Conversion of [19-2H2]fusicocca-2,10(14)-diene into its 8β-ol and fusicoccins by Phomopsis (Fusicoccum) amygdali

Article abstract of DOI:10.1016/S0040-4039(00)00173-8

Feeding experiments of (+)-[19-²H₂]fusicocca-2,10(14)-diene, a deuterated derivative of a new candidate for the initially-forming fusicoccane-hydrocarbon in the fusicoccin biosynthesis, with the fusicoccin- producing fungus Phomopsis (Fusicoccum) amygdali have revealed its incorporation into fusicoccins J and A through (+)-fusicocca-2,10(14)-dien- 8β-ol, whose occurrence in the mycelial extract has been confirmed by direct comparison of its ¹H NMR and GC-MS data with those of the synthetic sample, proving the early biosynthetic route leading to the aglycons of fusicoccins. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)00173-8

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 2401–2404
Conversion of [19-2H2]fusicocca-2,10(14)-diene into its 8β-ol
and fusicoccins by Phomopsis (Fusicoccum) amygdali
Takeshi Sassa,a,∗ Chang-Shan Zhang,b Mitsuyoshi Sato,a Naoto Tajima,a Nobuo Kato b and
Akira Mori b
aDepartment of Bioresources, Faculty of Agriculture, Yamagata University, Tsuruoka, Yamagata 997-8555, Japan
bInstitute of Advanced Material Study, Kyushu University, Kasuga, Fukuoka 816-8580, Japan
Received 20 December 1999; revised 20 January 2000; accepted 21 January 2000
Abstract
Feeding experiments of (+)-[19-2H2]fusicocca-2,10(14)-diene, a deuterated derivative of a new candidate for the
initially-forming fusicoccane-hydrocarbon in the fusicoccin biosynthesis, with the fusicoccin-producing fungus
Phomopsis (Fusicoccum) amygdali have revealed its incorporation into fusicoccins J and A through (+)-fusicocca-
2,10(14)-dien-8β-ol, whose occurrence in the mycelial extract has been confirmed by direct comparison of its 1H
NMR and GC–MS data with those of the synthetic sample, proving the early biosynthetic route leading to the
aglycons of fusicoccins. © 2000 Elsevier Science Ltd. All rights reserved.
Keywords: biosynthesis; glycosides; microbial reactions; terpenes and terpenoids.
Fusicoccins A (1)1,2 and J (2)3,4 are the main metabolites produced by the plant pathogenic fungus
Phomopsis (Fusicoccum) amygdali (Del.) Tuset and Portilla (Fig. 1).5 They and the structurally-related
cotylenins6–8 [cotylenin A (3) is shown as a representative], isolated from Cladosporium sp. 501-7W,
display a number of phytohormone-like effects for all higher plants via the activation of the plasma
membrane H+-ATPase.9 Currently, their action is attracting much attention as the fusicoccin-binding
14-3-3 proteins are regarded to be the key proteins in intracellular signal transductions in both plant10
and animal cells.11 It has been an accepted belief since the 1970s that fusicocca-1,10(14)-diene (4) is
an intermediary hydrocarbon at an early stage of the biosynthesis of fusicoccins.12,13 However, recently,
we have isolated and synthetically determined (+)-fusicocca-2,10(14)-diene (5), a double bond isomer
of 4, as the main hydrocarbon constituent from the mycelial extract of P. amygdali.14 Furthermore, the
intermediacy of 5 in the fusicoccin biosynthesis is strongly supported by the transformation of fusicocca-
2,10(14)-dien-8β-ol (6), provided synthetically as a deuterium-labeled derivative, into 2 by the fungus.15
Since this bio-transformation from 6 to 2 proceeds very efficiently, we suspected that the conversion of
5 into 6 is the real process in vivo as the first hydroxylation step leading to fusicoccins. Here, as direct
∗
Corresponding author. Tel: +81-235-28-2862; fax: +81-235-28-2862.
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)00173-8
tetl 16453

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proof of this postulation, we report the isolation of 6 from the mycelial extract of P. amygdali and the
transformation of 5 by this fungus into 2 and 1 via 6 using synthetic deuterium-labeled 5-d2 as a feeding
substrate.
Fig. 1.
The feeding substrate, [19-2H2]fusicocca-2,10(14)-diene (5-d2), and an authentic sample of fusicocca-
2,10(14)-dien-8β-ol (6) were synthesized from the previously reported fusicocca-2,10(14)-diene deriva-
tive 714 (Scheme 1); 5-d2 [a colorless oil, m/z (%) 274 (C20H30D2, M+, 14), 273 (M+−1, 5.8), and 135
(100); carbon signals distinguishable from those of non-deuterated 514 are δC (100 MHz, CDCl3) 20.40
(quintet, deuterated C-19, ∆δ −0.58), 21.21 (C-20, ∆δ −0.03), and 26.89 (C-15, ∆δ −0.15)] and 6 [a
20
colorless oil, [α] +75 (c 0.048, CHCl3); m/z (%) 288 (M+, 7.5), 245 (10), 227 (12), 151 (52), 122
D
(46), and 95 (100) by GC–MS (tR 10.779–10.818 min);† 1H NMR is broadened at ambient temperature
because of the conformational mobility, but there are several diagnostic signals at δH (400 MHz, CDCl3)
0.91 (3H, s, H-18), 0.92 (3H, d, J 6.8, H-19), 1.00 (3H, br d, J 6.8, H-20), 1.62 (3H, br s, H-16), 2.35
(dm, J 10.8, H-9), and 3.82 (dm, J 12.0, H-8)].
Scheme 1. Reagents and conditions. (i) Ref. 14; (ii) (ClCO)2, DMSO then Et3N, CH2Cl2; (iii) LiAlD4, THF (2 steps, 80%); (iv)
MsCl, Py; (v) LiEt3BD, THF (two steps, 88%); (vi) same as (ii); (vii) excess DIBAL-H, toluene (two steps, 79%); (viii) TsCl,
Py; (ix) LiEt3BH, THF (two steps, 71%)
At first, the natural occurrence of 6 in mycelia of P. amygdali F614 was investigated in detail. A pale
yellow hexane extract (ca. 4.0 g) of mycelia obtained from 200 culture flasks, each containing 100 cm3
of the culture medium (8.0% commercial sugar, 1.0% corn steep liquor, and 0.5% peptone in deionized
water),‡ was carefully separated by silica gel flash chromatography. From the fraction (1.63 g) eluted
with a mixture of hexane:EtOAc (10:1), a colorless oil {0.36 mg, [α]20 +50 (c 0.033, CHCl3)}, of which
D
the Rf value is consistent with that of authentic 6, was obtained by repeated chromatography on silica
gel using mixtures of CHCl3:EtOH (40:1), hexane:acetone (50:1), and CHCl3:EtOAc (50:1). Its entity
†
Shimadzu GC-17A/QP-5000; DB-1 capillary column, 30 m; flow rate of the carrier (He), 1.9 cm3/min; chamber temp.,
60–170°C (30°C/min) then 170–280°C (15°C/min).
‡
The fungus strain F6 grown for 5 days produced 120 µg/cm3 of fusicoccin J (2) as the main metabolite.

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was unambiguously established by direct comparisons of full-scanned GC–MS (tR 10.772–10.808 min)
and 1H NMR (400 MHz, CDCl3) data with those of authentic 6 (see above). Thus, 6, used as the feeding
substrate in the previous paper,15 is the real metabolite existing in mycelia of P. amygdali.
Then the feeding experiments were carried out using 5-d2 to get direct proof for the intermediacy of 5
as well as 6 in the biosynthesis of fusicoccins. Each EtOH solution of 5-d2 (3.0 mg in 0.2 cm3) was added
to six cultures (each 100 cm3 of a medium containing 2.0% glucose, 0.2% corn steep liquor, and 0.2%
peptone in deionized water§) of the fungus strain F6 which had been grown for 3 days at 25°C. After
additional cultivation for 37 h, mycelia were obtained by filtration and treated as above. The hexane
extract (82 mg) obtained was flash-chromatographed with a mixture of CHCl3:EtOH (80:1) to give a 6-
containing fraction (ca. 0.8 mg). GC–MS data of the 6-containing fraction and its mixture with synthetic
15
6-d1 as an internal standard clearly demonstrated that the fed 5-d2 was incorporated efficiently into 6;
the former m/z (%) 95 (100), 288 {[M(6)]+, 4}, 290 {[M(6-d2)]+, 8}; and the latter m/z (%) 95 (100), 288
{[M(6)]+, 2}, 289 {[M(6-d1)]+, 7}, 290 {[M(6-d2)]+, 5.5}.
Fusicoccins, 1 and 2, in the feeding experiment were chromatographically separated from the EtOAc
extract (11.4 mg from one culture flask described above) of the culture filtrate with the additional
cultivation for 48 h. The 1-containing fraction (0.8 mg) and the 2-containing fraction (1.1 mg) were
analyzed by FAB-MS; m/z (rel. int.%, the former//natural-1) 703 [(M+Na)+, 100//100], 704 [(M+1+Na)+,
50.5//39.3], 705 {[M+2+Na and M(d2)+Na]+, 46.3//10.6} and m/z (rel. int.%, the latter//natural-2) 603
[(M+Na)+, 100//100], 604 [(M+1+Na)+, 41.8//36.1], 605 {[M+2+Na and M(d2)+Na]+, 47.5//9.0}. The
incorporation of 5-d2 is estimated as ca. 30% in 2.
These and the previously reported (the ∆1 isomer of 6 was not converted into natural fusicoccins)15
results lead to the conclusion that the fusicoccane hydrocarbon leading to the aglycons of fusicoccins
should now be revised to 5 from 4. The results also reveal that the first hydroxylation of 5 occurs on the
non-allylic 8β-position to afford 6 in the early metabolic process (Scheme 2).
Scheme 2. The metabolic process from geranylgeranyl diphosphate (GGDP) to fusicoccins in Phomopsis (Fusicoccum)
amygdali
The direct formation of 6 from 5 was further supported from the feeding experiment using the 8-epimer
of 6 (8), which was synthesized easily from 7. The bio-transformation of 8 by the fungus afforded only
the corresponding ketone 9 (Scheme 3). This result clearly rules out the possibility of intervention of
either 8 or 9 in the formation of 6.
Consequently, as exemplified also by the case of the taxadiene biosynthesis,16 the structure of the
initially forming hydrocarbon proposed only on the basis of structure–similarity to that of the final
metabolites should not be accepted blindly without corroborative proof even for the skeleton which
obeys the ‘isoprene rule’. The conclusion on the fusicoccane-hydrocarbon is of primary importance for
§
In this medium, the production of fusicoccin J (2) dropped to 10–20 µg/cm3.

2404
Scheme 3. Reagents and conditions. (i) LiAlH4, THF (100%); (ii) TsCl, Py; (iii) LiEt3BH, THF (two steps, 20%);¶ (iv) P.
amygdali, 3 days (ca. 30%)
our current efforts to isolate a gene encoding the ‘fusicoccane synthase’ as a fungal novel diterpene
cyclase.17
Acknowledgements
We are grateful to Professor Antonio Graniti, University of Bari, Italy, for his help in providing the
fungus, P. amygdali, for T.S., and to Professor Koichi Ogiyama, Yamagata University, for GC–MS
measurements. This work was partially supported by the Ministry of Education, Science, Sports, and
Culture of Japan through Grant-in-Aid for Scientific Research to TS (No. 11460053). The financial
assistance of the Naito Foundation is also greatly appreciated.
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¶
The low yield of the reductate (8) was due to the elimination of the tosylate to form fusicocca-2,10(14),15(19)-trien-8α-ol.