Part I Study On Secondary Metabolites Of Endophytic Fungi Periconia Sp. F-31 Part II Stachybotrys Chartarum Biosynthesis Of The Scorpion Stachybocin A

This dissertation consists of two parts:the secondary metabolites from endophytic fungus Periconia sp.F-31 in part I and the biosynthetic pathway of meroterpenoid Stachybocin A in Stachybotrys chartarum in part II.Part I Studies on the secondary metabolites from endophytic fungus Periconia sp.F-31.Bioassay results revealed that the EtO Ac extract of an endophytic fungus Periconia sp.F-31,which was originally isolated from the medicinal plant Annona muricata,displayed potent cytotoxicity against several human cancer cell lines.Our previous chemical investigation of its secondary metabolites led to the identification of several novel cytochalasans Periconiasins A-H featuring an unusual 9/6/5 tricyclic ring system,rare polyketide synthase-nonribosomal peptide synthetase(PKS-NRPS)hybrid metabolites Pericoannosins A and B,and a sesquiterpenoid Periconianone A with unusual skeletal.Biological studies revealed Periconiasins A and B exhibited selective and significant cytotoxicity against the HCT-8 and BGC-823 human tumor cells and Periconianone A showed significant neural anti-inflammatory activity.With the goal of finding novel secondary metabolites with potent biological activities,a further chemical investigation on this fungal strain has been systematically conducted.A variety of chromatographic methods(silica gel column chromatography(CC),C18 CC,Sephadex LH-20 CC,MCI CC and normal/reverse phase semi-preparative HPLC)were used to separate metabolites from Periconia sp.F-31.The structures of isolated compounds were elucidated on the basis of extensive spectroscopic data analysis(IR,UV,NMR,MS,and CD)and X-ray single-crystal diffraction.Sixty-three metabolites were obtained from this fungal strain,and twenty-seven(1-27)of them are new compounds,including eleven polyoxygenated eremophilane sesquiterpenes derivatives Periconianones C-M(1-11);four PK-AA hybrid molecules Periconiasins I-L(12-15);four prenylated polyketones Periconones B-E(16-19);four monoterpenoids 2-Carene-5,8-diol(20),2-Carene-8,10-diol(21),2-Carene-8-acetamide(22),and 8-Hydroxy-1,7-expoxy-2-menthene(23);two lactone derivatives 5-Hydroxy-4-(1,2-dihydroxyhexyl)-oxepin-1(6H)-one(24)and 5-Hydroxy-4-(1-hydroxyhexyl)-oxepin-1(6H)-one(25);one Ergosta-4,6,8(14),22-tetraene-3?-adenine(26);one derivative of riboflavin 6,7-Dimethyl-1-(1'-D-ribity)-quinazoline-2,4(1H,3H)-dione(27).Compound 1 is the first isoeremophilane reported containing a linkage of C-8/C-11.The absolute configurations of all new compounds were determined by electronic circular dichroism(ECD),X-ray single-crystal diffraction,circular dichroism data of the[Rh2(OCOCF3)4]complex,Mo2(OAC)4-induced ECD,and calculated ECD.In addition,we have elucidated the plausible biogenetic pathway of the new compounds.All isolated new compounds were evaluated for in vitro cytotoxic,anti-HIV,and neural anti-inflammatory activities.Periconianone E(3),Periconiasin I(12),and Periconone E(19)showed cytotoxic activity against human MCF-7 cancer cells with an IC50 value of 17.9,4.8,and 4.2 ?M,respectively;Periconianone H(6)displayed low cytotoxic activity against the HeLa cancer cell line with an IC50 value of 16.5 ?M;Periconianone H(6),Periconiasin J(13),and Periconone B(16)displayed weak anti-HIV activity with an IC50 value of 11.0,25.0,and 18.0 ?M,respectively;Periconianone D(2),G(5),K(9)Periconiasin K(14),L(15)and Periconone E(19)exhibited anti-inflammatory activity indirectly by suppressing LPS-induced NO production in BV2 cells with inhibition rates of 10.2%,18.3%,16.1%,50.0%,18.3%,and 25.8%at a concentration of 1.0 ?M,respectively,which is comparable to that of curcumin,a positive control with an inhibition rate of 12.9%.In conclusion,the chemical investigation on the endophytic fungus Periconia sp.F-31 led to the isolation of sisity-three compounds including twenty-seven new compounds.Part of compounds exhibited good bioactivities.The results not only increase the structure diversity of metabolites from Periconia sp.F-31,but also pave the way for further biosynthesis study of these metabolites,meanwhile providing novel lead compounds in the discovery of innovation drug.Part ? Studies on the biosynthesis of meroterpenoid Stachybocin A in Stachybotrys chartarumStachybocin A is a dimeric meroterpenoid and derived from polyketide-terpenoid hybrid pathway,which was consisted of two same phenylspirodrimanes units fused to a C5 alkyl chain.Stachybocin A displayed endothelin antagonists,anti-HIV,and cytotoxic activities.In addition,the bioassay showed that Stachybocin A had the significant antagonistic effects in the N-methyl-D-aspartic(NMDA)receptor binding assay,which indicated a promising lead compound for the treatment of inflammatory pain.However,Stachybocin A has low natural yield,complicated chemical synthesis steps in harsh reaction conditions,which limited further research for innovative drugs.So revealing the biosynthetic pathway of Stachybocin A and achieving its efficient biosynthesis,which will solve the problem of the source of Stachybocin A and provide a new way to synthesize Stachybocin A derivatives.On the basis of finding of producing strain Stachybotrys chartarum CGMCC 3.5365 and whole genome sequencing,the biosynthetic pathway of Stachybocin A will be elucidated by gene disruption,isolation and structural identification of mutants,heterologous expression,and identification of enzyme funcation.Until now,we have completed the following works:(1)the biosynthetic gene cluster of Stachybocin A was confirmed;(2)the genetic manipulation system of S.chartarum was established;(3)four genes involving in the Stachybocin A biosynthesis by targeted gene deletions were elucidated and the prenyltransferase of the C-3 of orsellinic acid and carboxylic acid reductase of them by heterogenous expression were identified.In summary,our results will provide a basis to elucidate the biosynthesis of meroterpenoid Stachybocin A in S.chartarum CGMCC 3.5365.