One-step enantioselective synthesis of (4S)-isosclerone through biotranformation of juglone by an endophytic fungus

Article abstract of DOI:10.1016/j.tetlet.2012.12.038

We describe here a direct access to (4S)-isosclerone (+)-1, an important structural component of several natural products featuring a spirobisnaphthalene ring system. Starting with the commercially available 5-hydroxy-1,4- naphthalenedione (juglone), biotransformation by the isosclerone-producing endophytic fungus Paraconiothyrium variabile is described. The absolute configuration of (+)-1 was determined unambiguously using circular dichroism and by measurement of the optical rotation. Moreover, the biotransformations of other naphthalene derivatives were undertaken and led to the corresponding (4S)-hydroxy-1-tetralone. At last, this work brings some insights on the biosynthesis of natural tetralones.

Full text of DOI:10.1016/j.tetlet.2012.12.038

Tetrahedron Letters 54 (2013) 1189–1191
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
One-step enantioselective synthesis of (4S)-isosclerone through
biotranformation of juglone by an endophytic fungus
⇑
⇑
Soizic Prado , Didier Buisson , Idrissa Ndoye, Marine Vallet, Bastien Nay
Muséum National d’Histoire Naturelle, Unité Molécules de Communication et Adaptation des Micro-organismes (UMR 7245 CNRS-MNHN), 57 rue Cuvier (CP 54), 75005 Paris, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 July 2012
Revised 5 December 2012
Accepted 7 December 2012
Available online 24 December 2012
We describe here a direct access to (4S)-isosclerone (+)-1, an important structural component of several
natural products featuring a spirobisnaphthalene ring system. Starting with the commercially available
5-hydroxy-1,4-naphthalenedione (juglone), biotransformation by the isosclerone-producing endophytic
fungus Paraconiothyrium variabile is described. The absolute configuration of (+)-1 was determined
unambiguously using circular dichroism and by measurement of the optical rotation. Moreover, the bio-
transformations of other naphthalene derivatives were undertaken and led to the corresponding (4S)-
hydroxy-1-tetralone. At last, this work brings some insights on the biosynthesis of natural tetralones.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Biotransformation
Endophytic fungus
Naphthalenone pentaketide
Secondary metabolites
Circular dichroism
OH
O
OH
8a
O
4
In the course of a program focusing on the structure determina-
tion and ecological studies of metabolites from the endophytic
fungus Paraconiothyrium variabile (1E2a)1 isolated from the plum
yew Cephalotaxus harringtonia var. drupacea (Siebold & Zucc.)
Koidz, we isolated (+)-(4S)-isosclerone (1), a dihydronaphthale-
none commonly found in both enantiomeric forms in plants and
fungi. Its absolute configuration was determined by comparison
of the circular dichroism (CD) spectra with those available from
the literature.2 Indeed, isosclerone was first isolated from Sclerotina
sclerotinium as a new bioactive metabolite with plant growth
regulating properties3 and later in a large variety of plants and
fungi.4–6 Recently, the enantiomer (ꢀ)-(4R)-regiolone 2 was also
reported as a phytotoxin of Botrytis cinerea7 and configurational
assignment of its stereocenter was unambiguously provided by
ab initio computational prediction of its theoretical optical rotation
and electronic CD spectra (Fig. 1).8
8
1
2
3
7
6
4a
5
HO
H
H
OH
(+)-(4S)-isosclerone (1)
(-)-(4R)-regiolone (2)
Figure 1. Structure of isosclerone (1) and regiolone (2).
and 2 was achieved in four steps from 2-(acetoxy)-6-(bromo-
methyl)benzoate and 8.8% overall yield10 or in one step from the
chemical reduction of juglone (40% overall yield),4 but no stereose-
lective synthesis of (4S)-isosclerone (1) has been reported to date.
Microbial transformations of aromatic hydrocarbons like naph-
thalene have been the subject of extensive research.11 Endophytic
fungi are able to act as biocatalysts and can chemically modify
compounds.12 For instance, biotranformations of monoterpe-
noids,13,14 alkaloids,15 and taxoids,16 by endophytic fungi were re-
ported.17 More relevant to this work is the obtention of the racemic
4-hydroxy-1-tetralone by the biotransformation of naphthalene
using Streptomyces griseus NRRL 8090.11
We wish to report herein the first one-step enantioselective
synthesis of compound 1, which can be viewed as an ideal
synthesis, through direct biotransformation of juglone (3) by the
endophytic fungus Paraconiothyrium variabile. Moreover, biotrans-
formations of analogues were also undertaken.
Interestingly, the phytotoxic activity was correlated to the abso-
lute configuration of each isomer.2
These compounds are also of particular interest as building
blocks for more complex natural compounds featuring a spirobis-
naphthalene structure such as preussomerins, palmarumycins, or
diepoxins which have significant biological activities.9 Neverthe-
less compound 1 is weakly produced in fungal cultures (from 0.4
to 2 mg Lꢀ1).3,8 Moreover a racemic synthesis of compounds 1
⇑
Corresponding authors. Tel.: +33 1 40 79 31 19; fax: +33 1 40 79 31 35 (S.P.);
tel.: +33 1 40 79 81 24; fax: +33 1 40 79 31 35 (D.B.).
A kinetic study of the biotransformation of juglone by P. varia-
bile was first performed. The biomass was obtained by culturing
E-mail addresses: sprado@mnhn.fr (S. Prado), buisson@mnhn.fr (D. Buisson).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.12.038

1190
S. Prado et al. / Tetrahedron Letters 54 (2013) 1189–1191
OH
O
the other hand, for the oxymethine protons of 5 a coupling
constant of 8.1 Hz associated to the absence of NOE correlation
implied a trans relative configuration. Interestingly, these two
compounds have already been isolated as natural products from
fungi.21,22
Biosynthetically, this experiment is interesting if we consider
the fact that P. variabile is a natural producer of (+)-isosclerone
as previously shown by us.20 Indeed juglone may be an intermedi-
ate in aromatic pentaketide biosynthesis in fungi even though we
did not isolate it during our previous work from P. variabile. Of
course prior to the biotransformation experiment, we checked
and confirmed that the used resting cells did not contain any
constitutive isosclerone residue.
OH
O
O
OH
O
OH
O
i
+
+
OH
OH
OH
OH
5
OH
3
1 (50%)
4
11 % (1:5)
(minor products, non separable)
Scheme 1. Biotransformation of juglone (3). (i) P. variabile resting cells, 48 h,
phosphate buffer, 25 °C.
the fungus for 4 days in YMS culture medium and incubation was
conducted in resting cells in phosphate buffer.18 Juglone (3)
(17 mg) was then added and biotransformation reactions were
monitored by HPLC-MS. At 48 h, juglone was totally consumed
and the chromatogram revealed the presence of two distinct peaks.
Purification on silica gel (CH2Cl2:MeOH 95:5) led to compound
1 (8.6 mg, 50% yield) with high enantioselectivity (>98% ee)19 and
to the mixture of two constitutional isomers 4 and 5 (2 mg, 11%)
nonseparable in our hands (Scheme 1). Structure of the isolated
compound 1 was established on the basis of 1H and 13C NMR which
confirmed its structure as either isosclerone already isolated from
P. variabile20 or regiolone. The absolute configuration of 1 was as-
signed by circular dichroism and comparison to a pure sample of
natural isosclerone. As depicted in Figure 2, the CD spectra were
superimposable and showed characteristic strong positive Cotton
Eventually, the biotransformation was successfully conducted
at semi-preparative scale and led to relevant quantity of
isosclerone.23
The bioconversion pathway is interesting as juglone (3) has to
undergo two biocatalytic reductions through two possible path-
ways. The first one would involve the reduction of carbon–carbon
double bond followed in the second step by an infrequent reduc-
tion of polyhydroxynaththalene which has been only reported in
melanin biosynthesis.24 In contrast, the second pathway, would
involve the reduction of the carbonyl at C-4 followed by the one
of the double bond. Compounds 4 and 5 may thus arise from
nonstereoselective spontaneous hydration of the putative enone
intermediate, with compound 5 being thermodynamically favored.
Moreover, with the aim to better characterize the scope of the
biotransformation by this endophytic fungus and the biotransfor-
mation pathway, other naphthalene analogues were tentatively
transformed. In this context, 1,4-naphtoquinone (6) was added to
the cell suspension of P. variabile and (+)-(4S)-hydroxy-1-tetralone
(7) was promptly isolated in a good yield (42 mg, 48% yield) with
60% enantiomeric excess (Scheme 2).25 This result shows that the
reduction of a symmetric prochiral diketone is less stereoselective
than the hydroxylated one.
Addition of 1,4-dihydroxynaphtalene (8) to P. variabile also led
to compound (7). This was likely due to the conversion of the
1,4-dihydroxynaphtalene (8) into the corresponding quinone (6).
Unfortunately, this conversion was also observed under inert
atmosphere and did not permit to discriminate between the both
the proposed pathways above. Moreover a trial with tetralone (9)
did not lead to any product.
In conclusion, we have reported here an original and efficient
use of a biotransformation by an endophytic fungus for the enan-
tioselective synthesis of (+)-isosclerone (1) from juglone (3). Addi-
tional experiments led to the biotransformation of the 1,4-
naphthoquinone (6) into 4-hydroxy-1-tetralone (7) also with good
effect at 213 nm (
258 nm (
De = +15.90) and a strong negative band at
D
e
= ꢀ3.40) suggesting that compound (1) is (+)-(4S)-
isosclerone. Moreover, the configuration of C-4 deduced by the
CD spectrum is in good agreement with the value found experi-
mentally of optical rotation in CHCl3
+24.5).8
½
a 2D2
ꢁ
+18.5 (c, 3.25) (lit. ½a D27
ꢁ
Concerning the mixture of the two diasteroisomers 4 and 5, HR-
ESI-MS data pointed out a single protonated ion [M+H]+ at m/z
195.0649 corresponding to the molecular formula C10H11O4 (calcd
195.0658 for C10H11O4). This mixture was also analyzed by 1H NMR
and revealed the presence of two constitutional isomers with a ra-
tio 1:5 (4:5). The 13C NMR spectrum exhibited for each compound
10 carbon resonances including one carboxyl, two oxymethines
and three quaternary. The 1H–1H COSY spectrum was indicative
of two spin systems as represented in Figure 3. These substructures
were assembled from the HMBC data and led to the characteriza-
tion of 3,4,8-trihydroxy-1-tetralone 4 and 5.
The relative configuration of the diol moiety of 4 and 5 was
determined by the analysis of NOE correlations and coupling
constant values. Indeed for 4, a NOE correlation between the two
oxymethine protons was observed associated to a low coupling
constant (J = 3.1 Hz) suggesting a syn relationship for the diol. On
Figure 2. CD spectra of (1) (solid line) and pure sample isosclerone (hatched line) recorded in MeOH at 22 °C (c, 10ꢀ2).

S. Prado et al. / Tetrahedron Letters 54 (2013) 1189–1191
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OH
OH
COSY
HMBC
OH
OH
4
5
Figure 3. Key 1H–1H COSY (bold bonds) and HMBC correlations (H ? C) for 4 and 5.
O
O
O
i
OH
6
7 (48%)
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Scheme 2. Biotransformation of 1,4-naphtoquinone (6). (i) P. variabile resting cells,
8 h, phosphate buffer, 25 °C.
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yields. It should be noted that these compounds could be the struc-
tural components of several natural products featuring a spirobis-
naphthalene skeleton.
19. Enantiomeric excess was determined by GC analysis. Colonm CP-chirasil-Dex
CB 25 m 0.32 mm, 25 lm, carrier gas He 15 psi, oven temperature 140 °C
(49 min), gradient 5 °C/min 160 °C. Retention times (S)-isoscerone 52.7 min.
(R)-regiolone 53.4 min.
20. Combès, A.; Ndoye, I.; Bance, C.; Bruzaud, J.; Djediat, C.; Dupont, J.; Nay, B.;
Prado, S. PloS One 2012, 7, e47313.
Acknowledgments
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H.; Liu, H. W. Chem. Biodivers. 2011, 8, 1689–1700.
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16.
23. Two hundred milliliters of YMS-culture medium in a 500 mL flask containing
(g/L) glucose 16, yeast extract 4, malt extract 10, and soybean peptones 5 were
The French Embassy at Dakar, Senegal and Cheikh Anta Diop
University at Dakar are acknowledged for providing a research fel-
lowship to IN. This project was supported by the following grants:
‘Projets Innovants FRB 2009’ from the Fondation pour la Recherche
sur la Biodiversité (AAP-IN-2009-026), the Action Transversale du
Muséum ‘Biodiversité des microorganismes dans les écosystèmes
actuels et passés’ from the Museum national d’Histoire naturelle.
The authors wish to thank L. Dubost for the mass spectra, A. Deville
for NMR spectra, and S. Lacoste for providing the fungal strains and
P. Alberti for CD.
inoculated with 200 lL glycerol suspension of P. variabile and incubated at
27°C and 160 rpm in an orbital shaker. After 96 h biomass was collected by
filtration and cells (90 g) were then suspended in phosphate buffer (0.2 M, pH
6.5, 100 mL) and juglone (100 mg) dissolved in DMF (100 lL) was added.
Biotransformations were performed at 27°C in an orbital shaker. After 48 h of
incubation, the cell suspension was filtered. Thus, the filtrate was saturated
with NaCl, extracted with ethyl acetate (three times) and the organic phases
were combined, dried over Na2SO4. After evaporation, the product was purified
by flash chromatography (CH2Cl2/MeOH 9:1) (44.2 mg, 44%). ½a D20
ꢁ
+18.5 (c,
3.25, CHCl3).
Supplementary data
24. Thompson, J. E.; Fahnestock, S.; Farrall, L.; Liao, D. I.; Valent, B.; Jordan, D. B. J.
Biol. Chem. 2000, 275, 34867–34872.
25. Biomass (90 g) obtained as described above was suspended in phosphate
buffer (0.2 M, pH 6.5, 90 mL). 1,4 Naphtoquinone (90 mg) dissolved in DMF
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2012.
12.038.
(500 lL) was also added in buffer (90 mL) and this solution was poured to the
cell suspension. Biotransformations were then performed at 27°C in an orbital
shaker, stopped after 8 h and treated as above. After evaporation, the product
was purified by flash chromatography, elution with CH2Cl2/MeOH 97:3 (42 mg,
48%), ½a 2D0
ꢁ
+21.3 (c, 0.47, CHCl3) (Lit. ½a D20
ꢁ
+23.5, c, 0.7, CHCl3).26 Enantiomeric
excess determined by GC analysis. Colonm CP-chirasil-Dex CB 25 m 0.32 mm,
References and notes
1. Langenfeld, A.; Prado, S.; Nay, B.; Porcher, E.; Cruaud, C.; Lacoste, S.; Bury, E.;
Hachette, F.; Hosoyaf, T.; Dupont, J. Fungal Biol. in press.
2. Evidente, A.; Andolfi, A.; Cimmino, A. Chirality 2011, 23, 674–693.
25 lm, carrier gas He 15psi, oven temperature 140°C (20 min), gradient 3°C/
min 150 °C. Retention times (S)-isomer 26.2 min, (R)-isomer 27.3 min.
26. Joly, S.; Nair, M. S. Tetrahedron: Asymmetry 2001, 12, 2283–2287.