Thesis (M.S.)--University of Wisconsin--Madison, 1981. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 67-70).

The search for antifungal secondary metabolites from cruciferous plants exhibiting resistance to pathogenic fungi led to the investigation of <i>Eruca sativa </i>(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 <i>Cladosporium cucumerinum</i> in TLC biodetection assays.<p>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.<p>With the precursors in hand, the biosynthetic pathway(s) and biogenetic relationship between phytoalexins was investigated using the tuberous crucifers, <i>Brassica napus </i> L. ssp rapifera (rutabaga) and <i>B. rapa </i> (turnip), and detached leaves of <i>Erucastrum gallicum </i> (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).<p>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.<p>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.<p>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).

The search for antifungal secondary metabolites from cruciferous plants exhibiting resistance to pathogenic fungi led to the investigation of <i>Eruca sativa </i>(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 <i>Cladosporium cucumerinum</i> in TLC biodetection assays.<p>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.<p>With the precursors in hand, the biosynthetic pathway(s) and biogenetic relationship between phytoalexins was investigated using the tuberous crucifers, <i>Brassica napus </i> L. ssp rapifera (rutabaga) and <i>B. rapa </i> (turnip), and detached leaves of <i>Erucastrum gallicum </i> (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).<p>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.<p>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.<p>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).

Thesis (M. Phil.)--University of Hong Kong, 1981.

published_or_final_version / Botany / Master / Master of Philosophy

M.Sc. / On exposure to abiotic or biotic stresses, plants initiate a cascade of metabolic reactions, some of which lead to the biosynthesis of secondary metabolites with roles in self defense. Phytoalexins are a class of secondary metabolites synthesized de novo in response to microbial attack by activation of certain biosynthetic pathways. Cruciferae phytoalexins are all indole based with a carbon, nitrogen and sulfur containing constituent on the 3’ position of the indole ring. This common similarity of all Cruciferae phytoalexins suggests that the plants all share a common indole precursor. Camalexin is the primary phytoalexin of Arabidopsis thaliana. De novo synthesis of camalexin upon infection, as well as its antimicrobial nature supports its role in disease resistance. Evidence exists that suggests the inducible biosynthesis of camalexin involves steps of the tryptophan pathway, along with an increase in transcript and protein levels of the tryptophan pathway enzymes after microbial infection. Bacterial LPS (lipopolysaccharide) has been described as one of the microbe/pathogenassociated molecular patterns (M/PAMPs) capable of eliciting the activation of the plant innate immune system. LPS is an integral component of the cell surface of Gram-negative bacteria. It is a complex which is exposed to the external environment, and is thus involved with external interactions of the bacteria. The hypothesis investigated in this dissertation is that LPS, as a lipoglycan PAMP, results in activation of signal transduction pathways involved in defense that lead to the production of the defense metabolite, camalexin. Furthermore, that the genes CYP71B15, CYP79B2 and TSB are up-regulated in response to LPS during camalexin biosynthesis via the tryptophan pathway. To test this hypothesis, camalexin production was investigated through a combination of analytical techniques including thin layer chromatography (TLC), high performance liquid chromatography (HPLC), gas chromatography (GC), ultra pressure liquid chromatography-mass spectrometry (UPLC-MS) and fluorescence spectroscopy. Genes in the camalexin biosynthetic pathway were investigated by two-step reverse transcription polymerase chain reaction (PCR), GUS reporter gene assays and quantitative real time PCR (RT-qPCR).

Hop cell wall material (CWM) extracted from hop leaves (hamulus lupulus) was purified and characterized. The total sugar content, uronic acid content and monosaccharide composition of the CWM were determined. Galacturonic acid is the major component in the CWM. A mixture of unsaturated oligogalacturonides were released from purified hop CWM by autoclaving. The biological activity of these oligomers was tested for their ability to elicit phytoalexins. The oligomer with hexagalacturonic acid possessed the greatest biological activity. Column chromatography and high-pH anion exchange chromatography were used for the sample separation and purification. Fast-atom-bombardment mass spectrometry (FAB-MS) was used for the structure elucidation. The FAB-MS spectrum showed that the unsaturated galacturonosyl residue was located at the nonreducing terminus of the oligomer. / Graduation date: 1993

Chemical investigation of the transformation of the crucifer phytoalexins brassinin, brassilexin and camalexin by the phytopathogenic fungus <i>Alternaria brassicicola</i> was carried out. The objectives of this study included:<p> 1) the isolation and characterization of the metabolites of biotransformation of brassinin, brassilexin and camalexin by <i>A. brassicicola</i>;<p> 2) determination of the antifungal activity of these phytoalexins and their metabolites against <i>A. brassicicola.</i><p> The phytoalexins were synthesized and characterized using HPLC retention time tR, 1H NMR, 13C NMR, LC-MS and HRMS-ESI data. The metabolites of the biotransformation were also synthesized and characterized similarly. The metabolism of each phytoalexin and their metabolites was studied by analyzing broth extracts by HPLC. The percent inhibition of growth of <i>A. brassicicola</i> was determined by radial growth mycelial assays.<p> The biotransformation of brassinin by <i>A. brassicicola</i> afforded Nb-acetyl-3-indolylmethylamine via indole-3-methylamine intermediate. Brassilexin was metabolized to 3-(amino)methyleneindoline-2-thione by the reduction of the isothiazole ring. Camalexin did not appear to be metabolized or the metabolism was very slow. The results of biotransformation and bioassay studies established that the metabolism of brassinin by <i>A. brassicicola</i> was a detoxification process. However, these studies using brassilexin did not provide a rigorous conclusion. Camalexin showed strong inhibition of growth against <i>A. brassicicola</i> suggesting its importance in defense against this pathogen.

The focus of my research was on the secondary metabolites produced by crucifer plants under stress and their biological activity against fungi. Both cultivated and wild plants were investigated to isolate phytoalexins and phytoanticipins, and determine their metabolite profiles.<p>The first chapter of this thesis describes cruciferous plants and their most important pathogenic fungi. These plants are divided into three groups: oilseeds, vegetables and wild species. The metabolites isolated from these plants and their biosynthetic studies are reviewed. In addition economically important necrotrophic fungi such as <i>Leptosphaeria maculans</i>, <i>Alternaria brassicae</i>, <i>Sclerotinia sclerotiorum</i> and <i>Rhizoctonia solani</i> are also reviewed along with their phytotoxins. <p>The second chapter of this thesis describes the detection, isolation, structure determination, syntheses of stress metabolites and biological activity of these metabolites against <i>L. maculans</i>, <i>S. sclerotiorum</i> and <i>R. solani</i>. The investigation of cauliflower led to the isolation of seven phytoalexins: 1-methoxybrassitin (55), spirobrassinin (71), isalexin (64), brassicanal C (60), caulilexins A (106), B (107), and C (105). The phytoalexins caulilexins A (106), B (107) and C (105) were reported for the first time. Caulilexin A (106), having a disulfide bridge, showed the highest activity against S. sclerotiorum and R. solani among the known phytoalexins. Similarly four phytoalexins: 1-methoxybrassitin, brussalexins A (121), B (117) and C (118) along with four metabolites: ascorbigen (51), diindolylmethane (50), 1-methoxy-3,3-diindolylmethane (119) and di-(1-methoxy-3-indolyl)methane (120) were isolated from Brussels sprouts. The phytoalexins brussalexins A (121), B (117) and C (118) are new metabolites. Brussalexin A (121) is the only cruciferous phytoalexins having an allyl thiolcarbamate functional group. The metabolite 1-methoxy-3,3-diindolylmethane (119) is reported for the first time.<p>The investigation of brown mustard for polar metabolites led to the isolation of indole-3-acetonitrile (76) and spirobrassinin (71) along with isorhamnetin-3,7-diglucoside (134). Investigation of wild species such as Asian mustard, sand rocket, wallrocket, hedge mustard and Abyssinian mustard for production of stress metabolites led to the isolation of indole-3-acetonitrile (76), arvelexin (84), 1,4-dimethoxyindole-3-acetonitrile (137), rapalexins A (138) and B (142), methyl-1-methoxyindole-3-carboxylate (59) and metabolites bis(4-isothiocyanotobutyl)-disulfide (139), 5-(3-isothiocyanato-propylsulfanyl)-pentylisothiocyanate (136) and 3-(methylsulfinyl)-propylisothiocyanate (135). <p>Two metabolites were also isolated from Brussels sprouts and brown mustard; however, these structures are not yet determined. The metabolites 1,4-dimethoxyindole-3-acetonitrile (137) and 5-(3-isothiocyanato-propylsulfanyl)-pentylisothiocyanate (136) are reported for the first time.

Plants produce complex mixtures of secondary metabolites to defend themselves from pathogens. Among these defenses are metabolites produced de novo, phytoalexins, and constitutive metabolites, phytoanticipins. As a counter-attack, pathogenic fungi are able to transform such plant defenses utilizing detoxifying enzymes. This thesis investigates the metabolism of two important cruciferous phytoalexins (brassinin (33) and camalexin (39)) by the phytopathogenic fungus Botrytis cinerea and the metabolism of cruciferous phytoanticipins (glucosinolates and derivatives) by three economically important fungi of crucifers Alternaria brassicicola, Rhizoctonia solani and Sclerotinia sclerotiorum to investigate their role in cruciferous defense. In the first part of this thesis, the transformations of brassinin (33) and camalexin (39) by B. cinerea were investigated. During these studies a number of new metabolites were isolated, their chemical structures were determined using spectroscopic techniques, and further confirmed by synthesis. Camalexin (39) was transformed via oxidative degradation and brassinin (33) was hydrolyzed to indoly-3-methanamine (49). The metabolic products did not show detectable antifungal activity against B. cinerea, which indicated that these transformations were detoxification processes. Camalexin (39) was found to be more antifungal than brassinin (33). In the second part of this thesis, the metabolism of glucobrassicin (86), 1-methoxyglucobrassicin (87), 4-methoxyglucobrassicin (90), phenylglucosinolate (65), and benzylglucosinolate (66), the corresponding desulfoglucosinolates and derivatives by three fungal pathogens (A. brassicicola, R. solani and S. sclerotiorum) was investigated and their antifungal activity against the same pathogens was tested. Aryl iii glucosinolates 65 and 66 were metabolized by A. brassicicola but not by R. solani or S. sclerotiorum, whereas indolylglucosinolates were not metabolized by any pathogen. Indolyl desulfoglucosinolates (159 and 233) were transformed by R. solani and S. sclerotiorum to the corresponding carboxylic acids and indolyl acetonitriles 40, 102, and 103 were also metabolized to the corresponding carboxylic acids by all pathogens. None of the glucosinolates or their desulfo derivatives showed antifungal activity, but some of their metabolites showed low to very high antifungal activities. Among these metabolites, diindolyl-3-methane (113) showed the highest antifungal activity, and benzyl isothiocyanate (170) showed higher inhibitory effect against R. solani and S. sclerotiorum, but did not inhibit the growth of A. brassicicola. The cell-free extracts of A. brassicicola, R. solani, and S. sclerotiorum were tested for myrosinase activity against several glucosinolates. The cell-free extracts of mycelia of A. brassicicola displayed higher myrosinase activity for sinigrin (131), phenyl and benzyl glucosinolates 65 and 66, but lower activities for glucobrassicin (86) and 1-methoxyglucobrassicin (87); no myrosinase activity was detected in mycelia of either R. solani or S. sclerotiorum.