Phytoalexins from crucifers : structures, syntheses and biosyntheses

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)

Cyclo­linopeptide K butanol disolvate monohydrate

The title compound, C56H83N9O11S·2C4H10O·H2O, is a butanol–water solvate of the cyclo­linopeptide cyclo(Metsulfone1-Leu2–Ile3–Pro4–Pro5–Phe6–Phe7–Val8–Ile9) (henceforth referred to as CLP-K) which was isolated from flax oil. All the amino acid residues are in an l configuration based on the CORN rule. The cyclic nona­peptide exhibits eight trans peptide bonds and one cis peptide bond observed between the two proline residues. The conformation is stabilized by an α- and a β-turn, each containing an N—H⋯O hydrogen bond between the carbonyl group O atom of the first residue and the amide group H atom of the fourth (α-turn) and the third residue (β-turn), repectively. In the crystal, the components of the structure are linked by inter­molecular N—H⋯O and O—H⋯O hydrogen bonds into a two-dimensional network parallel to (001). The –C(H2)OH group of one of the butanol solvent mol­ecules is disordered over two sets of sites with refined occupancies of 0.863 (4) and 0.137 (4)

Novel flax orbitide derived from genetic deletion

Abstract Background Flaxseed orbitides are homodetic plant cyclic peptides arising from ribosomal synthesis and post-translation modification (N to C cyclization), and lacking cysteine double bonds (Nat Prod Rep 30:108-160, 2013). Screening for orbitide composition was conducted on the flax core collection (FCC) grown at both Saskatoon, Saskatchewan and Morden, Manitoba over three growing seasons (2009-2011). Two flax (Linum usitatissimum L.) accessions ‘Hollandia’ (CN 98056) and ‘Z 11637’ (CN 98150) produce neither [1−9-NαC]-linusorb B2 (3) nor [1−9-NαC]-linusorb B3 (1). Mass spectrometry was used to identify novel compounds and elucidate their structure. NMR spectroscopy was used to corroborate structural information. Results Experimental findings indicated that these accessions produce a novel orbitide, identified in three oxidation states having quasimolecular ion peaks at m/z 1072.6 (18), 1088.6 (19), and 1104.6 (20) [M + H]+ corresponding to molecular formulae C57H86N9O9S, C57H86N9O10S, and C57H86N9O11S, respectively. The structure of 19 was confirmed unequivocally as [1−9-NαC]-OLIPPFFLI. PCR amplification and sequencing of the gene coding for 18, using primers developed for 3 and 1, identified the putative linear precursor protein of 18 as being comprised of the first three amino acid residues of 3 (MLI), four conserved amino acid residues of 3 and/or 1 (PPFF), and the last two residues of 1 (LI). Conclusion Comparison of gene sequencing data revealed that a 117 base pair deletion had occurred that resulted in truncation of both 3 and 1 to produce a sequence encoding for the novel orbitide precursor of 18. This observation suggests that repeat units of flax orbitide genes are conserved and suggests a novel mechanism for evolution of orbitide gene diversity. Orbitides 19 and 20 contain MetO and MetO2, respectively, and are not directly encoded, but are products of post-translation modification of Met present in 18 ([1−9-NαC]-MLIPPFFLI)

Additional file 1: of Novel flax orbitide derived from genetic deletion

Table S1. Calculated flaxseed orbitide masses and sequences. (DOCX 22 kb

Additional file 3: of Novel flax orbitide derived from genetic deletion

Figure S2. 13C NMR spectrum of [1−9-NαC]-OLIPPFFLI (19). (PDF 145 kb

Additional file 6: of Novel flax orbitide derived from genetic deletion

Figure S5. 1H-13C HMBC spectrum of [1−9-NαC]-OLIPPFFLI (19). (PDF 189 kb

Additional file 4: of Novel flax orbitide derived from genetic deletion

Figure S3. 1H-1H COSY spectrum of [1−9-NαC]-OLIPPFFLI (19). (PDF 479 kb

Additional file 8: of Novel flax orbitide derived from genetic deletion

Figure S7. 1H-1H NOESY spectrum of [1−9-NαC]-OLIPPFFLI (19). (PDF 476 kb