Phytoalexins from Crucifers: Structures, Syntheses and Biosyntheses

by Owiti, Denis Paskal

The search for antifungal secondary metabolites from cruciferous plants exhibiting resistance to pathogenic fungi led to the investigation of Eruca sativa (rocket). Chemical analysis of extracts showed arvelexin (51) as the only inducible component. Bioassay guided isolation (FCC, PTLC) and characterization (NMR, MS) led to the identification of two phytoanticipins, 4-methylthiobutyl isothiocyanate (166) and bis(4-isothiocyanatobutyl)disulfide (167). Compounds 166 and 167 inhibited the germination of spores of Cladosporium cucumerinum in TLC biodetection assays.

Next, isotopically labeled compounds containing 2H and 34S at specific sites were synthesized for use in studying of the biosynthetic pathway of crucifer phytoalexins and indolyl glucosinolates. Among the synthesized precursors, [4',5',6',7'-2H4]indolyl-3-[34S]acetothiohydroxamic acid (174a), the first sulfur-34 containing indolyl derivative was synthesized. In addition, non-isotopically labeled compounds (containing 1-methyl, 1-boc and 1-acetyl groups), that is, substrates used for precursor-directed biosynthesis, were also prepared.

With the precursors in hand, the biosynthetic pathway(s) and biogenetic relationship between phytoalexins was investigated using the tuberous crucifers, Brassica napus L. ssp rapifera (rutabaga) and B. rapa (turnip), and detached leaves of Erucastrum gallicum (dog mustard). The biosynthetic relationship between indolyl glucosinolates and phytoalexins was investigated in rutabaga and turnip. The indolyl moiety of the phytoalexins cyclobrassinin (28), rutalexin (33), spirobrassinin (34), brassicanate A (43), and rapalexin A (53), as well as indolyl glucosinolates glucobrassicin (70), 4-methoxyglucobrassicin (156), and neoglucobrassicin (199) was confirmed to derive from L-tryptophan (78). The 1-methoxy-containing phytoalexins, erucalexin (38) and 1-methoxyspirobrassinin (35) were shown to derive from indolyl-3-acetaldoxime (112) through 1-methoxyindolyl-3-acetaldoxime (116). The 1-methoxy substituent of neoglucobrassicin was also shown to derive from 1-methoxyindolyl-3-acetaldoxime (116).

The incorporation of indolyl-3-acetothiohydroxamic acid (174) into the phytoalexins cyclobrassinin, rutalexin, brassicanate A, rapalexin A, and spirobrassinin, and into the glucosinolate glucobrassicin is reported for the first time. On the other hand, incorporation of 174 into 4-methoxyglucobrassicin and neoglucobrassicin was not detected under current experimental conditions. Cyclobrassinin was incorporated into spirobrassinin among the NH-containing phytoalexins, whereas sinalbin B (31) [biosynthesized from 1-methoxybrassinin (18)] was incorporated into erucalexin and 1-methoxyspirobrassinin. The efficient metabolism of [SC2H3]brassicanal A into [SC2H3]brassicanate A suggested a biogenetic relationship between these two phytoalexins, whereas absence of incorporation of indolyl-3-acetonitrile (49) into rutabaga phytoalexins or indolyl glucosinolates indicated that 49 is not a precursor of these secondary metabolites under the current experimental conditions.

The rutabaga and turnip tubers separately metabolized 1-methylindolyl-3-acetaldoxime (170) and 1-methylindolyl-3-acetothiohydroxamic acid (178) into 1-methylglucobrassicin (201); however, no 1-methyl-containing phytoalexins were detected in the extracts. Rutabaga tissues metabolized 1-(tert-butoxycarbonyl)indolyl-3-methylisothiocyanate (180) into 1-(tert-butoxycarbonyl)brassinin (181) and 1-(tert-butoxycarbonyl)spirobrassinin (196), whereas 1-acetylbrassinin (184) was the only detectable metabolic product of 1-acetylindolyl-3-methylisothiocyanate (183) in both rutabaga and turnip root tissues.

In conclusion, indolyl-3-acetothiohydroxamic acid (174) seems to be the branching point between brassinin and glucobrassicin. The biosynthetic pathway of NH-containing crucifer phytoalexins was mapped and follows the sequence L-tryptophan, indolyl-3-acetaldoxime, indolyl-3-acetothiohydroxamic acid, brassinin (possibly through indolyl-3-methylisothiocyanate), and other phytoalexins. The biosynthetic pathway of 1-methoxy-containing phytoalexins follows a similar sequence through 1-methoxyindolyl-3-acetaldoxime (biosynthesized from indolyl-3-acetaldoxime).