Tautomers of anthrahydroquinones: Enzymatic reduction and implications for chrysophanol, monodictyphenone, and related xanthone biosyntheses

Article abstract of DOI:10.1021/ja307151x

Reduction of emodin by sodium dithionite resulted in the formation of two tautomeric forms of emodin hydroquinone. Subsequent conversion by the short-chain dehydrogenase/reductase (SDR) MdpC into the corresponding 3-hydroxy-3,4-dihydroanthracen-1(2H)-one implies that deoxygenation is the first step in monodictyphenone biosynthesis. Implications for chrysophanol formation as well as reaction sequences in the related xanthone, ergochrome, and bianthraquinone biosyntheses are discussed.

Full text of DOI:10.1021/ja307151x

Communication
pubs.acs.org/JACS
Tautomers of Anthrahydroquinones: Enzymatic Reduction and
Implications for Chrysophanol, Monodictyphenone, and Related
Xanthone Biosyntheses
Michael A. Schatzle, Syed Masood Husain, Sascha Ferlaino, and Michael Muller*
̈
̈
Institut fur Pharmazeutische Wissenschaften, Albert-Ludwigs-Universitat Freiburg, Albertstr. 25, 79104 Freiburg, Germany
̈
̈
S
* Supporting Information
ABSTRACT: Reduction of emodin by sodium dithionite
resulted in the formation of two tautomeric forms of
emodin hydroquinone. Subsequent conversion by the
short-chain dehydrogenase/reductase (SDR) MdpC into
the corresponding 3-hydroxy-3,4-dihydroanthracen-
1(2H)-one implies that deoxygenation is the first step in
monodictyphenone biosynthesis. Implications for chrys-
ophanol formation as well as reaction sequences in the
related xanthone, ergochrome, and bianthraquinone
biosyntheses are discussed.
anthones represent a structurally diverse class of natural
X
products and are common among plants, bacteria, and
fungi (fungal genera, e.g., Aspergillus, Helminthosporium,
Penicillium, and Pyrenochaeta).1 Fungal xanthone rearrangement
of emodin (1) via a benzophenone intermediate, such as
monodictyphenone (2)2 or any derivative thereof, is believed to
represent a crucial step in the biosyntheses of ravenelin (3),3
shamixanthone (4),4 tajixanthone (5),3c,4 and ergochromes
(e.g., 6).3c,5 By analogy to sterigmatocystin formation in
aflatoxin biosynthesis,3c the formation of 2 has been proposed
to proceed by a complex sequence of epoxidation, rearrange-
ment, deoxygenation, Baeyer−Villiger (BV) oxidation, and
another deoxygenation.2c In previous studies, deoxygenation of
1 to chrysophanol (7) has been observed in cell-free and
partially purified preparations of Pyrenochaeta terrestris.6
Furthermore, it has been demonstrated that 7 is incorporated
into 4−6 (Figure 1),4c,d,5d suggesting that deoxygenation is the
first step in biosynthesis. However, contrary to these reports, it
has been assumed that 7 is not an intermediate on the paths to
2 and 4.2c,4e Recently, this issue has been revisited to provide a
full rationale of the biosynthetic pathway.7
Figure 1. Formations of monodictyphenone (2) and related xanthones
3−6 proceed by a deoxygenation reaction and BV oxidation.2−5,7
Chrysophanol (7) has been shown to be a precursor of 4−6.4c,d,5d,7
Each deoxygenation site is highlighted by a circle.
dehydration (SD) sequence in 1,8-dihydroxynaphthalene
(DHN) melanin biosynthesis.8 Thus, the striking parallels
between these enzymes suggested a DHN melanin-like
pathway.
From a chemical point of view, anthrahydroquinones exhibit
a pronounced keto−enol tautomerism,9 a putative requirement
for enzymatic reduction, as described for DHN melanin
biosynthesis.10 Because the cell is in a highly reduced state,
which prevents redox cycling of potentially toxic (anthra)-
quinones,11 the hydroquinones and their tautomers might also
represent the more prevalent forms in vivo. Hence, we tested
MdpC for its capability to reduce emodin hydroquinone (8).
The reduction of 1 by sodium dithionite to generate
hydroquinone 8 was investigated first. HPLC−MS analysis
revealed the formation of two products, both of which showed
a molecular mass corresponding to 8. Upon exposure to air,
these compounds were completely converted back into 1.
Detailed studies of 1- and 2-hydroxyanthrahydroquinones have
shown a preferential tautomerism to the oxanthrones.9 In our
Gene deletion studies have identified five gene products
(MdpB, -C, -J, -K, and -L) as essential for downstream
processing of 1 to 2 (Figure 1).2c For the formation of 4, the
involvement of one further monooxygenase (MdpD), as well as
of two prenyl transferases (XptA and -B), and one
oxidoreductase (XtpC) distant from the gene cluster has
been described.4e
MdpB is related to scytalone dehydratase (SD) (55% amino
acid similarity)8a from Magnaporthe grisea, while MdpC shows
high similarity to tetrahydroxynaphthalene reductase (T4HNR)
(64%)8b and trihydroxynaphthalene reductase (T3HNR)
(70%)8c from the same strain. These enzymes catalyze double
deoxygenation by a reduction (T4HNR, T3HNR) and
Received: July 21, 2012
Published: August 21, 2012
© 2012 American Chemical Society
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Communication
case, extraction into acetonitrile-d3 and NMR analysis revealed
the formation of tautomer 8a12 along with oxanthrone 8b,
while hydroquinone 8 itself was not detected (Scheme 1).
to transform 14 into (R)-3,9-dihydroxy-3,4-dihydroanthracen-
1(2H)-one (15) in 7% yield. 13 showed no conversion.
In addition to the observed overall transformation of 1,
deoxygenation as an initial step in the biosyntheses of 2 and
related xanthones is supported by several previously reported
observations: (i) Deletion of mdpC led to a strong increase in 1
and oxidized derivatives (e.g., ω-hydroxyemodin), whereas the
deoxygenated products (2, 7 and xanthones) were absent. (ii)
Deletion of mdpL (a putative BV monooxygenase) led to
accumulation of both 1 and the deoxygenated products 7 and
aloe-emodin. Moreover, 2 and related xanthones (e.g., 4) were
absent.2c,4e Altogether, this argues that deoxygenation precedes
BV oxidation.
Scheme 1. Tautomers of Anthrahydroquinones and Enzyme-
Catalyzed Reduction
Accordingly, we propose the following reaction sequence for
the biosyntheses of chrysophanol (7), monodictyphenone (2)
and related xanthones (Figure 2): Tailoring of the putative
As a next step, codon-optimized N-terminally His-tagged
mdpC was cloned into pET19b, overexpressed, and purified by
Ni-NTA affinity chromatography (see the Supporting In-
formation). In situ reduction of 1 with sodium dithionite and
subsequent conversion with MdpC-his, using glucose dehydro-
genase/D-glucose as an NADPH cofactor regeneration system,
gave a 58% yield of 3-hydroxy-3,4-dihydroanthracen-1(2H)-one
9 (Scheme 1). The absolute configuration was determined to
be R using CD spectroscopy. Incubation of 1 with MdpC-his
without the addition of sodium dithionite resulted in no
conversion. In contrast, Anderson and co-workers have
proposed the direct enzymatic reduction of 1 without any
involvement of the hydroquinone or its tautomers.6 As an
alternative substrate, 1,3-dihydroxyanthraquinone (10) was
reduced by sodium dithionite to a single product, which was
assigned by NMR analysis as 11a,12 a tautomer of hydro-
quinone 11. Subsequent conversion by MdpC-his gave 12 in
22% yield.13
Figure 2. Proposed biosynthetic pathway of 2 in Aspergillus nidulans
FGSC A4. Tailoring of PKS-derived 16 to give 8a/8b and conversion
by MdpC into 9 putatively represent the initial steps in the
biosyntheses of 7, 2, and related xanthones.
polyketide synthase (PKS) product atrochrysone carboxylic
acid (16) yields emodin anthrone (13) and emodin (1).2c,15
Reduction of 1 leads to 8a/8b, the tautomeric forms of emodin
hydroquinone (8). The enzymes responsible for the tailoring
steps, with the exception of an emodin anthrone oxygenase,16
have not yet been characterized. The oxidation of 13 might also
lead directly to 8a/8b. As in melanin biosynthesis,10,17 MdpC-
catalyzed addition of a hydride to any tautomeric form of 8
yields 3-hydroxy-3,4-dihydroanthracen-1(2H)-one 9. Dehydra-
tion of 9 by MdpB to chrysophanol hydroquinone (17) and
oxidation leads to chrysophanol (7).18 The further biosynthetic
course remains unknown. In analogy to aflatoxin biosynthesis,
epoxidation of 7 before BV oxidation has been proposed in the
formation of 3, 5, and 6;3c however, this mechanism cannot
explain the rearrangement of 7 to 2. The same is valid for
arugosin F, the aldehyde derivative of 2 and probable
intermediate in the biosynthesis of 4 and 5. Here, 2 seems to
represent a shunt metabolite.4d,7
Anthrones are in tautomeric equilibrium with their aromatic
anthrols and thus exhibit the putative requirement for
enzymatic reduction.14 Hence, we tested emodin anthrone
(13) and 1,3-dihydroxyanthrone (14) as substrates for MdpC-
his (Scheme 2). Although in both cases the hydroxyanthracene
was not detected by NMR analysis in acetone-d6, we were able
Scheme 2. Enzymatic Reduction of Anthrone 14
A substructure similar to that of 9, albeit in a different
oxidation state, can also be found in dihydrocatenarin B (18),
isolated from Penicillium islandicum (Figure 3). The rather
unstable 18 easily decomposes to islandicin (19),19 which in
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This paper is dedicated to Prof. Dr. G. Bringmann on the
occasion of his 60th birthday. Financial support of this work by
the Deutsche Forschungsgemeinschaft (IRTG 1038) is grate-
fully acknowledged. We thank O. Fuchs for skillful technical
support, V. Brecht for measurement of NMR spectra, and Prof.
Dr. G. Fuchs and Prof. Dr. H. G. Floss for helpful discussions.
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ASSOCIATED CONTENT
* Supporting Information
Experimental details and characterization data. This material is
available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
michael.mueller@pharmazie.uni-freiburg.de
■
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NOTE ADDED AFTER ASAP PUBLICATION
■
This paper was published ASAP on August 28, 2012. The table
of contents graphic has been updated. The revised version was
posted on August 30, 2012.
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