Secondary Metabolites From Two Endophytic Fungi Fusarium Sp. NJ-F5 And Paraphoma Sp. GR-F9

Endophytic fungi live in the tissues of healthy plants,and coexist and evolve with host plants for a long time.They constructed a special mutualistic symbiotic relationship with host plants,influencing the production of biologically active secondary metabolites.Fungal endophyes could synthesize small-molecule natural products with various structural types,such as terpenoids,polyketides,peptides and alkaloids.These compounds showed a variety of biological activities,such as antimicrobial,antitumor,antiviral,anti-inflammatory,hypolipidemic,and allelopathic activities.They are an important source of drug leads.In this study,50 strains of endophytic fungi were isolated from the plant Mahonia fortunei collected from Qingdao and Nanjing,and then small-scale fermentation was carried out based on the one strain many compounds（OSMAC）strategy.Liquid chromatography high-resolution mass spectrometry（LC-HRMS）technology was used to screen the chemical constituents of endophytic fungi.With the aid of Sci Finder database,an endophytic fungus Fusarium sp.NJ-F5 was found to produce gibberellins,a group of plant hormones with important agricultural values.The crude extracts of endophytic fungi were further evaluated for their antibacterial,antifungal,cytotoxic,anti-inflammatory,quorum sensing,and allelopathic activities.It was found that the crude extract of Fusarium sp.NJ-F5 had obvious cytotoxic activity.Meanwhile,another endophytic fungus Paraphoma sp.GR-F9 was screened out.It showed significant activity against Colletotrichum musae（ACCC 31244）.Therefore,in this thesis,the above-mentioned two fungal strains Fusarium sp.NJ-F5 and Paraphoma sp.GR-F9 were subjected to chemical investigation.Secondary metabolites of Fusarium sp.NJ-F5 were chemically studied herein.Under the guidance of nuclear magnetic characteristic signals of gibberellins in the carbon-13nuclear magnetic resonance spectrum(¹³C NMR),the targeted separation and purification of gibberellins were carried out by silica gel column chromatography,medium pressure liquid chromatography,and high performance liquid chromatography（HPLC）.Finally,16compounds were isolated and identified by MS,NMR and X-ray diffraction studies.These compounds included 13 diterpenoid-type gibberellins,3β,16α-dihydroxy-9,15-cyclo-gibberellin A₉（1）,7α-methoxy-6,7-lactone-gibberellin A₁₂（2）,7β-methoxy-6,7-lactone-gibberellin A₁₂（3）,16α-hydroxy-9-ene-gibberellin A₁₄（4）,gibberellin A₁₄（5）,gibberellin A₁₃（6）,gibberellin A₂₅（7）,fujenoic diacid（8）,6β,7β-dihydroxykaurenoic acid（9）,7β,11α-dihydroxy-kaurenolide（10）,7β,18-dihydroxy-kaurenolide（11）,（1α,2β,10β）-2-hydroxy-1-methyl-8-methylenegibb-5a-ene-1,10-dicarboxylic acid（12）,and（1α,2β,10β）-2-hydroxy-1-methyl-8-methylenegibb-4a-ene-1,10-dicarboxylic acid（13）,two esters or amides:2,3-dihydroxy-3-methylbutyl-2-（4-hydroxyphenyl）acetate（14）,and N-（2-phenylethyl）acetamide（15）,as well as one cyclic peptide:fusaripeptide A（16）.Compounds 1–4,12 and 14 are new compounds,and compound 10 is a new natural product.In the gibberellin family,compound 1 contains a special C-9/C-15 carbon-carbon bond,forming a complex and rare 6/5/6/5/3/5 six-membered ring system.Compounds 2 and3 are diastereoisomers with a special 6/7/6/5 tetracyclic skeleton,which is significantly different from the classic 6/5/6/5 tetracyclic gibberellin skeleton.Considering that gibberellins generally have carboxyl groups,and some compounds have aα,β-unsaturated ketone as the Michael reaction acceptor,and further considering the structural characteristic of"Cap-Linker-ZAB"in the histone deacetylase（HDAC）inhibitor Volinotar（SAHA）,theα,β-unsaturated ketone of gibberellins was designed as Cap in this study.By linking the ZAB part of SAHA with gibberellin（Cap）through a long chain（linker）,a novel dual-target inhibitor of Michael receptor and HDAC was tentatively constructed.We designed the target compound（17）with gibberellic acid（GA₃）as the starting point,and six synthetic intermediates WK-1（18）,WK-2（19）,WK-3（20）,WK-4（21）,WK-5（22）,and WK-6（23）were successfully obtained.Extensive bioactivity evaluation showed that compound 1 had obvious allelopathic activity,and its promoting effect on the root growth of Arabidopsis thaliana was comparable to that of agriculturally used GA₃.Compound 16 exhibited good cytotoxic activity with IC₅₀ values of 20.03,16.64 and 12.03μM against A549,Hep G2 and PC3 cells,respectively.The cytotoxic evaluation of the semi-synthetic products using five cancer cell lines showed that compounds 20 and 21 had selective cytotoxicity against human renal clear cell adenocarcinoma cells 786-O with IC₅₀ values of XX and XX,respectively.Secondary metabolites of Paraphoma sp.GR-F9 were also chemically studied in this thesis.Under the guidance of antifungal activity,the metabolites were separated by column chromatography on silica gel,and HPLC.Nine compounds were identified by NMR and related techniques,including five isocoumarins:（S）-8-hydroxy-6-methoxy-4,5-dimethyl-3-methyleneisochromen-1-one（24）,4,8-dihydroxy-6-methoxy-4,5-dimethyl-3-methyleneisochroman-1-one（25）,banksialactone B（26）,banksialactone C（27）,and clearanol F（28）,two lactam compounds:（2E,4E）-2-methyl-hexa-2,4-dienoic acid（2’S,3’S）-isoleucine amide（29）and（2E,4E）-2-methyl-hexa-2,4-dienoic acid（2′R,3′S）-isoleucinol amide（30）,two fatty acids:（8Z,11Z）-8,11-octadecadienoic acid（31）and（7Z,10Z）-7,10-octadecadienoic acid（32）.Compound 29 is a new compound.At present,the isolated compounds showed no activity against C.musae（ACCC 31244）,suggesting that we should continue to explore the trace compounds of Paraphoma sp.GR-F9.In conclusion,in this study,the endophytic fungus Fusarium sp.NJ-F5 with production of gibberellins,and the endophytic fungus Paraphoma sp.GR-F9 with antifungal activity against C.musae were screened out based on the OSMAC,metabolomics,and biological evaluation.26 secondary metabolites with various structural types were isolated and identified,including seven new compounds（1–4,12,14,and 29）and one new natural product（10）.Compound 1,the new member of gibberellin family,has a unique 6/5/6/5/3/5-fused ring system.It showed obvious allelopathic activity for promoting root growth.Natural product 16 exhibited good cytotoxic activity.In this thesis,GA₃,as an important member of gibberellins,was used as starting point to obtain six semi-synthetic products,the potential dual-target inhibitor of Michael receptor and HDAC.Compounds 20 and 21 showed selective cytotoxicity againt 786-O cells,deserving further research.This thesis not only enriches the structures and biological activity of gibberellins,but also lays a foundation for the subsequent research and application of gibberellins.It also suggests that endophytic fungi are an important source of bioactive natural products and worthy of further exploration.