Probing the Phytopathogenic Stem Rot Fungus with Phytoalexins and Analogues: Unprecedented Glucosylation of Camalexin and 6-Methoxycamalexin M. Soledade C. Pedras* and Pearson W. K. Ahiahonu Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon SK, Canada S7N 5C9 Received 19 February 2002; accepted 16 April 2002 Abstract—The remarkable metabolism of the cruciferous phytoalexins camalexin and 6-methoxycamalexin by the stem rot phyto- pathogen Sclerotinia sclerotiorum is reported. The biotransformations yielded camalexins glucosylated at N-1 or C-6 of the indole ring, with substantially lower antifungal activity than camalexins. A camalexin analogue with the positions N-1 and C-6 blocked was metabolized but at a much slower rate than the natural phytoalexins. The chemistry involved in the metabolism of natural camalexins and two new analogues, as well as their novel metabolites and respective antifungal activities is described. # 2002 Elsevier Science Ltd. All rights reserved. Introduction Camalexins are unique phytoalexins biosynthesized by a few wild crucifers. 1 Phytoalexins, induced plant chemi- cal defenses, can selectively inhibit plant pathogens and plant pathogens can react differently to phytoalexins. In instances where phytopathogens can circumvent phy- toalexins through metabolic detoxification, an envir- onmentally attractive strategy to control such plant pathogens could use selective inhibitors of the fungal detoxifying enzymes. 2 Towards this end, we have been investigating the metabolism of cruciferous phytoalexins by economically important phytopathogenic fungi. 1 Our previous work showed that camalexin (1) could be detoxified by the fungus Rhizoctonia solani Kuhn (Scheme 1) to 5-hydroxycamalexin (2), which was fur- ther transformed into metabolites 3 and 4, substantially less toxic to the pathogen than camalexin (1). 3,4 How- ever, we have also shown that not all plant pathogenic fungi or bacteria can transform camalexin (1). 5 In continuation of those studies, we examined the metabolism of naturally occurring camalexins 1 and 1a by the stem rot fungus Sclerotinia sclerotiorum (Lib.) de Bary. S. sclerotiorum causes stem rot disease in a vast range of plants and is considered a worldwide problem. 6 An evaluation of the biological activity of diverse phy- toalexins against S. sclerotiorum indicated that cama- lexin (1) could inhibit completely the growth of the pathogen. Subsequently, we established that metabolism and detoxification of camalexins 1 and 1a occurs via glyco- sylation, an unusual detoxification reaction in plant pathogenic fungi. 7 To probe the selectivity of these enzymatic reactions we designed camalexin analogues based on the structure of the detoxification products. The results of these studies are now reported for the first time. 8 Results and Discussion Camalexins 1–1c were synthesized from the respective indoles as described in the Experimental and previously reported. 9 Initial experiments established the minimum inhibitory concentration (5  10 4 M) of camalexin (1) and 6-methoxycamalexin (1a) to S. sclerotiorum. Subse- quently, camalexins 1 and 1a were incubated separately with S. sclerotiorum, culture samples were extracted and analyzed. HPLC analysis of the extracts of fungal cul- tures incubated with camalexins 1 and 1a indicated that both were completely metabolized in ca. 48h. After 24h incubation of S. sclerotiorum with camalexin (1), pro- ducts 5 and 5a were isolated, whereas 5, 5a and 5b were isolated after incubation with 6-methoxycamalexin (1a) 0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0968-0896(02)00208-0 Bioorganic & Medicinal Chemistry 10 (2002) 3307–3312 *Corresponding author. Tel.: +1-306-966-4772; fax: +1-306-966- 4730; e-mail: soledade.pedras@usask.ca