Acta Bot. Neerl. 29 (2/3), May 1980, p. 193-197. Brief communications Biological implications of the stereo- chemistry of isoflavonoid phytoalexins A. Fuchs and C.A.X.G.F. SichererI* Laboratoriumvoor Fytopathologie,Landbouwhogeschool,Binnenhaven 9,6709 PDWageningen** To properly evaluate the role of phytoalexins in plant disease resistance it is useful to know whether or not the pathogens involved are capable of metaboliz- ing and detoxifying these compounds. In studying this question, rather than the mechanism of sé the of in degradation per specificity fungi degrading phy- toalexins seems crucial. have the breakdown of For several years we been studying in vitro pterocar- panoid phytoalexins by various fungal pathogens (for recent literature, see with Fuchs & Hijwegen 1979; Fuchs et al. 1980a,b). The pterocarpan skeleton its numbering system is given infig. la; the most common sites of substitutionin the differentpterocarpans are the 2,3, and 4-positions in the A-ring, the 8,9, and 10-positions in the D-ring and the 6a-position. Due to the two asymmetric carbon atoms (centres of chirality) and the ef- fusionofthe B and C-ring, asevident fromNMR data (Pachler & Underwood of Fig. 1. a. Pterocarpan skeleton with preferred sites substitution (incircles); b. (-)-pterocarpan with 1 6aa, llaa-configuration(R = H: 6aR, laR; R = OH: 6aS, 1 laS); c. (+ )-pterocarpan with 6ap, 1 lap-configuration (R = H: 6aS, llaS; R = OH: 6aR, llaR). ‘Present address; Laboratorium voor Organische Chemie, Technische Hogeschool, Delft, The Netherlands. “Communicated at the Meeting ofthe section for Plant Pathology (Royal Botanical Society ofthe 1979. Netherlands) onNovember 22, 194 A. FUCHS AND C. A. X. G. F. SICHERER 1967), all pterocarpans occur in two enantiomeric forms (figs, lb, c). In general, absolute to designate configurations of pterocarpans the R, S nomenclature is used. With this nomenclatureit depends on the nature of the substituent at the C- whether is 6a position a certain absolute configuration indicated as 6aR, 11 aR or 6aS, llaS & Sicherer-Roetman With the (Sicherer 1980). a, p nomenclature, on the other hand, such a confusion is avoided, though the a, P nomenclatureis this not a priori unambigious. According to designation, irrespective of the of the C-6a in lb has the nature substituent at a pterocarpan as depicted fig. 6aa, 11 aa-configuration, with a negative valueof whereas the configuration as in 1c the 1 with value of given fig. represents 6a3, laP-configuration, a positive No (see tableI, which summarizes substituentsand absolute configuration at C- and C-l for of 6a la some common pterocarpans). Generally, each pterocarpan only one enantiomeris found in nature; however, in some instances two enanti- in omers co-occur one plant species (for instance, (+ )-inermin ( = maackiain) and ( + )-inermin (Perrin & Cruickshank 1969; Braz Filho et al. 1973). Literaturedata show that in some fungi differences in substitution pattern do interfere with the of not or hardly fungus’ ability to carry out a given type the degradation. For instance, alfalfa pathogen Stemphylium botryosum can its host & Millar convert phytoalexin medicarpin (Steiner 1974) as well as (-)- inermin (Higgins 1975) and phaseollin (Higgins et al. 1974) To their corres- ponding isoflavans by reductive opening of the dihydrofuran ring C (cf. fig. 2). Apparently, the substitution pattern at C-8 and 10 in the D-ring (cf. table I) does determinethe substrate of the involved in this not specificity enzyme(s) type of degradation. However, (+)- 6a-hydroxy-inermin, which has a substitution pattern in the A and differsfrom latter D-ring similarto thatof(-)-inermin, but the pterocarpanin its absolute configuration and in possessing a hydroxyl group at C-6a, is not Fig. 2. Conversion of the two enantiomers of inermin and 6a-hydroxy-inermin by Stemphylium botryosum (because of the different numberingsystem ofpterocarpans and isoflavans the 6a-position of pterocarpans is comparablewith the 3-position of isoflavans). IMPLICATIONS OF STEREOCHEMISTRY OF PHYTOALEXINS 195 Table I. Substituents and absolute configurationat C-6a and C-11 a ofsome common pterocarpans. i.p. = isopentenyl Pterocarpan C-2 C-3 C-4 C-6a abs. C-8 C-9 C-10 conf. (-) medicarpin H OH H H ot H OCH3 H (-) pterocarpin H OCH H H •a O - CH - O H 3 2 H H - CH - O H (±)inermin(= H OH a or p O maackiain) 2 - - H ( ±) 6a-hydroxy-inermin H OH H OH a or p O CH O 2 H OCH H OH O - ch - 0 H (+) pisatin 3 P 2 ( + ) variabilin H OCH H OH P H H 3 OCHj (+) tuberosin H OH H OH P chromene (-O) H I H H (—) glyceollin (O-) chromene OH a H OH II H H OH (—) glyceollin chromene(-O) OH a H H H H (—) phaseollin OH a H (O-)chromene H H H H OH (—) phaseollidin OH a i.p. converted to its corresponding 3-hydroxy-inerminisoflavan (cf. fig. 2) by this On the other the certain fungus. hand, depending on fungus involved a pterocar- pan might be converted along very different pathways, as exemplified by pha- seollin, for which at least five types of conversion are known at this moment {cf. Van Etten& Pueppke 1976; Bailey et al. 1977). However, the available know- ledge, although limited, on the various pathways of fungal degradation of be does pterocarpans (for synopsis, see Hijwegen & Fuchs, to published) suggest thatsubstitution at C-6a as well as absolute configuration are amongthe of key determinantsfor degradation pterocarpans by a given fungal species. Another aspect to be taken into account in the evaluation of the role of phytoalexins is the sensitivity of fungi to these compounds. It should be realized that low or non-sensitivity can be eitheran intrinsic featureof the fungal species of of the involved or a consequence rapid breakdown phytoalexin concerned. the does in itself However, ability to degrade a phytoalexin not imply non- whereas f. and F. f. sensitivity ; Fusariumoxysporum sp. lycopersici oxysporum sp. sensitive in pisi are equally to pisatin a thin-layer chromatographic bioassay (Homans & Fuchs 1970) as well as in amycelial growth test (Fuchs & DeVries, to under normal be published), in vivo conditions the pea pathogen apparently cir- cumvents the toxic action of the phytoalexin through rapid degradation {cf. Fuchs et al. 1 980b). Unlike substrate specificity offungi with respect to phytoale- xin breakdown, sensitivity to phytoalexins does not seem to depend on the absolu- te configuration at C-6aand C-1 la. Although Perrin& Cruickshank (1969) and Van Etten (1976) disagree with each other aboutthe occurrence of a structure- activity relationship with regard to antifungal activity of pterocarpans, a given absolute configuration evidently is no prerequisite for fungitoxicity: (4-)-, (± )- and (-)-inermin inhibitedthe growth of Moniliniafructicola to the same extent. From the considerations given above it is evident that in such studies as currently performed by us on pterocarpanoid phytoalexin degradation hn exact is knowledge about absolute configurations indispensable. Substitution patterns 196 A. FUCHS AND C. A. X. G. F. SICHERER well conformationaldata the skeleton well be derived as as on pterocarpan may from NMR-analysis. However, up till now absolute configurations ofpterocar- pans have usually been established with reference to the absolute configuration of trifolirhizin (= (-)-inermin-(3-D-glucoside) as determinedby Ito et al. (1965) via degradation to (-)-paraconic acid. Because of the possibly significant role of the absolute configuration at C-6a and C-lla, it seems desirable to use less indirect methods, such as anomalous X-ray scattering, to establish the absolute of Use of directmethodsis stereochemistry pterocarpans. also more appropriate in theelucidation of biogenetic relationships among pterocarpans (Sicherer & Sicherer-Roetman, 1980) as well as in establishing the stereochemistry of iso- flavan analogues of pterocarpans (cf. Kurosawa et al., 1968, 1978). In this context, it is interesting to note that the absolute configuration of(-)-phaseollin Van HeuvelcI al. and DeMartinis al. as reported by den (1974) et (1977,1978) is contrary to current views, though not based on experimental evidence (Cof- DeMartinis et al., fen, pers. comm.; pers. comm.). As soon as theabsolute configuration of one pterocarpan has unequivocally that of others be determined been established, may by comparison, by means of circular dichroism (CD) or optical rotatory dispersion (ORD), rather than by estimation of one optical rotation value. For CD we simple [ot] D instance, using confirmed that the absolute configuration of pisatin from both Pisum sativum and Lathyrus odoratus is opposite to that of phaseollin (from bean), glycecollin (from soy bean) and medicarpin (from Jack bean). REFERENCES Bailey, J. A., R. S. Burden, A. Mynett & C. Brown (1977): Metabolism of phaseollinby Septoria nodorum and other non-pathogensof Phaseolus vulgaris. Phytochemistry 16:1541-1544. BrazFilho,R.,0. R. Gottlieb, S. L. Vieira Pinho,F. J. Queiroz Monte& A. I.daRocha (1973); Flavonoids from Amazonian Leguminosae. Phytochemistry 12; 1184 1186. DeMartinis, C., M. F. Mackay, D. R. Perrin & B. J. Poppleton(1977);The molecular structure of phaseollin. Tetrahedron Lett. 2981-2982. — & B. —, J. Poppleton (1978): The crystal and molecular structure of (-)-phaseollin.Tetrahedron 34; 1849-1852. Fuchs, A. &T. Hijweoen (1979): Specificity in degradationofisoflavonoid phytoalexins. ActaBot. Neerl. 28; 227-229. F.W. de Vries, C. A. Landheer & A. vanVeldhuizen —, (1980a): 3-Hydroxy-maackiainisoflavan, metabolic Fusarium f. a pisatin produced by oxysporum sp. pisi. Phytochemistry 19: 000-000. —,— & M. PlateroSanz (1980b):The mechanism of pisatin degradationby Fusarium oxysporum f. sp. pisi. Physiol. Plant Path. 16: 119-133. Heuvel, J. van den, H. D. van Etten, J. W. Serum, D. L. Coffen & T. H. Williams (1974): Identification of 1a-hydroxy phaseollone,aphaseollinmetabolite produced by Fusarium solani. Phytochemistry 13; 1129-1131. Higgins, V. J. (1975): Induced conversion of the phytoalexin maackiain to dihydromaackiainby the alfalfa pathogen Stemphylium botryosum. Physiol. Plant Path. 6; 5-18. A. Stoessl & M.