Diene, and \Beta

$Diene, and \Beta$

Biosci. Biotechnol. Biochem., 68 (7), 1608–1610, 2004 Note Fusicocca-3(16),10(14)-diene, and - and -Araneosenes, New Fusicoccin Biosynthesis-related Diterpene Hydrocarbons from Phomopsis amygdali

y Takeshi SASSA,1;2; Hiromichi KENMOKU,2 Kayoko NAKAYAMA,1 and Nobuo KATO3

1Department of Bioresource Engineering, Faculty of Agriculture, Yamagata University, Wakaba-cho, Tsuruoka 997-8555, Japan 2Course of the Science of Bioresources, The United Graduate School of Agricultural Sciences, Iwate University (Yamagata University), Wakaba-cho, Tsuruoka 997-8555, Japan 3The Institute of Scientiﬁc and Industrial Research, Osaka University, Mihogaoka, Ibaraki 567-0047, Japan

Received March 8, 2004; Accepted April 10, 2004

Further isolation and examination of fusicoccane Fusicoccin A (FC A, Fig. 1),1) one of unique diterpene hydrocarbons biosynthetically related to fusicoccin glucosides produced by Phomopsis amygdali, shows not from Phomopsis amygdali allowed us to identify new only potent plant plasma membrane Hþ-ATPase-acti- fungal diterpene hydrocarbons of fusicoccadiene and vating activity,2) but also novel effects on amphibian araneosene. These were assigned as (þ)-fusicocca- embryogenesis.3) Its aglycon is a polyoxygenated de- 3(16),10(14)-diene, and (þ)- - and (þ)--araneosenes. rivative of fusicocca-1,10(14)-diene with a 5/8/5- These findings led to the experimental clarification of tricyclic skeleton.1) We have recently isolated a novel the structures of the biosynthetic hydrocarbon inter- (þ)-fusicocca-2,10(14)-diene (1, Fig. 1) from P. amyg- mediates presumed earlier. dali F6 as one of the important genuine hydrocarbon intermediates in the early stage of FC biosynthesis,4) and Key words: fusicoccadiene; araneosene; Phomopsis confirmed its structure by a total synthesis.4) In the amygdali; diterpene hydrocarbon; fungal course of isolating 1 from mycelia of the other FC- metabolite producing strains (Niigata 2 and 74) of P. amygdali,5)

Fig. 1. Structures of Fusicoccin A, (þ)-Fusicocca-2,10(14)-diene (1), (þ)-Fusicocca-3(16),10(14)-diene (2), (þ)--Araneosene (3), and (þ)-- Araneosene (4), and Their Biosynthetic Relationships. 1 and 3, and 2 and 4 are respectively shown with ﬁlled and unﬁlled arrows in the fusicoccin biosynthesis from geranylgeranyl diphosphate (GGDP), together with the chemical isomerization of 2 to 1 and 20 shown with normal arrows.

y To whom correspondence should be addressed. Fax: +81-235-28-2862; E-mail: [email protected] Fusicoccadiene and Araneosene from Phomopsis amygdali 1609

Fig. 2. Total Ion Chromatogram from the GC-MS Analysis of the n- Hexane-soluble Portion Obtained from the Mycelial Extract of a 7- Fig. 3. Observations of Differential NOEs in a C6D6 Solution of 2. 1 Day Culture of Phomopsis amygdali Niigata 2. H-NMR (C6D6, J(Hz)): 0.92 (d, 7, 17-H), 1.06 (s, 18-H), 1.56 Conditions for the GC-EIMS analysis: Aligent 6890N GC-5973N (ddd, 13,7,5, 12-H), 1.60 (dd, 14.5,2.5, 1-H), 1.88 (ddd, 13,8,8, MSD/Wiley 7n.1 database system; HP-1MS capillary column 12-H), 1.96 (dd, 14.5,11.5, 1-H), and 2.44 (br t, 9, 2-H). (0.25 mm 30 m), 60 C (2 min)–150 C (30 C/min)–180 C (10 C/min)–210 C(2C/min). 7, 15-H), 4.68 and 4.73 (each m, 16-H); C (CDCl3): 19.9 (br, C-17), 21.0 and 21.1 (C-19/20), 23.1 (br, C-9), we detected unknown hydrocarbons (2, 3 and 4) 27.0 (C-15), 27.3 (br, C-5), 27.7 (C-13), 28.2 (C-18), structurally related to 1 at tR 13.56 (1), 13.77 (2), 30.4 (C-4), 31.8 (br, C-8), 35.7 (C-12), 36.7 (br, C-7), 14.13 (3), and 17.30 (4) min by a GC-MS analysis 42.2 (C-1), 44.5 (br, C-6), 45.3 (C-2), 50.5 (C-11), 101.6 (Fig. 2). Compound 2 showed MS data similar to those (C-16), 138.7 (br, C-10), 140.5 (br, C-14), and 160.2 of 1,4) and 2 and 4 were detected as intense total ion (C-3). These observations and comparisons of its NMR chromatogram (TIC) peaks together with the strong peak data with those of 1 revealed 2 to be the 3(16)-double- of 1 in the course of fungal growth. These findings bond isomer of 1.The1H-NMR spectrum of 2 in acid- stimulated us to isolate and examine these hydrocarbons non-removed CDCl3 for a few hours at ambient temper- from the fungus. We report here the isolation and ature varied in a large part to that of 1; H (CDCl3, structural determination of (þ)-fusicocca-3(16),10(14)- J(Hz)): 0.86 (d, 7, 17-H), 0.92 and 0.98 (each d, 7, H- diene (2), and the identification of (þ)--araneosene (3) 19/20), 0.93 (s, 18-H), 1.63 (br s, H-16), 2.62 (septet, 7, and (þ)--araneosene (4) from this fungus (Fig. 1). H-15), 1.95 and 2.30 (each d, 13, H-1), 2.48 (m, H-6).4) The n-hexane extract (2.1 g), including the basic and Furthermore, differential NOEs in a C6D6 solution of 2 neutral substances, was prepared in the usual way from at 50 C were observed as shown in Fig. 3, indicating mycelia obtained from the culture (48 Sakaguchi flasks) the cis configuration between -oriented 6-H and 2-H in of P. amygdali Niigata.5) This extract was carefully 2. Thus, the structure of 2 was assigned to be (þ)- separated by silica gel (Wakogel FC 40) flash chroma- fusicocca-3(16),10(14)-diene having the 2/6-cis config- tography, using n-hexane as the eluent, to give 9 uration (Figs. 1 and 3). A detailed NMR analysis of the fractions. Fractions 2, 3, 5 and 7 gave 1 (46 mg), 2 mixture of 1 and 2 in acid-non-removed CDCl3 just (12 mg, including a small amount of 1), 3 (colorless described revealed the presence of a new fusicoccadiene 0 0 solid, 1.2 mg), and 4 (colorless oil, 5 mg), respectively. (2 ) in the respective signal ratio of 3:1:1; 2 H (CDCl3, Fraction 3 was further purified by rechromatography J(Hz)): 0.88 (d, 7, 17-H), 0.94 and 1.00 (each d, 7, 19/ by a similar method to give 2 (colorless oil, 7 mg). 20-H), 1.03 (s, 18-H), 1.66 (br s, H-16), 2.70 (br septet, Compound 2 had ½D þ19 (c 0.18, CHCl3), and 7, H-15), 5.30 (br s, H-4)). These characteristic NMR 0 molecular formula C20H32 based on its HR-EIMS signals suggested that 2 was fusicocca-3,10(14)-diene þ data (m=z (M ): calcd. for C20H32, 272.2486; found, as one of the endo-cyclic double-bond isomers of 2 272.2484). Its EIMS data [m=z (%): 272 (Mþ, 10), 257 (Fig. 1). Isomeric hydrocarbons 1 and 20 derivable from (12), 229 (45), 201 (4), 187 (6), 173 (11), 161 (14), 135 2 were newly prepared by an acid treatment with p- 4) 1 (100), 121 (85)] were similar to those of 1. Its H- and toluenesulfonic acid in C6D6/(CD3)2CO at 55 C for 13C-NMR signals were fully assigned by COSY, DEPT 10 hr; an olefinic proton at 5.37 as the diagnostic signal and HMBC methods by using a 600 MHz NMR showing the presence of 20 was observed in the NMR spectrometer; H (CDCl3, J(Hz)): 0.88 (d, 7.1, 17-H), spectrum. A GC-MS analysis of the reaction mixture 0.93 and 0.99 (each d, 7, 19/20-H), 1.10 (s, 18-H), 1.48 clearly indicated the presence of a deuterated mixture of 0 and 1.90 (each dd, 15, 2.0, 1-H, and 15, 11.5, 1-H), 1 (tR 13.56 min) and 2 (tR 13.17 min) together with non- 1.53 and 1.81 (each ddd, 13, 7, 7, 12-H, and 13, 8.5, deuterated 2 (tR 13.77 min) in the respective ratio of 8.5, 12-H), 2.17 (m, 6-H), 2.35 (m, 2-H), 2.69 (septet, 8:1:1. The MS data for 1 showed principally those of 1610 T. SASSA et al. [16-2H]-1; molecular ions m=z (rel. intensity): 273 (100, derivation of 20 from 2 suggested the presence of the [16-2H]-1), 274 (24) and 275 (3). The spectrum of 20 fusicocca-10(14)-en-3-yl cation as the final carbocation also showed that of [16-2H]-20; molecular ions m=z (rel. in the formation of intermediate 1, which is generated by intensity): 273 (100) and 274 (38). Non-deutrated 20 three consecutive 1,2-hydride shifts during the further newly prepared by an H2SO4/(CH3)2CO treatment of 2 cyclization of (þ)--araneosene to fusicoccadienes. The showed MS data similar to those of 2 (Fig. 1). A further results from this study clarify the biosynthetic route of 1 acid (H2SO4/(CD3)2CO) treatment of this hydrocarbon from GGDP in the early stage of FC biosynthesis as mixture at room temperature led to a decrease of the shown in Fig. 1. olefinic proton signal at 5.37 in the NMR spectrum. A GC-MS analysis of the reaction mixture gave a single Acknowledgments TIC peak showing the presence of a deuterated mixture of 1; its molecular ions were also observed at m=z (rel. This work was partially supported by the Ministry of intensity) 273 (100), 274 (30) and 275 (5), and ions 274 Education, Culture, Sports, Science and Technology of and 275 increased in their relative intensity being Japan though grant-in-aid for scientific research (No. 2 3 partially due to [4,16- H2]-1 and [4,4,16- H2]-1, re- 13306009 to T. S.). spectively. On the other hand, a careful analysis of the GC-MS data from the mycelial extract of the fungus did References not show the presence of 20 in the diterpene hydrocarbon fraction. 1) Ballio, A., Brufani, M., Casinovi, C. G., Cerrini, S., Compound 4 showing MS data similar to those of Fedeli, W., Pellicciari, R., Santurbano, B., and Vaciago, -araneosene (dolabella-3(E),7(E),12(18)-triene in the A., The structure of fusicoccin A. Experientia, 24, 631– 6) GC-MS database (Fig. 2)) demonstrated ½D þ106 (c 635 (1968). 1 13 0.30, CHCl3), and H- and C-NMR spectra identical to 2) Wurtele, M., J.-Ottmann, C., Wittinghofer, A., and 6) those of ()--araneosene (½D 102 (c 1.0, CHCl3); Oecking, C., Structural view of a fungal toxin acting on a 14-3-3 regulatory complex. EMBO J., 22, 987–994 4 C(CDCl3): 142.5, 134.8, 132.5, 129.3, 126.0, 122.0, 48.4, 42.1, 40.3, 40.0, 38.7, 38.2, 28.3, 27.9, 24.3, 23.6, (2003). 21.7, 21.2, 16.3, and 15.3.6) Thus, 4 was determined to 3) Bunney, T. D., De Boer, A. H., and Levin, M., Fusicoccin signaling reveals 14-3-3 protein function as a novel step be (þ)--araneosene (Fig. 1). The isolation and identi- in left-right patterning during amphibian embryogenesis. fication of 4 from the mycelial extract allowed us to find Development, 130, 4847–4858 (2003). new congener 3 by a GC-MS analysis. Compound 3 4) Kato, N., Zhang, C.-S., Matsui, T., Iwabuchi, H., Mori, 6) showed MS data similar to those of -araneosene. It A., Ballio, A., and Sassa, T., Isolation of (þ)-fusicocca- 1 showed ½D þ133 (c 0.090, n-hexane), and H- and 2,10(14)-diene, a 5-8-5 tricyclic diterpene hydrocarbon 13C-NMR spectra identical to those of ()--araneosene biosynthetically related to the fusicoccin aglycon from 6) (½D 127 (c 3.04, n-hexane); 3 C (CDCl3): 143.7, Fusicoccum amygdali, and confirmation of its structure by 138.8, 137.8, 129.3, 127.5, 124.7, 51.5, 40.9, 38.2, 37.0, total synthesis. J. Chem. Soc. Perkin Trans. 1, 2473–2474 34.7, 27.5, 27.4, 27.1, 26.1, 21.8, 21.4, 20.7, 16.9, and (1998). 15.7).6) Thus, 3 was determined to be (þ)--araneosene 5) Tajima, N., Kume, H., Kanematsu, S., Kato, N., and (Fig. 1). Sassa, T., Chemical identification of fusicoccins from a Japanese isolate Niigata 2 of peach Fusicoccum canker An FC biosynthesis has been investigated in detail by 0 13 2 fungus (Phomopsis amygdali) and production of 3 - using [3- C,4- H2]mevalonolactone, demonstrating a deacetylfusicoccin A by the fungus. J. Pesticide Sci., 1,2-deuteride shift in the formation of the bicyclic 27, 64–67 (2002). hydrocarbon intermediate and then two consecutive 1,2- 6) Jeny, L., and Borschberg, H.-J., Synthesis of the dola- deuteride shifts during its further cyclization to FC A bellane diterpene hydrocarbon ()--araneosene. Helv. having a fusiccocca-1,10(14)-diene skeleton (cf. Fig. 1).7) Chim. Acta, 78, 715–731 (1995). We have recently isolated not only 1, but also its 8-ol 7) Banerji, A., Hunter, R., Mellows, G., Sim, K.-Y., and from the fungus and demonstrated that their synthetic Barton, D. H. R., Use of deuterium as a tracer with 13C deuterides were clearly incorporated into both FCs A nuclear magnetic resonance spectroscopy in following and J.8) The structural elucidation of 2 and 20, and the deuteride migration in terpenoid biosynthesis: mechanism identification of 3 and 4 just described led to putative of geranylgeranyl pyrophosphate cyclization in fusicoccin biosynthesis. J. Chem. Soc. Chem. Commun., 843–845 structures for the FC-biosynthetic intermediates to (1978). clarify their stereostructures in the formation of 1 from 8) Sassa, T., Zhang, C.-S., Sato, M., Tajima, N., Kato, N., GGDP (Fig. 1). Two 11/5-bicyclic intermediates in- 2 and Mori, A., Conversion of [19- H2]fusicocca-2,10(14)- cluded in the biosynthesis of 1 are thus assigned to be diene into its 8-ol and fusicoccins by Phomopsis the (þ)--araneosen-15-yl cation and (þ)--araneosene (Fusicoccum) amygdali. Tetrahedron Lett., 41, 2401– based on the identification of 3 and 4. Furthermore, the 2404 (2000). occurrence of 2 together with 1 and the chemical