Fungal metabolism of naphthoflavones

Article abstract of DOI:10.1016/j.molcatb.2015.04.007

Naphthoflavones (benzoflavones) are synthetic flavonoids commonly used in drug metabolism studies as selective activators or inhibitors of cytochrome P-450 enzymes. Nowadays they are also used as a component of food supplements for body builders. There is

Full text of DOI:10.1016/j.molcatb.2015.04.007

Accepted Manuscript
Title: Fungal metabolism of naphthoflavones. Effect of
substitution in the B-ring
´
Author: Jarosław Popłonski Sandra Sordon Tomasz Tronina
´
Agnieszka Bartmanska Ewa Huszcza
PII:
S1381-1177(15)00105-8
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2015.04.007
MOLCAB 3147
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date:
Revised date:
Accepted date:
2-2-2015
1-4-2015
10-4-2015
´
´
Please cite this article as: J. Poplonski, S. Sordon, T. Tronina, A. Bartmanska,
E. Huszcza, Fungal metabolism of naphthoflavones. Effect of substitution
in the B-ring, Journal of Molecular Catalysis B: Enzymatic (2015),
http://dx.doi.org/10.1016/j.molcatb.2015.04.007
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Fungal metabolism of twelve naphthoflavone compounds was investigated
Effect of different substituent in B-ring was evaluated
Products of 4’-hydroxylation and 4’-O demethylation have been obtained
Aspergillus and Verticilium strains were most effective biocatalyst
Fungal metabolism do not coincides with human or rodent metabolism
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Fungal metabolism of naphthoflavones. Effect of substitution in the B-ring.
Jarosław Popłoński, Sandra Sordon, Tomasz Tronina, Agnieszka Bartmańska, Ewa Huszcza
Department of Chemistry, Wrocław University of Environmental and Life Sciences, Norwida 25,
50-375 Wrocław, Poland
Corresponding author: Jarosław Popłoński, e-mail: jaroslaw.poplonski@up.wroc.pl
Abstract
Naphthoflavones (benzoflavones) are synthetic flavonoids commonly used in drug
metabolism studies as selective activators or inhibitors of cytochrome P-450 enzymes. Nowadays
they are also used as a component of food supplements for body builders. There is no data
regarding naphthoflavone microbial metabolism. In the present studies sixty-three fungal strains
have been screened for their ability to transform α-naphthoflavone (7,8-benzoflavone) or β-
naphthoflavone (5,6-benzoflavone). Five strains belonging to the genera Penicillium,
Cladosporium, Aspergillus and Verticillium transformed α-naphthoflavone and β-naphthoflavone to
the corresponding 4’-hydroxy derivatives. These selected fungi have been used in a further study on
biotransformation of naphthoflavones with a differently substituted B-ring. Only 4’-methoxy
derivatives have been transformed to the related 4’-hydroxy products. Selected strains are good
biocatalysts to obtain 4’-hydroxy naphthoflavones in the one step reaction.
keywords: naphthoflavones, benzoflavones, biotransformation, hydroxylation, demethylation, fungi
1. Introduction
Naphthoflavones are synthetic derivatives of flavones, a group of naturally-occurring
flavonoids widely distributed in the plant world. At the beginning of the 1970s α-naphthoflavone
(α-NF, 7,8-benzoflavone, 1) was found to be in the spotlight of researchers because of its effect on
the metabolism of carcinogens [1]. The subsequent studies showed that this compound inhibits the
activation of procancerogenic polycyclic hydrocarbons and activates aflatoxin B1 to mutagenic
metabolites in vitro [2,3]. Interestingly, β-naphthoflavone (β-NF, 5,6-benzoflavone, 2), an isomer of
α-NF (1), also affects the activity of microsomal polycyclic hydrocarbon hydroxylases, however, it
acts as an inducer of these enzymes activities in vitro [4, 5]. A few years later Kellis and coworkers
discovered that α-NF (1) is a potent inhibitor of aromatase, the enzyme responsible for
transformations of androgens into estrogens [6]. They also showed that β-NF (2) has no inhibitory
activity. Nowadays the properties of these two naphthoflavones, that rely on the selective
modulation of the activity of particular cytochrome P-450 monooxigenases, are widely applied in
drug metabolism studies [7, 8].
The biological activity of naphtoflavones was tested in parallel with their metabolism (Table
1). The much greater interest in α-NF (1) compared to β-NF (2) may be associated with the fact that
some metabolites of α-NF (1) have a higher inhibitory potency [9, 10]. Further studies on the
metabolism of naphtoflavones seem to be mandatory especially due to the action of aromatase-
inhibiting α-NF (1) that is used as a component of dietary supplements sold for bodybuilding. In a
consequence humans may be exposed to gut microbial metabolites of α-NF (1) and such may differ
from those already identified.
For years whole cell microorganism biotransformations have been used to mimic
mammalian metabolism or to obtain human particular metabolites at reasonable quantities using
simple methods [11]. To our best knowledge there is no data concerning naphthoflavones microbial
metabolism, therefore we decided to study this phenomenon.
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Table 1. Comparative data from different studies on α-NF (1) and β-NF (2) metabolism.
Metabolites of
α-NF (1)
β-NF (2)
Long-Evans rat liver
microsomes and reconstituted
Cytochrome P-450 System
[12]
5,6-oxide, 5,6-dihydrodiol, 7,8-
dihydrodiol, 6-hydroxy,7-
hydroxy, 9-hydroxy and one
unknown compound
5,6-dihydrodiol, 7,8-
dihydrodiol, 8-hydroxy, 5-
hydroxy and five unknown
compounds
rat liver microsomes and
reconstituted selection of
Cytochrome P-450 System
[13]
5,6-oxide, 5,6-dihydrodiol, 7,8-
dihydrodiol, 6-hydroxy and
one unknown metabolite
Charles River CD rat liver
microsomes [14]
5,6-oxide, 5,6-dihydrodiol,6-
hydroxy, 9-hydroxy
Charles River CD rat, mouse, 5,6-oxide, 5,6-dihydrodiol, 7,8-
rabbit and Syrian golden
hamster liver microsomes [15]
dihydrodiol, 6-hydroxy,7-
hydroxy, 9-hydroxy
Sprague Dawley rat liver
microsomes [16]
5,6-oxide, 5,6-dihydrodiol,
9,10-dihydrodiol, 6-hydroxy
hepatic microsomes from the
marine fish Stenotomus
versicolor [17]
unidentyified dihydrodiol
5,6-oxide, 5,6-
dihydrodiol,9,10-dihydrodiol,
5-hydroxy
liver microsomes [18]
Syrian golden hamster liver
microsomes [19]
6-hydroxy,7-hydroxy and six
other metabolites
5,6-dihydrodiol, 7,8-
dihydrodiol, 6-hydroxy,7-
hydroxy
Syrian golden hamster
hepatocytes [20]
Syrian golden hamster liver
and kidney microsomes [21]
5,6- dihydrodiol, two isomeric
hydroxyderivatives
5,6-oxide, 5,6-dihydrodiol, 7,8-
dihydrodiol, 6-hydroxy,7-
hydroxy
human liver microsomes [22]
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Table 1. Comparative data from different studies on α-NF (1) and β-NF (2) metabolism.
Metabolites of
recombinant human CYP3A4
and NADPH:CYP reductase
with cytochrome b5 [23]
5,6-oxide, 7,8-dihydrodiol
2. Experimental
2.1 Materials
α-NF (1), β-NF (2), 1-hydroxy-2-acetonaphthone, 2-hydroxy-1-acetonaphthone, 2-
methoxybenzaldehyde, 3-methoxybenzaldehyde, 4-methoxybenzaldehyde, 4-methylbenzaldehyde
and 4-fluorbenzaldehyde were purchased from Sigma Aldrich. Ten naphthoflavone derivatives were
obtained from corresponding hydroxy-acetonaphthones and benzaldehydes as a starting materials.
Two stage synthesis was used in which first step was base aldol condensation of hydroxy-
acetonaphthones and benzaldehydes to afford chalcones. Second step was oxidative-cyclisation
of chalcones with iodine in DMSO to afford flavones (Scheme 1) [24].
Scheme 1: Synthesis of naphthoflavone derivatives (a)KOH, EtOH; b)I2, DMSO)
The structures of the obtained compounds were confirmed by NMR spectra analysis.
Spectroscopic data of methoxy-derivatives (1a-c, 2a-c) are with agreement with literature data [24].
Data of other compounds (1d-e, 2d-e) are as follows:
1
4’-methyl-α-naphthoflavone (1d). H NMR (600 MHz, DMSO-d6) δ: 2.39 (3H, s, H-Me),
7.11 (1H, s, H-3), 7.40 (2H, m, H-3’, H-5’), 7.80 (2H, m, H-8, H-9), 7.97 (1H, m, H-5), 7.90 (1H,
13
m, H-6), 8.08 (2H, m, H-2’, H-6’), 8.10 (1H, m, H-7), 8.63 (1H, m, H-10). C NMR (150 MHz,
DMSO-d6) δ: 21.05 (C-Me), 107.45 (C-3), 119.59 (C-13), 119.97 (C-5), 122.23 (C-10), 123.47 (C-
11), 125.29 (C-6), 126.24 (C-2’, C-6’), 127.66 (C-9), 128.22 (C-7), 128.36 (C-1’), 129.48 (C-8),
129.79 (C-3’, C-5’), 141.91 (C-4’), 135.40 (C-14), 152.65 (C-12), 162.04 (C-2), 176.76 (C-4).
4’-fluoro-α-naphthoflavone (1e). 1H NMR (600 MHz, DMSO-d6) δ: 7.21 (1H, s, H-3), 7.47
(2H, m, H-3’, H-5’), 7.83 (2H, m, H-8, H-9), 7.95 (1H, m, H-6), 8.00 (1H, m, H-5), 8.12 (1H, m, H-
7), 8.33 (2H, m, H-2’, H-6’), 8.69 (1H, m, H-10); 13C NMR (150 MHz, DMSO-d6) δ: 108.07 (C-3),
116.29 (d, J = 22.00 Hz, C-3’, C-5’), 119.53 (C-13), 119.93 (C-5), 122.29 (C-10), 123.43 (C-11),
125.41 (C-6), 127.67 (C-9), 127.77 (d, J = 2.90 Hz, C-1’), 128.22 (C-7), 129.07 (d, J = 9.00 Hz, C-
2’, C-6’), 129.55 (C-8), 135.44 (C-14), 152.74 (C-12), 161.05 (C-2), 164.09 (d, J = 250.3 Hz, C-4’),
176.74 (C-4).
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1
4’-methyl-β-naphthoflavone (2d). H NMR (600 MHz, DMSO-d6) δ: 2.41 (3H, s, H-Me),
7.16 (1H, s, H-3), 7.42 (2H, m, H-3’, H-5’), 7.69 (1H, m, H-7), 7.78 (1H, m, H-6), 7.90 (1H, d, J =
9.0 Hz, H-10), 7.97 (1H, m, H-8), 8.06 (2H, m, H-2’, H-6’), 8.36 (1H, d, J = 9.0 Hz, H-9), 9.97 (1H,
13
m, H-5). C NMR (150 MHz, DMSO-d6) δ: 21.05 (C-Me), 109.12 (C-3), 116.18 (C-12), 118.16
(C-10), 126.08 (C-5), 126.11 (C-2’, C-6’), 126.55 (C-7), 127.51 (C-1’), 128.57 (C-8), 129.01 (C-6),
130.36 (C-13), 129.69 (C-14), 129.75 (C-3’, C-5’), 135.69 (C-9), 141.85 (C-4’), 156.94 (C-11),
160.39 (C-2), 179.18 (C-4).
4’-fluoro-β-naphthoflavone (2e). 1H NMR (600 MHz, DMSO-d6) δ: 7.20 (1H, s, H-3), 7.45
(2H, m, H-3’, H-5’), 7.68 (1H, m, H-7), 7.78 (1H, m, H-6), 7.89 (1H, m, H-10), 8.10 (1H, m, H-6”),
8.23 (2H, m, H-2’, H-6’), 8.36 (1H, m, H-9), 9.95 (1H, m, H-5). 13C NMR (150 MHz, DMSO-d6) δ:
109.65 (C-3), 116.11 (C-12), 116.25 (d, J = 22.0, C3’, C-5’), 118.13 (C-10), 126.04 (C-5), 126.60
(C-7), 127.30 (d, J = 2.9 Hz, C-1’), 128.58 (C-8), 128.85 (d, J = 9.0 Hz, C-2’, C-6’), 129.06 (C-6),
129.63 (C-14), 130.37 (C-13), 135.78 (C-9), 156.90 (C-11), 160.35 (C-2), 164.07 (d, J = 250.4, C-
4’), 179.15 (C-4).
2.2. Analytic procedures
TLC was carried out on Merck silica gel 60, F254 (0.2 mm thick) plates using
chloroform/methanol (9:1 v/v) or hexane/acetone/2-propanol (8:1:1 v/v) as developing solvents.
After drying, spots were visualized under short-and long-wavelength UV light, the plates were then
sprayed with methanol-sulfuric acid (1:1 v/v) solution. HPLC was performed on a Waters 2695
Alliance instrument with a photodiode array detector Waters 2996 (detection from 220 to 500 nm
wavelength) using the analytical HPLC column Cosmosil Cholester 5 μm (4.6 x 250 mm) at the
flow rate of 1 ml/min. A linear solvent gradient from 45% to 95% aq MeOH containing 0.05%
13
HCOOH of over 39 min was used. 1H NMR, 13C NMR, DEPT 135°, 1H–1H NMR (COSY),1H– C
NMR (HMQC and HMBC)) were recorded on a DRX Bruker Avance TM 600 (600 MHz)
instrument in DMSO-d6, UV spectra were run on a Spectrofotometer Cintra 303, GBC, in methanol.
Positive and negative-ion ESI-MS spectra were taken on a Bruker microTOF-Q spectrometer.
2.3. Microorganisms
Sixty-three strains of fungal cultures from the culture collection of the Department of
Chemistry at the Wrocław University of Environmental and Life Sciences (Poland) have been used
in the screening procedure. The strains used came from the Department of Pharmaceutical Biology
and Botany of the Wrocław Medical University, Poland (indexed AM), Department of Forest
Pathology of the Agricultural University of Kraków, Poland (indexed ARK), Department of
Chemistry of the Wrocław University of Environmental and Life Sciences, Poland (indexed KCh),
Department of Plant Protection of the Wrocław University of Environmental and Life Sciences,
Poland (indexed UPF). The strains used in the screening procedure are as follows: Absidia coerulea
AM 93, A. glauca AM 177, Acremoniella atra AM 12, Armillaria mellea AM 296, A. mellea AM
477, Aspergillus sp. AM 14, A. glaucus AM 211, A. nidulans AM 243, A. ochraceus AM 370, A.
ochraceus AM 456, A. candidus AM 386, A. niger UPF 702, A. niger UPF 709, A. niger UPF 724,
A. fumigatus UPF 703, A. versicolor UPF 703, Beauveria bassiana AM 278, Botrytis cinarea AM
235, Chalara sp. ARK 16664, Cladosporium avellaneum AM 135, Coryneum betulinum ARK
16534, Crumenulopsis sororia ARK 15940, Cunninghamella japonica AM 472, Disculina betulina
ARK 16538, Fusarium culmorum AM 10, F. culmorum AM 196, F. culmorum AM 282, F. scirpi
AM 199A, F. oxysporum AM 2, F. oxysporum AM 145, F. oxysporum UPF 727, F. tricinctum AM
395, Laetiporus sulphurens AM 525, Morteriella vinacea AM 149, M. isabellina AM 212, Mucor
hiemalis UPF 729, Penicillium albidum AM 47, P. purpurogenum AM 49, P. vermiculatum AM 13,
P. vermiculatum AM 50, P. camembertii AM 51, P. urticae AM 54, P. thomi AM 91, P. vinaceum
AM 110, P. lilacinum AM 111, P. chrysogenum AM 112, P. chermesinum AM 113, P. spinulosum
AM 114, P. frequentas AM 351, P. frequentas AM 359, P. diversum AM 388, P. notatum KCh
904, P. notatum UPF 725, Piptoporus betulinus AM 475, Pleurotus ostreatus AM 482, P. ostreatus
UPF 721, Rhizopus nigricans UPF 701, Spicaria divaricata AM 423, Stemphylium botryosum AM
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279, Syncephalastrum racemosum AM 105, Trametes versicolor AM 536, Trichothecium roseum
UPF 700, Verticillium sp. AM 424. Microorganisms were maintained on Sabouraud medium (3%
glucose and 1% peptone) agar slants at 5°C.
2.4. Conditions for biotransformations
The cultures were shaken on rotary shakers (130 speed, 6.5 amplitude) at 28°C in 100-ml
Erlenmeyer flasks with 30 ml of the medium in the screening studies and in 300-ml Erlenmeyer
flasks with 100 ml of the medium in the preparative scale transformation. In all experiments fungi
were cultivated on Sabouraud medium (3% glucose and 1% peptone). Agar slant cultures were used
to obtain the preculture, and then 3-day precultures were transferred to the main cultures media (1
ml to the 30 ml and 3 ml to the 1500 ml). Substrates were added after 5 days of culturing. Substrate
control for all naphthoflavones consisted of the substrate and a sterile growth medium incubated
without microorganisms.
In the screening studies 20 mg of substrate (1, 1a-e, 2, 2a-e) dissolved in 2 ml of DMSO
were equally distributed among 4 flasks (0,5 ml each). Biotransformations were carried out for 7
and 14 days in duplicate, acidified with 1M HCl to pH around 5 (if necessary) and then reaction
mixtures were extracted with ethyl acetate (2 x 10 ml).
In the preparative biotransformations, 60 mg of substrate (1, 1a, 2 or 2a) dissolved in 4 ml
of DMSO was equally distributed among four flasks with 5-day fungal cultures (1 ml each).
Reactions were carried out for 21 days, acidified with 1M HCl to pH around 5 (if necessary), and
then reaction mixtures were extracted with ethyl acetate (3 x 40 ml).
Extracts were dried over anhydrous magnesium sulphate and the solvent was filtered and
evaporated under vacuum. Residues were dissolved in methanol and analysed by TLC and HPLC.
2.5. Products isolation and analysis
The products of naphtoflavones biotransformation were separated by column
chromatography on silica gel 60 (230 – 400 mesh, Merck) using chloroform/methanol (9:1 v/v) as
an eluent. Products structures were elucidated by NMR and MS spectroscopy methods. Spectral
data of products are as follows:
4’-hydroxy-α-naphthoflavone (3).1H NMR (600 MHz, DMSO-d6) δ: 7.00 (2H, m, H-3’, H-
5’), 7.04 (1H, s, H-3), 7.83 (2H, m, H-8, H-9), 8.01 (1H, s, H-5), 7.95 (1H, m, H-6), 8.12 (2H, m,
13
H-2’, H-6’), 8.13 (1H, m, H-7), 8.71 (1H, m, H-10), 10.33 (1H, s, C4’-OH); C NMR (150 MHz,
DMSO-d6) δ: 105.98 (C-3), 116.07 (C-3’, C-5’), 119.51 (C-13), 120.04 (C-5), 121.70 (C-1’),
122.24 (C-10), 123.50 (C-11), 125.14 (C-6), 127.64 (C-9), 128.23 (C-7), 128.32 (C-2’, C-6’),
129.50 (C-8), 135.37 (C-14), 152.58 (C-12), 160.88 (C-4’), 162.49 (C-2), 176.64 (C-4); HRESI-MS
[M-H+] (calculated/found) (m/z 257.0990/257.0979).
4’-hydroxy-β-naphthoflavone (4).1H NMR (600 MHz, DMSO-d6) δ: 6.96 (2H, m, H-3’, H-
5’), 7.02 (1H, s, H-3), 7.67 (1H, m, H-7), 7.77 (1H, m, H-6), 7.86 (1H, m, J = 9.0 Hz, H-10), 8.01
(2H, m, H-2’, H-6’), 8.09 (1H, m, H-8), 8.34 (1H, m, J = 9.0 Hz, H-9), 9.97 (1H, m, H-5), 10.33
(1H, s, C4’-OH).13C NMR (150 MHz, DMSO-d6) δ: 107.67 (C-3), 115.98 (C-3’, C-5’), 116.06 (C-
12), 118.14 (C-10), 121.19 (C-1’), 126.14 (C-5), 126.45 (C-7), 128.14 (C-2’, C-6’), 128.54 (C-8),
129.78 (C-14), 128.91 (C-6), 130.34 (C-13), 135.47 (C-9), 156.81 (C-11), 160.78 (C-2), 160.80 (C-
4’), 179.10 (C-4); HRESI-MS [M-H+] (calculated/found) (m/z 257.0990/257.0983).
3. Results
3.1. Screening studies
Sixty-three fungal strains were screened for their ability to transform α-NF (1) and β-NF (2).
The selection of some species was done based on literature data concerning flavone
biotransformations [25]. Progress of the biotransformation was determined by TLC and HPLC
6 of 6
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analysis of culture extracts. Five fungal strains: Penicillium thomi AM 91, Cladosporium
avellaneum AM 135, Verticillium sp. AM 424, Aspergillus glaucus AM 211 and A. niger UPF 724
were capable of transforming α-NF (1) and/or β-NF (2). These microorganisms were chosen for
screening studies for their metabolic potential to transform derivatives of 1 and 2, and for further
transformations in a preparative scale.
3.2. Preparative transformations of naphtoflavones
Scale-up biotransformations of naphtoflavones: α-NF (1), β-NF (2), and ten derivatives of
these compounds (Scheme 2) afforded two products: 4’-hydroxy-α-naphthoflavone (3), 4’-hydroxy-
β-naphthoflavone (4) (Scheme 3). To obtain the highest transformation rates, we prolonged the
reaction time up to 21 days. However, the conversion rates measured by HPLC in comparison with
the results obtained in the screening tests slightly differed towards an increased yield. Also, if any
products were formed, they were observed after only one day of substrate exposition.
Scheme 2: Synthetized naphthoflavone derivatives used in the metabolism studies.
All five selected strains were able to metabolize 1 while only three were able to transform 2
(Table 2). The most effective biocatalysts were Aspergillus glaucus AM 211 and Verticillium sp.
AM 424 which transformed 1 to 3 with an isolated yield of 43% and 63%, respectively. These
strains were also the most efficient in transforming 2 to 4 with a 24% and 17% isolated yield. Out
of the ten tested derivatives of naphtoflavones 1 and 2, only two have been efficiently converted.
These were the 4'-methoxy-α-NF (1a) and 4'-methoxy-β-NF (2a).
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Scheme 3. Transformations of naphthoflavones by fungi.
One strain, Penicillium thomi AM 91, exhibited some substrate specificity because it was
able to transform only flavones with 7,8-benzo moiety, whereas Aspergillus niger UPF 724 was the
only strain capable of the O-demethylation of both 1a and 2a with isolated yields of 71% and 34%
exceeding the hydroxylation of naphthoflavones. Also the O-demethylation of 1a by Penicillium
thomi AM 91 resulted in compound 3 with a higher isolated yield (43%) then hydroxylation of 1
(24%).
Table 2. The yield of naphthoflavones metabolites obtained by selected fungal strains.
Strain
3 [%]* from
4 [%]* from
α-NF (1)
4’-OMe-α-
β-NF (2)
4’-OMe-β-
NF (1a)
traces**
71
NF (2a)
traces**
34
Aspergillus glaucus AM 211
Aspergillus niger UPF 724
43
17
36
24
traces**
9
Cladosporium avellaneum
AM 135
nd
nd
Penicillium thomi AM 91
Verticillium sp. AM 424
24
63
43
nd
traces**
17
nd
nd
*all yields are of pure isolated compounds
** traces refer to values under 2% of the conversion rate detected by HPLC
nd – not determined
8 of 8
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4. Discussion
Screening studies performed on sixty-three fungal strains resulted in the selection of five
strains and afforded two products. Biotransformations in which the conversion rate exceeded 2%
(HPLC, λ = 290 nm) were transferred to a larger scale. All the other microorganisms that produced
traces of products 3 and 4 (< 2%) belonged to the Aspergillus, Fusarium and Penicillium genus. It
is possible that optimization of the biotransformation conditions could increase its efficiency.
Comparing the data concerning the metabolism of naphtoflavones in animals, summarized in Table
1, with the results of our studies on microbial transformations, it can be seen that the enzyme
systems of mammals and fish modify naphthalene moiety, while microbial transformation products
were changed in the B-ring. Although the hydroxylation of the B-ring of unsubstituted flavones,
particularly 4’-hydroxylation, have been reported previously [25, 26], we found it interesting to test
the substrate specificity of the selected strains, especially since flavone microbial transformations in
only Aspergillus and Penicillium strains yielded positive transformation results in our preliminary
studies [27].
Further reactions with differently substituted derivatives were carried to check the impact of
additional steric hinderance in the B-ring and altered groups at position 4’ on the reaction results.
Reactions with 4’ substituted methoxy naphthoflavones 1a and 2a provided two parallel products: 3
and 4. Demethylation reactions for the flavonoid compounds with flavone or flavanone structure
have already been described, but not for compounds with additional benzyl moiety like
methoxynaphthoflavones. There are many literature examples of microbial O-demethylation of
flavones among which 4’-O-demethylation being the most frequent, most often using the fungi of
the genus Aspergillus [28]. Aspergillus niger UPF 724 was the only strain capable of the O-
demethylation of both 1a and 2a with the high isolated yields 71% and 34%, respectively (Table 2,
Scheme 3), exceeding the hydroxylation of naphthoflavones, which corresponds with literature
where the majority of the demethylation tends to occur at 3’ or 4’ of flavon or flavanone derivatives
and exceeds the related hydroxylation yields [28].
The obtained results indicate that different enzymes may be associated with hydroxylation
and demethylation in the selected strains. The lack of O-demethylation or 4’-hydroxylation products
for other methoxy derivatives may suggests different substrate specificity, but without further
research it will remain as an assumption. Moreover, taking into account α-NF (1) and β-NF (2)
activities regarding cytochrome enzymes, it is possible that its naphthoflavones derivatives inhibit
or do not induce certain enzymes required for transformations.
There are no data about the biological activities of 4’-hydroxynaphthoflavones, therefore
another step in our research will be the determination of these compounds’ cytotoxicity. Moreover,
the products obtained are more related to prokaryote flavonoid transformations and such
transformations seem to be expected, also for gut microflora exposed for α-NF (1) during
supplementation with dietary supplements for body builders containing α-NF (1) [26].
5. Conclusions
Among sixty-three fungal strains screened for ability to transform naphtoflavones only five
were efficient biocatalysts. Preparative scale experiments yielded the 4’-hydroxy derivatives of both
α-NF (1), β-NF (2) and its differently substituted analogues. Verticillium sp. and Aspergillus
glaucus proved to be the best biocatalyst to obtain 4’-hydroxynaphtoflavones through direct
hydroxylation, while Aspergillus niger through demethylation of 4’-methoxy substrates. The
process of demethylation was the most efficient. Our results indicate that fungi are useful
biocatalysts to obtain a 4’-hydroxy derivatives of naphthoflavones in the one step reaction. Fungal
metabolism of naphthoflavones do not coincides with mammalian metabolism.
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Acknowledgements
This work was financed by the (Polish) National Science Centre, Grant No. DEC-
2013/09/N/NZ7/01478.
Manuscript preparation was supported by the Wroclaw Centre of Biotechnology, programme The
Leading National Research Centre (KNOW) for the years 2014-2018.
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*Graphical Abstract (for review)
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