated, taking as an example the biotransforma-
tion of chalcone 1. Samples were collected each
12 h and the reaction monitored by HPLC which
gave information about changes in the ratio of prod-
ucts during the course of the process (Figure 1). In
the first 24 h only two products were formed:
metabolites 8 and 15. The maximum levels of 8
(32%) and 15 (19%) were achieved at 72 h. On the
third day metabolites 9, 16, and 17 appeared, and
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