Biosynthesis of indole diterpenes, emindole, and paxilline: Involvement of a common intermediate

Article abstract of DOI:10.1021/ol049115o

The key step for construction of the carbon skeleton in the indole diterpenes, paxilline, and emindole DA was examined. Intact incorporation of multiply ²H-labeled 3-geranylgeranylindole into two different fungal metabolites proves 3-geranylgeranylindole to be a biosynthetic intermediate. These results give evidence that indole diterpenes are biosynthesized via epoxidation of a common intermediate, and the subsequent cationic cyclization, analogous to those in the steroid biosynthesis.

Full text of DOI:10.1021/ol049115o

ORGANIC
LETTERS
2
004
Vol. 6, No. 16
697-2700
Biosynthesis of Indole Diterpenes,
Emindole, and Paxilline: Involvement of
a Common Intermediate
2
†
†
‡
,†
Shuhei Fueki, Tetsuo Tokiwano, Hiroaki Toshima, and Hideaki Oikawa*
DiVision of Chemistry, Graduate School of Science, Hokkaido UniVersity,
Sapporo 060-0810, Japan, and Department of Bioresource Science,
College of Agriculture, Ibaraki UniVersity, Tsukuba, Ibaraki 305-8642, Japan
hoik@sci.hokudai.ac.jp
Received May 14, 2004
ABSTRACT
The key step for construction of the carbon skeleton in the indole diterpenes, paxilline, and emindole DA was examined. Intact incorporation
2
of multiply H-labeled 3-geranylgeranylindole into two different fungal metabolites proves 3-geranylgeranylindole to be a biosynthetic intermediate.
These results give evidence that indole diterpenes are biosynthesized via epoxidation of a common intermediate, and the subsequent cationic
cyclization, analogous to those in the steroid biosynthesis.
Indole diterpenes1 isolated from fungi show structural
nominine (5), emeniveol (6), and aflavinine (7) (Figure
1). On the basis of the carbon skeletons, it is proposed that
these metabolites are biosynthesized by epoxidation of a
common intermediate, 3-geranylgeranylindole (8) and sub-
9
3
10
1
diversity and various bioactivities such as tremogenic,
2
3
insecticidal, and pollen growth inhibitory activity. Trem-
4
ogenic mycotoxins such as paxilline (1) share a common
carbon framework as shown in emindole SB (2) (Figure 1).
5
11
sequent cyclization (Scheme 1) similar to cationic cycliza-
On the other hand, there are a number of structural variations
such as emindoles DA (3), SA (2′-epimer of 3), PA (4),
tion in the biosynthesis of terpenes and steroids. Other
structural types of indole diterpenes represented by petro-
6
7
8
1
2
13
mindole (9) and radarin C (10) are also found in nature
Figure 1). These compounds would be biosynthesized via
*
To whom correspondence should be addressed: Phone: +81-11-706-
(
2
622. Fax: +81-11-706-3448.
†
Hokkaido University.
Ibaraki University.
terminal epoxide of 8.
‡
In the biosynthetic studies of 1 and its structurally related
(
1) (a) Betina, V. Mycotoxins: Chemical, Biological, and EnVironmental
11,14
Aspects; Elsevier: New York, 1989; Vol. 9. (b) Turner, W. B.; Aldridge,
D. C. Fungal Metabolites II; Academic Press: London, 1983.
metabolites,
involvement of tryptophan for the construc-
(
2) Recent example: Li, C.; Gloer, J. B.; Wicklow, D. T.; Dowd, P. F.
Org. Lett. 2002, 4, 3095-3098 and references therein.
3) Kimura, Y.; Nishibe, M.; Nakajima, H.; Hamasaki, T.; Shigemitsu,
N.; Sugawara, F.; Stout, T. J.; Clardy, J. Tetrahedron Lett. 1992, 33, 6987-
990.
4) Springer, J. P.; Clardy, J.; Wells, J. M.; Cole, R. J.; Kirksey, J. W.
Tetrahedron Lett. 1975, 16, 2531-2534.
5) Nozawa, K.; Yuyama, M,; Nakajima, S.; Kawai, K.; Udagawa, S. J.
Chem. Soc., Perkin Trans. 1 1988, 2155-2160.
6) Nozawa, K.; Nakajima, S.; Kawai, K.; Udagawa, S. J. Chem. Soc.,
Perkin Trans. 1 1988, 1689-1694.
7) Nozawa, K.; Udagawa, S.; Nakajima, S.; Kawai, K. Chem. Commun.
987, 1157-1159.
8) Kawai, K.; Nozawa, K.; Nakajima, S. J. Chem. Soc., Perkin Trans.
1994, 1673-1674.
(9) Gloer, J. B.; Tepaske, M. R.; Sima, J. S.; Wicklow, D. T.; Dowd, P.
F. J. Org. Chem. 1988, 53, 5457-5460.
(
(10) Gloer, J. B.; Rinderknecht, B. L.; Wicklow, D. T.; Dowd, P. F. J.
Org. Chem. 1989, 54, 2530-2532.
6
(
(11) (a) Acklin, W.; Weibel, F.; Arigoni, D. Chimia 1977, 31, 63. (b)
De Jesus, A. E.; Gorst-Allman, C. P.; Steyn, P. S.; Vanheerden, F. R.;
Vleggaar, R.; Wessels, P. L.; Hull, W. E. J. Chem. Soc., Perkin Trans. 1
1983, 1863-1868.
(
(
(12) Ooike, M.; Nozawa, K.; Udagawa, S.; Kawai, K. Chem. Pharm.
Bull. 1997, 45, 1694-1696.
(
(13) Laakso, J. A.; Gloer, J. B.; Wicklow, D. T.; Dowd, P. F. J. Org.
Chem. 1992, 57, 138-141.
1
(
(14) Mantle, P. G.; Weedon, C. M. Phytochemistry 1994, 36, 1209-
1217.
1
1
0.1021/ol049115o CCC: $27.50 © 2004 American Chemical Society
Published on Web 07/16/2004

Figure 1. Representative structures of indole diterpenoids.
tion of the indole diterpene core was proposed (Scheme 1).
Alternatively, in the biosynthetic study of nolispirodulic
acid,15 which has the same carbon skeleton as that of 1, a
series of feeding experiments demonstrated that the indole
Scheme 1a
a
Proposed biosynthetic pathway of indole diterpenes, paxilline (1), and emindoles SB (2) and DA (3). To avoid confusion, the numbering
of compounds 1 and 3 is the same as that in 8.
2698
Org. Lett., Vol. 6, No. 16, 2004

1
2
Figure 2. NMR spectra of paxilline (1) and emindole DA (3). (a, c) H NMR of 1 and 3, respectively; (b, d) H NMR of 1 and 3, derived
from [5,1′,1′- H ]-3-geranylgeranylindole 8a, respectively.
3
2
diterpene core is constructed via anthranilic acid and D-ribose,
indicating the intermediacy of indole-3-glycerol phosphate
prompted us to explore the substrate of enzymes responsible
for epoxidation and cyclization of indole diterpenes. Here,
we report the first direct evidence of the common inter-
mediacy of 3-geranylgeranylindole (8) in the biosynthesis
of indole diterpenes 3 and 1.
(Scheme 1). In 2001, Scott and co-workers identified the
biosynthetic gene cluster of paxilline (1) in Penicillium
16
paxilli. Gene disruption and chemical complementation of
the intermediate to the mutants lacking paxG and paxP
proved that two cytochrome P450 monooxygenases (PaxP
and PaxQ) are responsible for final conversion from paspa-
To exclude the possibility of degradation and reincorpo-
ration of the labeled compound, the deuterium labels were
1
7
line to 1. The presence of a gene encoding geranylgeranyl
diphosphate synthase in the biosynthetic gene cluster of 1
indicates that geranylgeranylated compound 8 is a plausible
intermediate. Rainier and Smith demonstrated a biomimetic
synthesis of emindole DA (3),18 supporting this proposal.
Recently, an alternative cyclization of a model epoxide to
afford a paxilline-like product has been reported by Clark
and co-workers.1 Matsuda and co-workers have reported
biomimetic synthesis of petromindole with plant origin lupeol
Scheme 2
9
20
synthase. This enzymatic synthesis provided further support
for epoxidation and cyclization of 3-geranylgeranylindole (8)
to give indole diterpenes. Although these biomimetic syn-
theses showed the feasibility of this route, direct evidence
of the intermediacy of 8 is still lacking. Our interest in
2
1
cationic cyclization to form complex natural products
(
15) Byrne, K. M.; Smith, S. K.; Ondeyka, J. G. J. Am. Chem. Soc. 2002,
24, 7055-7060.
16) Young, C.; McMillan, L.; Telfer, E.; Scott, B. Mol. Microbiol. 2001,
9, 754-764.
17) McMillan, L. K.; Carr, R. L.; Young, C. A.; Astin, J. W.; Lowe, R.
1
3
(
(
G. T.; Parker, E. J.; Jameson, G. B.; Finch, S. C.; Miles, C. O.; McManus,
O. B.; Schmalhofer, W. A.; Garcia, M. L.; Kaczorowski, G. J.; Goetz, M.;
Tkacz, J. S.; Scott, B. Mol. Genet. Genomics 2003, 270, 9-23.
(
18) Rainier, J. D.; Smith, A. B. Tetrahedron Lett. 2000, 41, 9419-
9
423.
19) Clark, J. S.; Myatt, J.; Wilson, C.; Roberts, L.; Walshe, N. Chem.
Commun. 2003, 1546-1547.
20) Xiong, Q. B.; Zhu, X. W.; Wilson, W. K.; Ganesan, A.; Matsuda,
(
(
S. P. T. J. Am. Chem. Soc. 2003, 125, 9002-9003.
Org. Lett., Vol. 6, No. 16, 2004
2699

introduced at two different positions of 8. Starting from
3 from 8a showed three signals at 7.12, 3.10, and 2.71 ppm
corresponding to deuteriums at 5-H and diastereotopic 1′-
CH with nearly the same integral ratio (1.0:0.8:0.8) (Figure
2
commercially available 5-bromoindole, lithiation followed
2
2
by quenching with H
2
2
O gave 5- H-indole, which was
2
converted to bromide 13 in two steps in 94% yield. Ester
4 was reduced with LiAl H to give an alcohol which was
4
2a,b). These observations indicate that 8a was incorporated
into 1 and 3 in an intact manner via epoxide 11 (Scheme 1).
Specific incorporation of 8a into 1 and 3 was relatively low
23
2
1
converted to bromide 15 in 49% yield using standard
conditions (Scheme 2). The resulting bromide 15 was treated
2
(0.82 and 0.16%, respectively, as determined from the H
21
with 3-lithioindole derived from 13 followed by desilylation
to afford 8a in acceptable yield (16%). Feeding experiments
of the labeled precursor were carried out with two different
fungi producing indole diterpenes. The labeled compound
NMR spectra with reference to natural abundance solvent
peaks of CH Cl and CH OH). With the authentic 8 in hand,
2 2 3
the mycelial extracts of both fungi were examined by HPLC.
None of the precursor 8 was detected in the extracts. This
observation indicated that 8 is not accumulated in the mycelia
and rapidly converted into paxilline (1) and emindole DA
(3).
To identify the enzymes responsible for cyclization of
indole diterpenes, we are currently working on functional
analysis of the corresponding gene products.
8
a was administered into P. paxilli (ATCC 26601), which
2
produces paxilline (1). The H NMR spectrum in CH
of the resultant paxilline (1) exhibited three signals at 6.87,
.51, and 2.28 ppm corresponding to deuteriums at 5-H and
diastereotopic 1′-CH with nearly the same integral ratio (1.0:
.1:1.0) (Figure 2c,d). Similar experiment with an emindole
DA-producing fungus, Emericella desertorum (IFO 80840),
3
OH
2
2
1
2
2
2 2
afforded H-labeled 3. The H NMR spectrum in CH Cl of
Acknowledgment. We are grateful to M. Nishizawa,
Tokushima Bunri University, and Dr. M. Shiono, Kuraray
Co., Ltd., for the generous gift of (E,E,E)-geranylgeraniol,
to Prof. Y. Ito, Shinshu University, for a generous gift of a
strain P. paxilli, and to Dr. T. Kan, University of Tokyo,
for helpful comments for the synthesis of the precursor. This
work was supported by Grants 16710150 and 16208012 from
the Ministry of Education, Science, Sports and Culture of
Japan and by Nagase Science and Technology Foundation.
(21) (a) Oikawa, H.; Toyomasu, T.; Toshima, H.; Ohashi, S.; Kawaide,
H.; Kamiya, H.; Ohtsuka, M.; Shinoda, S.; Mitsuhashi, W.; Sassa, T. J.
Am. Chem. Soc. 2001, 123, 5154-5155. (b) Oikawa, H.; Nakamura, K.;
Toshima, H.; Toyomasu, T.; Sassa, T. J. Am. Chem. Soc. 2002, 124, 9145-
9
2
153. (c) Tokiwano, T.; Fukushi, E.; Endo, T.; Oikawa, H. Chem. Commun.
004, 1324-1325.
(22) Amat, M.; Hadida, S.; Sathyanarayana, S.; Bosch, J. J. Org. Chem.
1
994, 59, 10-11.
(
23) Ester 14 was prepared from ethyl acetoacetate in three steps as
follows: (i) NaH, n-BuLi, THF, then farnesyl bromide; (ii) 3 M NaOH,
THF, rt-60°C (62%, two steps); (iii) (EtO)2P(O)CH2CO2Et, NaH, THF, 0
°
C (66%).
OL049115O
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Org. Lett., Vol. 6, No. 16, 2004