Agric. Biol. Chem., 48 (6), 1471 ~ 1477, 1984 1471 Fungal Transformation of the Antifungal Isoflavone Luteone Satoshi Tahara, Shiro Nakahara, Junya Mizutani and John L. Ingham* Department of Agricultural Chemistry, Faculty of Agriculture, Hokkaido University, Kita-ku, Sapporo 060, Japan *Phytochemical Unit, Department of Botany, University of Reading, Whiteknights, Reading RG6 2AS, England Received November 4, 1983 An antifungal isoflavone, luteone [5,7,2/,4/-tetrahydroxy-6-(3,3-diniethylallyl)isoflavone] is metabolised by cultures of Aspergillus flavus and Botrytis cinerea into 2",3"-dihydro-3"- hydroxyluteone (AF- 1), 2//,3 '/-dihydrodihydroxyluteone, a dihydrofuranoisoflavone (BG-1) and a dihydropyranoisoflavone. The structures of the metabolites were elucidated by physico-chemical and chemical procedures. The major metabolites, AF-1 and BC-1 are much less toxic than luteone against Cladosporium herbarum. The possible metabolic pathways are briefly discussed. SJ^^'-Tetrahydroxy^-^B-dimethyl- demethylating or reducing the molecule.6'7* allyl)isoflavone (luteone, 1) was first isolated In the case of 5,7,2',4'-tetrahydroxy-8-(3,3- from the young fruits of Lupinus luteus L. dimethylallyl)isoflavanone (kievitone) and 3,9- (yellow lupin) and found to be strongly dihydroxy- 1 0-(3, 3-dimethylallyl)pterocarpan antifungal.1) Considerable quantities of 1 and (phaseollidin), it was found that metabolism the related fungitoxin 5,7,4'-trihydroxy-6- by Fusalium solani f. sp. phaseoli involved a (3,3-dimethylallyl)isoflavone (wighteone) also straightforward hydration of the sidechain to occur on the surface of lupin Ieaves2'3) where, yield kievitone hydrate8) and phaseollidin hy- in conjunction with other isoflavones [e.g. drate,9* respectively. Both these modification 5,7,4'-trihydroxyisoflavone (genistein) and products were much less antifungal than either its 2'-hydroxy analogue3)], they may offer kievitone or phaseollidin.8'9* some degree of protection against potential We have recently isolated several fungal fungal pathogens. It has long been recognized, metabolites of luteone (1) by the incubation of however, that some fungi have the ability to this lupin-derived isoflavone with cultures of metabolise and detoxify isoflavonoid com- Aspergillus flavus and Botrytis cinerea. The pounds,^ and in certain cases this process spectroscopic and chemical evidence to show seems to be closely linked with pathogen- that four of the metabolites have structures 2 icity.4'5) Because 1 may function as a pre- (luteone hydrate), 3,4 and 5a (in Scheme 1) are infectional antifungal agent (prohibitin) on presented in this paper. leaves of L. luteus and other Lupinus species,3) it would be useful to determine if this isofla- MATERIALS AND METHODS vonoid was also susceptible to detoxification by micro-organisms. Previous studies have de- Substrate. Luteone (1) used in this experiment was monstrated that several Fusarium fungi me- isolated from L. albus and L. luteus as previously.3* MS m/z (%): 355 (M++l, 22), 354 (M+, 88), 339 (6.9), 312 tabolise simple isoflavones such as biochanin (6.9), 311 (M+ -43, 100), 300 (5.6), 299 (M+ -55, 80), 298 A (5,7-dihydroxy-4/-methoxyisoflavone) and (5.5), 165 (ll), 134 (5.1). XH-NMR <5^.one-d6 (100 MHz): formononetin (7-hydroxy-4'-methoxyisofla- 1.65 and 1.78 (each 3H, two br s, 4"- and 5"-H3), 3.37 (2H, vone) by first hydroxylating, methylating, brd,.7=7.3Hz, 1"-H2), 5.28 (1H, brt, J= 7.3Hz, 2"-H), 1472 S. Tahara et al. 6.44 (1H, dd, 7=8.9 and 2.4Hz, 5'-H), 6.48 (1H, incom- circular zone (13 ~ 14mmi.d.) of the test material adsorb- pleted, 3'-H), 6.53 (1H, s, 8-H), 7.12(1H,d,.7=8.9Hz, 6'- ed homogeneously. After the acetone had evaporated, a H), 8.14 (1H, s, 2-H), 13.05 (s, 5-OH). spore suspension of Cladosporium herbarum Fr. AHU 9262 in a mediumwas sprayed onto the plate and in- Epoxyluteone tetraacetate (IAc). The prenyl sidechain of cubated in a moist atmosphere for 2~3 days at 25°C luteone tetraacetate1* was oxidized to the corresponding according to Homans and Fuchs.n) epoxide according to Gupta et al.10) ra-Chloroperbenzoic acid (26mg) was added to a chilled (ice-bath) solution of Properties of metabolites. AF-1 (2): Colorless needles luteone tetraacetate (42mg) in CHC13 (4ml) and the from acetone,.mp 224~226°C. UV, MS and XH-NMR reaction mixture was stirred at 15°C for 6 hr. After diluting data are shown in Table I. to 25 ml with CHC13,the mixture was washed successively AF-2 (3). MS m/z(%): 388 (M+, 16), 370 (3.9), 330 (14), with 5% aqueous NaHCO3and brine. The organic layer 329 (97), 300 (25), 299 (100), 298 (4.5), 167 (4.7), 165 (39), was dried over Na2SO4and concentrated to near dryness 134 (5.0). UV l^?Hnm: 264, 293sh; +NaOMe, 276, in vacuo. The concentrate was subjected to preparative 340sh; +A1C13, 274, 315, 373; +NaOAc, 272, 338 TLC (PTLC) in benzene-EtOAc (4: 1) to isolate IAc as a (the addition of solid boric acid regenerated the MeOH colorless oil (23mg). MS m/z (%): 538 (M+, 0.9), 496 (5.9) spectrum). ^-NMR ^Sone"d6 (100 MHz): 1.26 and 1.28 454, (47), 412 (33), 395 (8.3), 379 (6.5), 371 (8.1), 370 (100), (each 3H, both s, 4"- and 5"-H3), 2.62 (1H, dd, J= 14 and 354 (6.7), 353 (10), 352(9.8), 312 (29), 311 (16), 177 (8.4), 9.8Hz, 1"-Ha), 3.25 (1H, dd, 7=14 and 2.0Hz, 1"-Hb), 134 (6.7), 59 (56). ^-NMR ^S0"6"'6 (100 MHz): 1.21 and 3.65 (1H, dd, J= 9.8 and 2.0Hz, 2"-H), 6.44(1H, dd,/= 1.38 (each 3H, both s, 4"- and 5"-H3), 2.ll, 2.28, 2.35 and 10 and 2.4Hz, 5 -H), 6.49 (1H, incomplete d, 3'-H), 6.49 2.38 (each 3H, 4xs, 4xCH3CO), 2.62~3.04 (3H, m, 1"- (1H, s, 8-H), 7.13 (1H, d, J=10Hz, 6'-H), 8.15 (1H, s, 2- H2 and 2"-H), 7.08 (1H, d, /=2.2Hz, 3 -H), 7.09 (1H, dd, H), 13.23 (s, 5-OH). .7=8.9 and 2.2Hz, 5 -H), 7.41 (1H, d,.7=8.9Hz, 6'-H), BC-1 (4). Pale yellow needles from MeOH, mp 7.43 (1H, s, 8-H), 8.16 (1H, s, 2-H). 229~231.5°C. [a£3-10.6° (c=0.094, MeOH). UV, MS and XH-NMRdata are shown in Table I. Metabolic experiments. Cultures of Aspergillus flavus BC-2 (5a). MS m/z (%): 371 (M++l, 16), 370 (M+, AHU7049 and Botrytis cinerea AHU9424 were grown in 100), 300 (4.0), 299 (58), 298 (12), 165 (35), 134 (31). UV a liquid medium consisting of glucose (5 g), peptone (1 g), A^Hnm: 227sh, 263, 292sh; +NaOMe, 247, 262, 279; yeast extract (0.1 g) and H2O(100ml). The medium was +AICI3, 230sh, 273, 316, 372; +NaOAc, 263, 292sh. 1H- dispensed into 500 ml shaking flasks (100 ml/flask), steri- NMR ^Sone"d6 (100MHz): 1.34 and 1.40 (each 3H, both lized (120°C for 12min) and then inoculated with 1 ml of s, 4"- and 5"-H3), 2.62 (1H, dd, /=17 and 7.1 Hz, 1"-HJ, mycelial suspension of the appropriate fungus. The cul- 2.98 (1H, dd, /=17 and 5.4Hz, r'-Hb), 3.87 (1H, dd, /= tures were placed on a reciprocal shaker (lOOrpm) and 7.1 and 5.4Hz, 2"-H), 6.40 (1H, s, 8-H), 6.44 (1H, incubated (25°C) for 4 days, at which point a solution of 1 incomplete dd, 5'-ft), 6.48 (1H, incomplete d, 3'-H), 7.13 in ethanol (5mg/ml) was added to give a final luteone (1H, d,.7=8.8Hz, 6'-H), 8.16 (1H, s, 2-H), 13.13 (s, 5- concentration of about 50 /ig/ml. After a further 3 days OH). incubation, an equal volumeof acetone wasaddedto each Tetraacetyl BC-2 (5b). The tetraacetate (5b) was formed flask, the contents immediately filtered by suction, and the by treating BC-2 (5a) with a mixture of acetic anhydride- filtrate concentrated in vacuo (30°C) to remove the ace- pyridine (1 : 1). MS m/z (%): 538 (M+, 4.1), 496 (9.2), 437 tone. The concentrate was then acidified to pH 3 (HC1), (ll), 436 (84), 422 (10), 421 (93), 394 (15), 380 (9.9), 379 shaken ( x 3) with ethyl acetate, and the combined ethyl (100), 352 (7.8), 337 (27), 203 (13), 165 (6.0), 134 (7.1). acetate fractions were washed first with 5% aqueous ^å NMR <5xMsone'd6 (100 MHz): 1.46 (6H, br s, 4"- and NaHCO3and then with a saturated solution of NaCl. 5//-H3), 2.7-3.0 (2H, m, 1"-H2), 5.14 (1H, t, 7=4.6Hz, Evaporation of the ethyl acetate gave a residue from which 2"-H), 6.87 (1H, s, 8-H), 7.07 (1H, d, /=2.2Hz, 3/-H), the A. flavus- or B. cinerea-derived metabolites of 1 (AF- 7.08 (1H, dd, 7=9.0 and 2.2Hz, 5 -H), 7.39 (1H, d, J= 1 and AF-2 or BC-1, BC-2 and BC-3) were isolated 9,0 Hz, 6'-H), 8.07 (1H, s, 2-H). Four acetyl methyl groups by PTLCusing pre-coated silica gel plates (Merck, Silica were found at S 2.02, 2.10, 2.28 and 2.31 as four singlets Gel 60 F-254, 0.25 or 0.5mm thickness) and the fol- (each 3H).