Phytoalexins and other antifungal metabolites from crucifers: isolation, synthesis and biosynthesis

Phytoalexins and phytoanticipins are antimicrobial natural products involved in plant defence pathways against plant pathogens and other stresses. Most cruciferous phytoalexins are indole containing compounds with various side chains (dithiocarbamates, isothiocyanate, isonitrile, acetonitriles etc.). Many phytoanticipins of crucifers are glucosinolates and their metabolites, which have diverse structures and precursors, including aliphatic, phenyl or indolyl containing amino acids. Indole glucosinolates are derived from tryptophan, which is also a biosynthetic precursor to cruciferous phytoalexins, however the biosynthetic relationship between cruciferous phytoalexins and indole glucosinolates has not been clarified. In this work, investigation of antifungal metabolites from wild crucifers, synthesis of antifungal metabolites and potential perdeuterated biosynthetic precursors, biosynthesis of metabolites of salt cress and that of rutabaga will be described. Investigation of the wild crucifers Brassica tournefortii, Crambe abyssinica, Diplotaxis tenuifolia and Diplotaxis tenuisiliqua for production of elicited antifungal metabolites, resulted in the discovery of a new phytoalexin, 1ꞌ,4ꞌ-dimethoxyindolyl-3ꞌ-acetonitrile, from D. tenuisiliqua. 1ꞌ,4ꞌ-dimethoxyindolyl-3ꞌ-acetonitrile is the first dimethoxy substituted phytoalexin with strong antifungal activity against plant fungal pathogens. The remaining plant species produced known phytoalexins which were initially discovered in wild and cultivated species; all of them produced arvelexin. A novel phytoalexin isocyalexin A, was isolated from rutabaga roots irradiated with UV-light; this is the first isocyanide of plant origin. The second section of the thesis deals with the biosynthesis of metabolites of salt cress (T. salsuginea) and their biosynthetic relationships with indole glucosinolates. In that regard, non-isotopically labeled compounds and perdeuterated biosynthetic intermediates such as [2,2,4ꞌ,5ꞌ,6ꞌ,7ꞌ-2H6]glucobrassicin, [2H3CS;4ꞌ,5ꞌ,6ꞌ,7ꞌ-2H4]-1ꞌ-methoxybrassinin, L-[2ꞌ,4ꞌ,5ꞌ,6ꞌ,7ꞌ-2H5]tryptophan, [2H3CO]-1ꞌ-methoxyindolyl-3ꞌ-acetaldoxime, L-[2H3CS]methionine, [4ꞌ,5ꞌ,6ꞌ,7ꞌ-2H4]brassinin and 1ꞌ-methoxy-2ꞌ-methylbrassinin were administered to salt cress leaves. For the first time, the biosynthetic relationship between indole glucosinolates and cruciferous phytoalexins was established. Intact incorporations of hexadeuterated glucobrassicin ([2,2,4ꞌ,5ꞌ,6ꞌ,7ꞌ-2H6]glucobrassicin) into wasalexins A, B and biswasalexins A1 and A2 were observed. Based on the feeding experiment results, for the first time a biosynthetic route that includes both indole glucosinolates (glucobrassicin and 1ꞌ-methoxyglucobrassicin) and 1ꞌ-methoxybrassinin was proposed. The third section of the thesis is about biosynthesis of metabolites of rutabaga (Brassica napus). Rutabaga produces phytoalexins that differ on their side chains. Biosynthetic origin of their side chains was investigated by administering fully labeled tryptophan (L-[U-13C11,U-15N2]Trp) and other perdeuterated precursors to rutabaga roots which revealed that the carbon and nitrogen atoms of cyclobrassinin, rapalexin A, isocyalexin A and spirobrassinin are fully derived from tryptophan, and also both rapalexin A and isocyalexin A incorporated deuterium from glucobrassicin. [4',5',6',7'-2H4]-4'-Methoxybrassinin was incorporated into 4ꞌ-methoxycyclobrassinin and 4ꞌ-methoxydehydrocyclobrassinin but not into rapalexin A, isocyalexin A and isalexin. The biosynthetic pathway that leads to isalexin, rapalexin A and isocyalexin A was further investigated using perdeuterated biosynthetic precursors such as (R,S)-[2H3CO,5',6',7'-2H3]-4'-methoxyindolyl-3'-glycine, [2H3CO,5',6',7'-2H3]-4'-methoxyindole-3'-carboxaldehyde oxime, [2H3CO,5',6',7'-2H3]desulfoglucorapassicin and etc. It has been confirmed that the pathway involves series of rearrangements that allow transformation of side chain of tryptophan into the side chains of rapalexin A and isocyalexin A without any degradations. In conclusion, cruciferous phytoalexins are derived from glucobrassicin which is a precursor for 1ꞌ- and 4ꞌ-methoxyglucobrassicins. 1-Methoxylated phytoalexins are biosynthesized through 1ꞌ-methoxyglucobrassicin via 1ꞌ-methoxybrassinin. Similarly, 4-methoxy phytoalexins are derived from 4ꞌ-methoxyglucobrassicin through two distinct pathways: via 4ꞌ-methoxybrassinin and 4ꞌ-methoxyindolyl-3ꞌ-glycine.