Biosynthetic studies on citreohybridones, metabolites of a hybrid strain KO 0031 derived fromPenicillium citreo-virideB. IFO 6200 and 4692

摘要

J. CHEM. SOC. PERKIN TRANS. 1 1994 135 Biosynthetic Studies on Citreohybridones, Metabolites of a Hybrid Strain KO 0031 Derived from Penici//ium citreo-viride B. IF0 6200 and 4692 Seiji Kosemura,a Hiroshi Miyata,e Shosuke Yarnamura,*Sa Kumyul Albone! and Thomas J. Simpson *A a Department of Chemistry, Faculty of Science and Technology, Keio University, Hiyoshi, Yokohama 223, Japan School of Chemistry, University of Bristol BS8 ITS, UK incorporation of '3C-labelled acetate, formate and ethyl 3,5-dimethylorsellinate into citreohybridones using a hybrid strain KO 0031 derived from Penicillium citreo-viride B. IF0 6200 and 4692 has establishedthat their biosynthesisproceeds via a mixed polyketide-terpenoid (meroterpenoid) pathway. The novel metabolites, anditomin 1,' austin 22 and terretonin citreohybridones A 8 and B 9,' isocitreohybridones A 10 and B 3, were isolated from Aspergillus variecolor, Aspergillus ustus, 11,6 citreohybriddiones A 12 and B 13,6 and citreohybridonol and Aspergillus terreus (NRRL 6278) respectively.Based on 14, from the mycelium of the hybrid strain KO 0031 derived stable isotope incorporation studies involving labelled acetate from Penicillium citreo-viride B. IF0 6200 and 4692. and methionine. it has been shown that these metabolites Interestingly, they display antifeedant and insecticidal activities against Plutella xylostella. Initial biogenetic analysis suggested I that these metabolites could be formed by successive methyl migration and skeletal rearrangement of a sesterterpenoid containing five isoprene units or alternatively from a degraded triterpenoid.However, we now report biosynthetic experiments on citreohybridonol 14 ' using sodium 1,2-'3C2acetate, sodium '3Cformate, and ethyl ~arboxy-6-'~C,-3,5-di-methylorsellinate, which indicate that this metabolite also is formed via a mixed polyketide-terpenoid (meroterpenoid) Anditomin 1 Austin 2 biosynthetic pathway (see Scheme 1). Results and Discussion Initial biosynthetic experiments on citreohybridones were carried out using sodium 1,2-' 3C2acetate and sodium'3Cformate. According to essentially the same procedure described the ethyl acetate extract of the culture medium was directly chromatographed on silica gel. Further separation and purification by repeated preparative TLC Terretonin 3 afforded citreohybridonol 14 (0.92).Citreohybridonol 14 exists as an equilibrium between two different ring D tautomers originate from a mixed polyketide-terpenoid (meroterpenoid) in both CDCl, as well as CD30D, which causes difficulties in biosynthetic path~ay.~The key step in their biosynthesis signal assignments in the 13C NMR spectrum. Therefore, 14 involves C-alkylation of the tetraketide-derived intermediate, was treated with acetic anhydride-pyridine to afford citreo-3,5-dimethylorsellinic acid 4, with farnesyl pyrophosphate, hybridone A 8 and the I3CNMR assignment (Table 1) is based giving 5 as shown in Scheme 1. This intermediate is then on complete 'H NMR decoupling experiments coupled with cyclized and undergoes oxidation and other modifications to 2D-INADEQUATEexperiments on 1,2-' 3C2acetate-labelled produce the above metabolite^.^ citreohybridone A 8.The average level of enrichment was Recently, we isolated seven new metabolites named estimated to be 12.7 based on the relative heights of the Table 1 3CNMR dataDfor the incorporation of 1,2-"C,acetate into citreohybridone A 8 Carbon 6 JIHz Carbon 6 JIHz Carbon 6 JIHz 1 20.89 11 123.13 41.5 21 19.23 2 22.12 36.0' 12 134.04 22 22.12 36.0' 3 75.78 36.4 13 59.88 37.6 23 178.74 49.0 4 34.52 35.3 14 69.23 57.0 24 26.47 5 55.35 33.2 15 169.45 82.0 25 22.12 36.0' 6 76.71 33.2 16 131.96 82.9 26 170.62 59.5 7 37.70 17 198.96 27 20.89 59.5 8 41.10 36.5 18 9.52 28 52.32 9 51.64 41.5 19 169.65 57.0 MeCO 164.91 10 43.70 49.0 20 17.31 36.6 MeCO 21.55 I3CNMR spectra were taken on a JEOL JNM-GX 400 NMR spectrometer.Relative to TMS in CDCl,. Overlapped with other satellites' signals. 136 J. CHEM. SOC. PERKIN TRANS. 1 1994 / COSCoA Me -C02Na OH 0 C02R HO HO 6 / 0 / 0 0 OH 0 00 OH Austin 2 Terretonin 3 Citreohybridones Me-C02Na Methionine0 Scheme 1 I I I 1 ~i"''"''',~,.-TI111.711111~m~-ml--m~p-m-~~, 169.7 169.6 169.5 169.4 169.3 169.2 169.1 169.0 PPm Fig. 1 100.4 MHz "C NMR spectrum of citreohybridone A labelled from ethyl carboxy-6-' 3C,-3,5-dimethylorsellinate J.CHEM. SOC. PERKIN TRANS. I 1994 coupling satellites and natural abundance signals.? No sig-nificant differences in enrichment levels between the farnesyl and orsellinate derived carbons were observed. The 3Cformate-labelled sample showed signals resulting from three highly enriched carbons (300) corresponding to C-18 (S 9.52), C-21 (S 19.23) and C-28 (S 52.32). The resulting labellingpattern of citreohybridone A is summarized in Scheme 1, and suggests that this metabolite is formed uia a mixed polyketide-terpenoid biosynthetic pathway with the same biosynthetic intermediate 7 proposed as a precursor of terretonin 3. Further evidence for this was provided from incorporation of 3C-labelled ethyl 3,5-dimethylorsellinate into citreohybridonol 14 which was converted into citreo-hybridone A 8 as above.The ~arboxy-6-'~C,1-3,5-dimethylorsellinate-labelled citreohybridone A 8 showed signals resulting from two highly enriched carbons (1 100) corresponding to C-15 (S 169.45, 2Jcc 3.9 Hz) and C-19 (S 169.65, 2Jcc 3.9 Hz) (see Fig. 1). The labelling pattern of citreohybridone A 8 is summarized in Scheme 2 and confirms the intact incorporation of 33-dimethylorsellinate. The pathway shown in Scheme 2 is consistent with the pathway proposed for the biosynthesis of terretonin 3.4sRing contraction of the tetracycliccarbocation 6 t Enrichments were calculated by dividing the total intensity of the coupling satellites by the intensity of the non-coupled, natural abundance resonance.No systematic variation of intensities was observed. 0 HO OR OR HO HO I I 602Me OH A II @OR'0 0 AcO'. , 0 Cb2Me0 . #8 OH Terretonin Ciireohybridones Scheme 2 21 d 24 R = Ac; Citreohybridone A 8 R = Ac; lsocitreohybridoneA 10 R = Me; Citreohybridone B 9 R = Me; lsocitreohybridoneB 11 0 . CitreohybriddioneA 12 Citreohybriddione6 13 Citreohybridond14 is common to both pathways with the subsequent alternative proton losses indicated in Scheme 2 producing the exocyclic methylene observed in terretonin or the endocyclic double bond in the citreohybridones. Thus the citreohybridones represent a further extension of the meroterpenoid pathway * of which andibenin 15, a co-metabolite of anditomin 1 in A.uariecolor, was the first representative. Interestingly, austin 2 has also been isolated from A. uariecolor. Metabolites related to austin have been isolated from Penicillium diuersum 4c and Emericella dentata,g and two unrelated metabolites which are almost certainly further products of the meroterpenoid path-way, fumigatonin 16 and paraherquonin 17 have been isolated from Aspergillus fumigatus and Penicillium paraherquei," respectively. The meroterpenoid pathway can now be seen to be relatively widespread in fungi. Andibenin 15 I --OAC 0 0 Fumigatonin 16 Paraherquoinin 17 J. CHEM.SOC. PERKIN TRANS. I 1994 Experimental Dimethyl 1,3-' 3C,malonate was obtained from Amersham International plc UK, ',C-labelled formate and acetate from ISOTEC inc., USA.Melting points were determined on a Mitamura Riken apparatus and uncorrected. Optical rotations were measured with a JASCO DIP-360 polarimeter and are recorded in units of lo-' deg cm2 g- '. IR spectra were recorded on a JASCO A-202 spectrophotometer. 'H NMR and 13C NMR spectra were recorded on a JEOL JNM-GX 400 NMR spectrometer in C2H4methanol or 2Hlchloroform; J values are recorded in Hz. Thin layer chromatography was performed using preparative (20 x 20 cm) glass plates coated with a 0.5 mm layer of silica gel (Merck Art. 5744 Kieselgel 60 PF254. UV light of wavelength 254 nm was used to visualize chromatograms. Incorporation of Sodium 1,2-' 3C2Acetate and Sodium ',C Formate into Citreohybridono1.-Polished rice (540 g) in deionized water (1.4 dm3) including sodium 1,2-' ,C,acetate (1 g) or sodium ',Cformate (1 g) was cooked using an electric rice cooker (100 "C, 20 min), and then transferred into an Erlenmeyer flask (3 dm3 x 5).This was sterilised (121 "C, 20 min at 2.1 atm) and then inoculated with a suspension of mycelium of the hybrid strain KO 0031 in sterilised water and incubated at room temperature for 30 days. The culture was extracted with acetone and then ethyl acetate. The combined extracts were partitioned between ethyl acetate and water. The ethyl acetate extract (1 0.0 g) was directly chromatographed on silica gel (40 g, silica gel 60 K070, 70-230 mesh, Katayama Chemical). After elution of higher fatty acids and their esters with benzenes, further elution with benzene ethyl acetate (3 :1) afforded a pale yellow oil (1 80 mg), which was further separated by repeated preparative TLC (Kieselgel PF254) using acetone- CHCl, (1 :20 -30), acetone-hexane (1 :1.5 -2) and then ethyl acetate-benzene (1 :3) to give citreohybridonol 14 as a colourless oil (92.2 mg, 0.92): a;' +67.3 (c 0.066, CHCI,); (M+, 500.240; C,,H,,O, requires M, 500.241); v,,,(film)/cm~' 3200, 1770, 1740 and 1620; major tautomer (60): G(CDC1,) ethyl acetate (3: 1) afforded a pale yellow oil (560 mg), which was further separated by repeated preparative TLC (Kieselgel PF254) using acetone-CHCl, (1 :20-30), acetone- hexane (1 :1.5-2) and then ethyl acetate-benzene (1 :3) to give citreohybridonol in 1.5yield.Acetylation of Citreohybridonol 14.-A solution of com-pound 14 (74.5 mg) in acetic anhydride (10 cm3)-pyridine (20 cm3) was stirred at 0 "C for 16 h after which work-up gave an oil which was subjected to preparative TLC using benzene- ethyl acetate (5:2) to afford citreohybridone A (54) and isocitreohybridone A (1 l), respectively. Citrephybridone A 8 formed colourless prisms, m.p. 261.5-263 "C in a sealed tube (from benzene-hexane); ah9 -25.4 (c 1.00, CHCl,) (M+, 542.249. C,,H3,09 requires M, 542.251); v,,,(film)/cm~' 1775, 1740, 1715, 1660 and 1240;G,(CDCl,) 0.90 (3 H, s, 25-H,), 0.93 (3 H, s, 24-H,), 1.27 (3 H, s, 2O-H,), 1.14 (3 H, s, DH,), 1.42 l H,ddd,J13.4, 13.4,6.2,I-Ha(ax),1.63(3H,s,18-H3),1.71 l H,s,SHa(ax), 1.65-1.82(2H,m,2-H,), 1.75 3H,dd, J2.4, 1.5, 21-HP (eq), 2.16 (1 H, m, 1-H), 2.16 (3 H, s, 3-OAc), 2.29 l H,d, J 14.7,7-Ha(ax), 2.31 (3 H, s, 15-OAc), 2.51 l H, dq, J 2.4,2.4,9-Ha(ax),2.741 H,dd, J14.7,4.3,7-HP(eq), 3.66(3 H, s, 14-CO,Me), 4.65 (1 H, dd, J3.4, 1.8, 3-H), 4.71 (1 H, d, J 4.3, 6-H) and 5.68 (1 H, s, 11-H); S,(CDCl,) 9.52 (9, C-18), 17.31 (9, C-20), 19.23 (9, C-21), 20.89 (t and q, C-21 and C-27), 21.44 (9, C-30), 22.12 (t, q and q, C-2, C-22 and C-25), 26.47 (9, C-24), 34.52 (4, C-4), 37.70 (t, C-7), 41.10 (s, C-8), 43.70 (s, C-lo), 51.54 (d, C-9), 52.32 (9, C-28), 55.35 (d, C(5), 59.88 (s, C-13), 69.23 (s, C-14), 75.68 (d, C-3), 76.71 (d, C-6), 123.13 (d, C- 1 I), 131.96 (s, C-16), 134.0 (s, C-12), 164.91 (s, C-29), 169.45 (s, C-15), 169.65 (s, C-19), 170.62 (s, C-26), 178.74 (s, C-23) and 198.96 (s, C- 17).Isocitreohybridone A 10 was a colourless oil: ah9., +22.6 (c 1.O, CHCl,) (M', 482.228. C30H3809-CH3C02H requires 482.230); v,,,(film)/cm-' 1770, 1740, 171 0, 1670, 1245, 1225, 1175 and 1 135;6(C,D,) 0.73 (3 H, S, 25-H,), 0.76 (3 H, S, 24-H,), 1.19(1 H, ddd, J 13.4, 13.4,6.2, 1-Ha), 1.25 (3 H, S, 2O-H,), 1.58 (3 H, S, 17-OAc), 1.62 (3 H, S, 18-H3), 1.65 (3 H, S, 3-OAc), 1.67 5.67(1H,brs,ll-H),4.72(1H,d,J3.9,6-Ha),4.65(1H,dd,J(2 H, m, 2-H,), 1.69 (3 H, dd, J2.4, 1.6, Zl-H,), 1.80 (3 H, s, 2.5, 3-HP), 3.67 (3 H, S, B-H,), 3.63 (1 H, d, J 14.2, 7-Ha), 2.51 (1 H, dd, J 14.2,4.4,7-Hp), 2.02 (3 H, S, 3-OAC), I .87 (3 H, s, 2l-H,), 1.33 (3 H, s), 1.32 (3 H, s), 0.94 (3 H, s) and 0.89 (3 H, s); other signals (S 2.25-1.25, 9 H) are overlapped one with another; minor tautomer (40): G(CDC1,) 5.83 (1 H, br s, 11-H), 4.78 (1 H, d, J3.9,6-Ha), 4.68 (1 H, dd, J2.5,2.5,3-Hp), 3.61 (3 H, S, Z-H,), 2.93 (1 H, d, J 14.2, 7-Ha), 2.75 (I H, dd, J 14.2,4.4, 7-Hp), 2.40 (1 H, dd, J2.4,9-H), 2.07 (3 H, S, 3-OAc), 1.87 (3 H, s), 1.43 (3 H, s), 1.25 (3 H, s), 0.97 (3 H, s) and 0.91 (3 H, s); other signals (S 2.25-1.25, 8 H) are overlapped one with another. Incorporation of Ethyl 3,5-Dimethylorsellinate into Citreo- hybridono1.-Ethyl ~arboxy-6-'~C,-3,5-dimethylorsellinate (96.0 atom 13C; 90.8 mg) was dissolved in hot distilled water (80 cm3) containing 'Tween 80' detergent (8 cm3).This sterilised solution was distributed evenly, by injection through the mycelial mat, into 4-day old stationary cultures of the hybrid strain KO 0031 (360 g of cooked rice in each of four 3-dm-, Erlenmeyer flasks). The cultures were then incubated for a further 31 days at 25 "C. The acetone extract was concentrated under reduced pres- sure to an acetone-free aqueous solution (1 dm3) and then extracted with ethyl acetate (8 dm3). The combined extracts (dark brown syrup, 4.9 g) were partitioned between ethyl acetate and water. The ethyl acetate extract (4.9 g) was directly chromatographed on silica gel (60 g, silica gel 60 K070, 70-230 mesh Katayama Chemical). After elution of higher fatty acids and their esters with benzene, further elution with benzene- 22-H,), 1.99(1 H,s, 5-H), 2.26(1 H,ddd, J 14.3,4.8,2.0, 1-Hp), 2.39 (1 H, dq, J2.4, 2.4, 9-H), 2.77 (1 H, dd, J 14.3, 4.4, 7-H/3), 3.25(3H,s,19-OMe),3.70(1H,d,J14.4,7-Ha),4.46(1H,d,J 4.4, 6-H), 4.69 (I H, dd, J3.3, 1.8, 3-H) and 5.91 (1 H, s, 1 I-H); G(CDCl,)7.91 (q), 17.61 (q),20.67(q),20.98(t),21.04(q),21.13 (q), 22.07 (t), 22.33 (q), 25.23 (q), 26.27 (q), 34.30 (s), 36.51 (t), 43.61 (s), 43.96 (s), 51.54(d), 51.72 (q), 52.74(s), 54.49 (d), 72.39 (s), 76.05 (d), 77.67 (d), 124.97 (d), 125.82 (s), 137.00(s), 164.96 (s), 169.19(s), 170.17(s), 174.18(s), 179.05 (s) and 203.90 (s).Ethyl 2,7-' 3C2-2,4-Dihydroxy-3,5,6-trimethylbenzoate (33- Dimethylorsellinate).-The '3C2-labelled 3,5-dimethylorsel- hate was prepared as previously described ' from diethyl 1,3-3C,malonate (1 .O g, 6.17 mmol) and 4-methylhex-4-en-3-one (730 mg, 6.52 mmol).The crude brown oily product was puri- fied by flash chromatography eluting with a 3-7 ethyl acetate in light petroleum (b.p. 40-60 "C) to give a pale yellow solid (230 mg, I .02 mmol, 16.5); dH(CDC13) 1.40 (3 H, t, J 7, CH,CH,), 2.11 (3 H, s, S-ArCH,), 2.16 (3 H, s, 5-ArCH3), 2.42 (3 H, s, 2-ArCH3), 4.39 2 H, q (d), J7 ('JCH3), CH,CH,, 5.30 (1 H, br s, 4-ArOH) and 1 1.56 (1 H, s (d) *JCH4, 2 ArOH; Sc(CDCl,), 7.9 (2 ArCH,), 11.8 (5 ArCH,), 14.2(3 ArCH,), 18.7 (CH,CH3), 61.2 (CHZCH,), 106.1 (C-1), 107.2 (C-2), 115.0 (C-3), 137.0 (C- 5), 159.4 (C-4), 159.5 (C-6) and 172.2 (CO,C,H,); m/z 226 (M+, 30), 180 (91), 152 (lo), 151 (100) and 120 (11) References 1 T.J. Simpson and M. D. Walkinshaw, J. Chem. Soc., Chem. Cornrnun., 1981,914. J. CHEM. SOC. PERKIN TRANS. 1 1994 2 K. K. Chexal, J. P. Springer, J. Clardy, R. J. Cole, J. W. Kirksey, J. W. Dorner, H. G. Cutler and W. J. Strawter, J. Am. Chem. SOC., 1976,98,6748. 3 J. P. Springer, J. W. Dorner, R. J. Cole and R. H. Cox, J. Org. Chem., 1979,44,4852. 4 (a)J. S. E. Holker and T. J. Simpson, J. Chem. SOC.,Chem. Commun., 1978, 626; (b) C. R. McIntyre, F. E. Scott, T. J. Simpson, L. A. Trimble and J. C. Vederas, J. Chem. SOC.,Chem. Commun., 1986, 502; (c)C. R. McIntyre, T. J. Simpson, D. J. Stenzel, A. J. Bartlett, E. O’Brien and J. S. E. Holker, J.Chem. SOC.,Chem. Commun., 1982, 78 1; (d) T. J. Simpson, Tetrahedron Lett. , 1981, 22, 3785; (e) S. A. Ahmed, F. E. Scott, D. J. Stenzel and T. J. Simpson, J. Chem. Soc., Perkin Trans. I, 1989, 807; (f)C. R. McIntyre, F. E. Scott, T. J. Simpson, L. A. Trimble and J. C. Vederas, Tetrahedron, 1989, 45, 2307. 5 S. Kosemura, K. Matsunaga, S. Yamamura, M. Kubota and S. Ohba, Tetrahedron Lett., 1991,32, 3543. 6 S. Kosemura, K. Matsunaga and S. Yamamura, Chemistry Lett., 1991, 1811. 7 S. Kosemura, H. Miyata, K. Matsunaga and S. Yamamura, Tetrahedron Lett. ,1992,33,3883. 8 T. J. Simpson, Chem. SOC.Rev., 1987,16, 123. 9 Y. Maebayashi, E. Okuyama, M. Yamasaki and Y. Katsube, Chem. Pharm. Bull., 1982,30, 191 1. 10 E. Okuyama, M. Yamasaki andY. Katsube, Tetrahedron Lett., 1984, 25, 8233. 11 E. Okuyama, M. Yamasaki, K. Kobayashi and T. Sakurai, Tetrahedron Lett., 1983,24, i113. 12 A. J. Bartlett, J. S. E. Holker E. O’Brien and T. J. Simpson, J. Chem. Soc., Perkin Trans. I, 1981, 198. Paper 3/03094F Received 1st June 1993 Accepted 28th September 1993