Yeast-mediated xanthone synthesis through oxidative intramolecular cyclization

Article abstract of DOI:10.1021/ol302283p

Benzoylphloroglucinol derivatives are natural products showing diverse biological activities that could be modulated by structural modifications. For this purpose, we studied the biotransformation of guttiferone A and of maclurin using a combinatorial approach for screening active microorganism strains. We found a novel and unexpected yeast-catalyzed oxidation that has selectively given a new oxy-guttiferone A and norathyriol.

Full text of DOI:10.1021/ol302283p

ORGANIC
LETTERS
2012
Vol. 14, No. 19
5054–5057
Yeast-Mediated Xanthone Synthesis
through Oxidative Intramolecular
Cyclization
Yann Fromentin,†,‡ Philippe Grellier,† Jean Duplex Wansi,§ Marie-Christine
Lallemand,‡ and Didier Buisson*,†
ꢀ
Unite Molecules de Communication et Adaptation des Micro-organismes, UMR 7245
CNRS-MNHN, 57, Rue Cuvier, CP54, 75005, Paris, France, Laboratoire de
ꢀ
ꢀ
ꢀ
Pharmacognosie, UMR 8638 CNRS-Universite Paris Descartes Sorbonne Paris Cite,
Faculte de Pharmacie, 75006 Paris, France, and Departement de Chimie Organique,
ꢀ
ꢀ
ꢀ ꢀ
Universite de Douala, Faculte des Sciences, P.O. Box 24157, Douala, Cameroon
buisson@mnhn.fr
Received August 16, 2012
ABSTRACT
Benzoylphloroglucinol derivatives are natural products showing diverse biological activities that could be modulated by structural modifications.
For this purpose, we studied the biotransformation of guttiferone A and of maclurin using a combinatorial approach for screening active
microorganism strains. We found a novel and unexpected yeast-catalyzed oxidation that has selectively given a new oxy-guttiferone A and
norathyriol.
In recent years, biotransformation entities derived from
natural products have gained importance in the search for
therapeutic drugs.1 In this context, we have investigated
the study of benzoylphloroglucinol derivatives, present in
various species and parts of plants. The simplest com-
pounds, like maclurin, are the precursors of polysubstituted
compounds such as polycyclic polyprenylated acylphloro-
glucinols (PPAPs familly), and especially guttiferones.2
Guttiferone A 1 was isolated from Symphonia globuli-
fera roots3 and recently from Garcinia livingstonei4 and
Garcinia macrophylla.5 This natural product exhibits a
wide pharmacological profile, with anti-HIV,3 cytotoxic,4ꢀ6
trypanocidal,7,8 antiplasmodial,8ꢀ10 leishmanicidal,8,11,12
and antibacterial13 effects. In an interesting way, guttifer-
one A shows good radical scavenging activity and could
(6) Merza, J.; Mallet, S.; Litaudon, M.; Dumontet, V.; Seraphin, D.;
Richomme, P. Planta Med. 2006, 72, 87–89.
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E.; Huerta-Reyes, M.; Reyes-Chilpa, R. Biol. Pharm. Bull. 2004, 27,
141–143.
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T.; Maes, L.; Cos, P. Phytother. Res. 2011, 25, 458–462.
(9) Ngouela, S.; Lenta, B. N.; Noungoue, D. T.; Ngoupayo, J.;
Boyom, F. F.; Tsamo, E.; Gut, J.; Rosenthal, P. J.; Connolly, J. D.
Phytochemistry 2006, 67, 302–306.
(10) Mangoyi, R.; Hayeshi, R.; Ngadjui, B.; Ngandeu, F.; Bezabih,
M.; Abegaz, B.; Razafimahefa, S.; Rasoanaivo, P.; Mukanganyama, S.
J. Enz. Inhib. Med. Chem. 2010, 25, 854–862.
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Sewald, N. Molecules 2007, 12, 1548–1557.
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Barbieri, C. L.; Beijo, L. A.; Doriguetto, A. C.; D’Martin, E. C.; dos
Santos, M. H. Phytomedicine 2010, 17, 339–345.
(13) Dias, K. S. T.; Januario, J. P.; D’ Dego, J. L.; Dias, A. L. T.; dos
Santos, M. H.; Camps, I.; Coelho, L. F. L.; Viegas, C. Bioorg. Med.
Chem. 2012, 20, 2713–2720.
† UMR 7245 CNRS-MNHN.
‡
ꢀ
ꢀ
UMR 8638 CNRS-Universite Paris Descartes Sorbonne Paris Cite.
§
ꢀ
ꢀ
Departement de Chimie Organique, Universite de Douala.
(1) Venisetty, R. K.; Ciddi, V. Curr. Pharm. Biotechnol. 2003, 4, 153–
167. Simeo, Y.; Sinisterra, J. V. Mini-Rev. Org. Chem. 2009, 6, 128–134.
(2) Ciochina, R.; Grossman, R. B. Chem. Rev. 2006, 106, 3963–3986.
(3) Gustafson, K. R.; Blunt, J. W.; Munro, M. H. G.; Fuller, R. W.;
Mckee, T. C.; Cardellina, J. H.; Mcmahon, J. B.; Cragg, G. M.; Boyd,
M. R. Tetrahedron 1992, 48, 10093–10102.
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M. J.; Weinstein, I. B.; Kennelly, E. J. J. Agric. Food Chem. 2010, 58,
4749–4755.
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Wisse, J. H.; Kingston, D. G. I. Planta Med. 2003, 69, 864–866.
r
10.1021/ol302283p
Published on Web 09/17/2012
2012 American Chemical Society

Table 1. Screening of Active Microorganisms in Guttiferone A Biotransformations
incubations
culture of
total number
entry
1
microorganisms
step 1
step 2
4 assays
of assays
selected microorganism (yielda)
8 strains
15 strains
12 strains
2 mixtures of 4 strains:
1 gave biotransformation
3 mixtures of 5 strains:
1 gave biotransformation
4 combinatorial mixtures:
1 gave biotransformation
6
Rhodotorula buffonii MUCL29812 (48%)
2
3
5 assays
8
4
Candida pinus MUCL 27856 (19%)
Pichia anomala NRRL Y40 (17%)
no assay required
a Yield in compound 2 after purification.
be a lead in the search for new antimalarial drugs.9,14
Several actions of guttiferone A toward cell pathologies
and specific enzymatic activities have also been reported.11,15
Concerning the antiparasitic activities, it is worth noting
that Malaria remains one of the most important infectious
diseases causing 1 million deaths every year. Due to the
rising prevalence of Plasmodium falciparum resistance, the
treatment of malaria is becoming increasingly difficult.
There is therefore an urgent need to diversify the antima-
larial therapeutic arsenal. Most of the antimalarial drugs
are natural products and natural product analogues.
For this reason, our group investigated the antiparasitic
potency of guttiferone A analogs. Because it is isolated
from fruits, guttiferone A is an interesting renewable
starting material for semisynthesis. Within the field of
natural products, pharmacomodulation has provided sig-
nificant results.16
Then to improve selected activity, we wanted to generate
a library of guttiferone A derivatives for performing
structureꢀactivity relationship studies. Among the strate-
gies used, we studied the biotransformation to obtain
selective modifications, and yeast-mediated transforma-
tions have been investigated with the hope to stereospeci-
fically reduce one of the carbonyl groups.
We present here the results of original approaches in the
screening of microorganism strain collections, the finding
of an unexpected yeast-mediated oxidation reaction, and
its use in the efficient synthesis of the natural product and
analog.
Microbial reduction of ketones especially with baker
yeast is largely documented;17 however the reduction is
strongly influenced by the bulkiness of substituents of the
carbonyl group. It turns out that the baker yeast is not
effective in benzophenone reduction, so we screened the
35 yeasts in our collection.
Usually a selection of active microorganisms was per-
formed by incubation of an individual strain with a sub-
strate to be transformed resulting in a number of tests
equal to the number of strains to be tested. In order to
reduce the number of assays, the selection of active yeasts
was performed using a new approach, based on the
implementation of biomass mixtures (Table 1). At first,
we tested mixtures of four (entry 1) and five (entry 2)
different strains (step 1), and biotransformations were
monitored by HPLC and mass spectrometry. Then the
microorganisms that were present in the mixtures able to
transform guttiferone A were tested individually (step 2),
and R. buffonii and C. pinus were selected among eight and
fifteen strains through 6 and 8 assays, respectively.
To improve our strategy, mixtures were carried out
using combinatorial distribution (entry 3). This approach
allowed identifying the active microorganisms without
further incubation (for details, see Supporting Information),
and the yeast P. anomala was selected among twelve
strains through only four assays. Our combinatorial
approach is an efficient strategy; the number of assays is
reduced by three compared to traditional screening.
HPLC chromatographic profiles of biotransformation
media with the three selected microorganisms showed one
peak having the same retention time, which is longer than
the one of the starting material. A mass spectrometry
spectrum showed a m/z 601 for [MþH]þ corresponding
to a loss of two protons regarding guttiferone A. These
results suggested an oxidative reaction rather than an
expected reduction reaction, and the product has been
identified as 3,16-oxyguttiferone 2 by NMR analysis.
The loss of one proton in the aromatic part on the
NMR spectra of product 2 leads to the structure of an
(14) Acuna, U. M.; Figueroa, M.; Kavalier, A.; Jancovski, N.; Basile,
M. J.; Kennelly, E. J. J. Nat. Prod. 2010, 73, 1775–1779.
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Tsutsui, K.; Tsutsui, K.; Yamada, M.; Fujimori, S. Chem. Pharm. Bull.
1997, 45, 418–420. Muleya, V.; Hayeshi, R.; Ranson, H.; Abegaz, B.;
Bezabih, M. T.; Robert, M.; Ngadjui, B. T.; Ngandeu, F.; Mukanga-
nyama, S. J. Enzyme Inhib. Med. Chem. 2008, 23, 391–399. Martins,
F. T.; Assis, D. M.; dos Santos, M. H.; Camps, I.; Veloso, M. P.; Juliano,
M. A.; Alves, L. C.; Doriguetto, A. C. Eur. J. Med. Chem. 2009, 44,
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Naal, Z.; Curti, C.; Andreu, G. L. P. J. Pharmacol. Sci. 2011, 116, 36–46.
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Org. Lett., Vol. 14, No. 19, 2012
5055

oxy-guttiferone. In fact the phenolic oxidative coupling
may engender four compounds due to the enolization of
the triketone (C-1 or C-3) and due to both aromatic
positions (C-12 or C-16), which could be involved in the
reaction. The presence of two singlets (H-12 and H-15)
showing no correlation between each other in COSY and
HMBC was in agreement with a cyclization in the position
16 on the catechol ring (Table 2).
HMBC (Figure 1) presents a strong correlation between
H-7 and carbon 1 at 194.38 ppm. The position of this
ketone is also confirmed with the correlation of H-29 with
carbon 1. In the other part, the HMBC correlation of H-17
with the specific quaternaries carbon 5 (51.96 ppm) and
carbon 3 (179.52 ppm) demonstrated the cyclization with
the enol in the position C-3.
Table 2. Spectral Data of 3,16-Oxyguttiferone A (2)a
13C
1H
J
no. δ (ppm)
δ (ppm)
(Hz) COSY
HMBC
1
2
3
4
5
6
7
8
9
194.38
118.91
179.52
65.02
51.96
41.58 1.88 (m, 1H)
39.24 2.08 (t, 2H)
66.75
7,24
4,8
5.54
6
1,5,6,8,24
207.39
10 174.49
11 118.02
12 151.15
13 103.92 6.96 (s, 1H)
14 154.96
11,12,14,15
15 147.15
16 109.52 7.43 (s, 1H)
10,12,14,15
3,4,5,18,19
17
27.40 2.84/2.96 (m/m, 2H)
18
17
18 119.67 4.63 (m, 1H)
19 136.17
20
21
22
23
24
18.65 1.73 (s, 3H)
25.90 1.41 (s, 3H)
20.41 1.28 (s, 3H)
18,19,21
18,19,20
4,5,6,23
4,5,6,23,35,36
5,25
37.89 1.42/1.53 (m/d, 2H) 7.31 34
30.42 1.77/1.92 (m/m, 2H) 6,25
25 124.60 4.85 (m, 1H)
26 134.09
24
27,28
Figure 1. Key HMBC correlation of 3,16-oxyguttiferone 2.
27
28
29
17.95 1.37 (s, 3H)
26.10 1.64 (s, 3H)
30.30 2.51 (t, 2H)
25,26,28
25,26,27
1,8,30,31
32,33
7.45 30
7.59 29
The active microorganisms were tested on a preparative
scale (20 mg, 0.1 mg/mL), and the most efficient, R. buffoni,
gave 2 in 48% yield (Scheme 1). Four oxy-guttiferones were
isolated from plants, but this is the first time that oxy-
guttiferone A was obtained. The other strains did not
transform guttiferone A in those conditions, and the
starting material was recovered unchanged, excluding a
spontaneous formation of oxy-guttiferone A.
30 120.83 5.21 (t, 1H)
31 135.54
32
33
34
18.12 1.70 (s, 3H)
26.03 1.70 (s, 3H)
23.91 1.98 (m, 2H)
30,31,33
30,31,32
23,35 23,35,36
35 124.98 5.10 (t, 1H)
36 132.98
6.73 34
37,38
37
38
17.79 1.64 (s, 3H)
26.32 1.70 (s, 3H)
35,36,38
35,36,37
a 13C (75 MHz), 1H (400 MHz) in MeOD.
Scheme 1. Biotransformation of Guttiferone A by R. buffoni
P. falciparum.19 Better activities than those observed for
guttiferone A were obtained but with increasing toxicity
toward Vero cells (Table 3).
Table 3. Trypanocidal, Antiplasmodial, and Cytotoxic Activ-
ities of Guttiferone and 3,16-Oxy-guttiferone
Vero cells
% inhibition
T. brucei P. falciparum
Biological activities of compound 2 were evaluated by
its ability to inhibit the growth of Trypanosoma brucei
bloodstream forms18 and the intraerythrocytic growth of
IC50
IC50
(μM/mL)
(μM/mL)
10ꢀ5 M 10ꢀ6 M
guttiferone A 1
2.95
2.08
3.32
1.25
89 ( 1 3 ( 3
94 ( 1 17 ( 3
3,16-oxy-guttiferone A 2
(18) Lethu, S.; Bosc, D.; Mouray, E.; Grellier, P.; Dubois, J.
J. Enzyme Inhib. Med. Chem. 2012 DOI: 10.3109/14756366.2011.643302.
(19) Grellier, P.; Sarlauskas, J.; Anusevicius, Z.; Maroziene, A.;
Houee-Levin, C.; Schrevel, J.; Cenas, N. Arch. Biochem. Biophys.
2001, 393, 199–206.
In order to extend the reaction, we investigated the
biotransformation of maclurin, which has phenol groups
5056
Org. Lett., Vol. 14, No. 19, 2012

that are able to react in the oxidative cyclization instead of
enol groups, by the selected yeasts. Furthermore, as men-
tioned before, maclurin is the biogenetic precursor of
guttiferone A; both compounds could react in the same
way. Incubation of maclurin in the presence of R. buffonii
afforded the natural xanthone derivative, norathyriol 4,
in 16% yield. Investigations of this biotransformation
showed that the enzyme was excreted in culture medium.
The best yield (26%) was obtained when incubation was
performed in culture medium (40 mg, 0.1 mg/mL) after
centrifugation (scheme 2), whereas heating of the culture
medium before addition of maclurin resulted in a loss of
activity.
The natural occurrence of xanthones with the corre-
sponding benzophenones could be in agreement with
the mechanism of cyclization proposed by Sang et al. for
garcinol.28 In some cases, xanthones are a mixture of isomers.
Maclurin, norathyriol (1,3,6,7-terahydroxy-xanthone), and
its isomer 1,3,5,6-terahydroxy-xanthone have been isolated
from Symphonia globulifera.29 Likewise, oxy-guttiferones K,
K2, I, and M together with their corresponding guttiferones
have been isolated from Garcinia cambogia.28
However oxyguttiferones are not systematically ob-
served in the presence of guttiferone suggesting an
enzymatic-catalyzed cyclization. Mechanisms for the cy-
clization in biosynthetic pathways of xanthones have been
postulated.30 A xanthone synthase has been identified as
cytochrome P450.31 However, cyclization of hydroxyben-
zophenones by laccase from Polystictus versicolor and
horse radish peroxidase have been reported, but yields in
these enzymatic oxidations were very low (5%) and the
enzymes did not act on maclurin.
Scheme 2. Biotransformation of Maclurin by Enzymatic
Activity from R. buffoni
In this work, we show the relevance in the screening of
microorganisms able to transform a molecule since it
allows finding unexpected enzymatic activity which could
be used in organic synthesis. For this purpose, our combi-
natorial approach is a suitable strategy to decrease the
number of assays. The newly found oxidative activity acts
like peroxidase and lacase, and just as those enzymes, it is
excreted. It must be noted that no lacase and peroxidase
have been described in wild-type strains of yeast. This
strategy has been used in the preparation of new oxygutti-
ferone and in the first enzymatic synthesis of norathyriol.
Norathyriol has been recently described for different
biological activities20,21 and as a safe chemopreventive
agent effective against development of UV-induced skin
cancer.22 It is the aglycone of mangiferin, which exhibits a
wide spectrum of pharmacological effects.23 So syntheses
of norathyriol, mangiferin, and derivatives have recently
received attention,21,24 and our method could serve as a
straightforward alternative to multistep syntheses.
Acknowledgment. We want to strongly thank Caroline
Bance for technical support in microbiology, Lionnel Dubost
for mass spectra, and Elisabeth Mourray for the technical
support in parasitology (UMR 7245, MNHN). Financial
Such chemical cyclizations were reported; benzophe-
nones were converted into xanthone photochemically25
or by using chemical reageant potassium ferricyanide.26
A
ꢀ
support for a PhD fellowship (Y.F.) by Universite Paris
Descartes, Sorbonne Paris Cite is gratefully acknowledged.
mechanistic study showed that reaction of garcinol with
the stable DPPH radical generates a conjugated radical
whose cyclization involving the catechol ring leads to the
tetracyclic xanthone derivatives.27
ꢀ
Supporting Information Available. Screening of active
microorganisms, preparation of oxy-guttiferone A and
norathyriol, 1D and 2D NMR spectra of products, and
protocols of measurement of biological activities. This
material is available free of charge via the Internet at
http://pubs.acs.org.
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Interact. 2010, 186, 287–294.
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I.; Carper, A.; Langfald, A.; Oi, N.; Kim, M. O.; Zhu, F.; Sosa, C. P.;
Zhou, K. Y.; Bode, A. M.; Dong, Z. G. Cancer Res. 2012, 72, 260–270.
(23) Hou, S. Y.; Wang, F.; Li, Y. M.; Li, Y.; Wang, M. Q.; Sun, D. J.;
Sun, C. H. Food Chem. 2012, 132, 289–294.
(24) Hu, L.; Hu, H.; Wu, W.; Chai, X.; Luo, J.; Wu, Q. Bioorg. Med.
Chem. Lett. 2011, 21, 4013–4015.
(25) Jefferson, A.; Scheinmann, F. J. Chem. Soc. C 1966, 175–8.
(26) Atkinson, J. E.; Lewis, J. R. Chem. Commun. 1967, 803.
(27) Sang, S. M.; Pan, M. H.; Cheng, X. F.; Bai, N. S.; Stark, R. E.;
Rosen, R. T.; Lin-Shiau, S. Y.; Lin, J. K.; Ho, C. T. Tetrahedron 2001,
57, 9931–9938.
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The authors declare no competing financial interest.
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