Studies On Secondary Metabolites Of Three Different Marineenvironment-derived Fungi: Structures And Bioactivities

Microbes could survive in diverse ecological niches. Due to the differences intheir living environments, competitions between species and other external factors,microbe could co-evoluted with ecological niches which leaded to microbial diversity.These microoganisms even belonging to the same species could have a stable,different gene expression when they were isolated in lab. It is indicated that the stableexpression in lab conditions could be applied for new drugs discovery.Based on the different marine environment-derived strain libraries established byour lab, we selected three different living environment marine-derived fungi, andcarried out study on secondary metabolites, biosynthesis and biological activity. Weperformed on the following works: culture condition optimization using OSMACstrategy; structural separation, identification and stereochemical studies, biosynthesisof novel structures, preliminary bioactive evaluation of some new compounds;molecular target screening, molecular docking experiment.We firstly applied an OSMAC strategy for optimizing the target strain fermentationconditions. Optimization experiments showed that the strain of Penicillium crustosumPRB-2were sensitive to the different culture systems, and finally we determined theoptimal scale fermentation condition in39kinds of culture conditions. We alsoobserved that the strain of Cladosporium sphaerospermum2005-01-E3could producedifferent secondary metabolites cultivating in rice and soybean medium, respectively.In all,70compounds were separated and purified, from the extracts of the3aimedstrains, by means of silica gel column, including Sephadex LH20, ODS-C18, PHPLC,MPLC and etc. From mangrove-derived fungus Aspergillus versicolor pjx-9,27compounds (1-27) were isolated; from Antarctic deep-sea derived fungus Penicilliumcrustosum PRB-2,36compounds (28-63) were isolated; from PacificDeep-Sea-Derived Fungus, Cladosporium sphaerospermum2005-01-E3,7 compounds (64-70) were isolated.Basing on their physico-chemical properties and spectral data (IR, UV, MS, NMR,CD, and X-ray), combined with conformational analysis and marfey method,structures of the70compounds were respectively determined. Among them therewere15dimeric xanthones (1-15),4anthraquinones (16-19),2steroids (22-23),16compounds containing a or two clavatol moieties (28-43),20alkaloids (20-21,24-25,55-70),13benzol derivatives and other structural types (26-27,44-54).Thirty-three compounds were new, including6dimeric xanthones (1-12,15)characterized by diverse linkages between two monomers and novel monomeric units,1-4and15with a rare2-4′or4-4′linkages and novel unreported monomeric units;two novel carbon skeletons, first class included two compounds (28-29) with a rare3,3a,9,9a-tetrahydro-1H-furo[3,4-b]chromen-1-one core, which possessed antipodalabsolute stereochemistries. The second class (42-43) had an novel6a-hydroxy-6,6a,12,12a-tetrahydro-5H-benzo[a]xanthen-5-one core, and the twocompounds were an inseparable mixture, respectively. two new chemical types:monoclavatol α-furanone adducts (31-34,36), monoclavatol α-pyranone (38-39)。Compounds64-67,69-70represented a novel class of tetramic acid congenerscharacterized by previously unknown6(3)-enamino-8,10-dihydroxy-or6(3)-enamino-7(8)-en-10-ol side chains and the biogenetically related cladosin E (68).We also obtained three new natural products (14,44-45). In the study, relative andabsolute configutations of1were established by X-ray. We established the relativeand absolute configurations of2-14by the combination of NOESY, comparing ECD,heating test. Structures of28and29were elucidated by spectroscopic methods, andtheir absolute configurations were assigned by singlecrystal X-ray diffraction and CDanalyses. Absolute configurations of42-43were going on based on a combination ofHPLC-CD and CD calculations. Absolute configurations of30-36were deduced bythe combination of X-Ray, ECD (comparison ECD, ECD calculation). Theirstructures (6468) were elucidated through a combination of spectroscopic data,chemical conversion, and both Mosher’s and Marfey methods for stereochemicalassignment. In the light of biosynthetic origin, we demonstrated that14likely played animportant precursor for producing other related dimeric xanthones via productgeneration time experiment and PH simulating test. We first investegated thebiosynthetic origin of the novel skeleton28and biosynthetically related35usingstable isotope labeling experiments. Compounds28and35were assembled by rarehybrid PKS and TCA cycle pathway, while assembly of38was verifyed via PKSpathway.The antitumor activities against eight cancer cell lines of these compounds wereassayed by MTT, SRB methods. Among them,8compounds (2,5,6,8,9,12-14) withsignificant bioactivities (cytotoxic, IC500.4-10μM) were found. Atropisomers8and9showed selective antitumor activities. They only showed potent cytotoxic activitiesagainst803cell line and8(IC50=4.60μM) had a stronger cytotoxic activity than9,while they both were no activity against normal cell line HEK293(IC50＞50μM). Amolecular target study indicated2,8,9inhibited toposiomerose I potently. Interestingly, atropisomers8and9differed dramatically in inhibiting Top I activitiesin the molecular level and the mechanism in cell level were underway. Among thethree compounds, compound2showed the most potent inhibiting Top I activity. Compounds64-70were evaluated for antiviral activity against the influenza A H1N1virus, as well as antitumor, antitubercular, and NF-κB inhibitory activities. Onlycompound66exhibited activity with an IC50=276μM against the influenza A H1N1virus (ribavirin as positive control, IC50131μM).In this study,33new secondary metabolites and3new natural products wereisolated and identified from3different-ecological niches fungi. A novel class ofdimeric xanthones, two class novel skeleton and a novel class of tetramic acidcongeners were discovered. We established the absolute configurations of newcompounds by multiple techniques, especially key chemical conversions. Wediscussed the biosynthetic origins of dimeric xanthones and novel skeletons. Dimericxanthones showed potent cytotoxic activities and might provide new structuretemplates for designing Topo I inhibitors.