Studies On The Antitumor Metabolites Of Two Fungal Mutants,BD-1-6 And BD-1-3,from The Diethyl Sulfate Mutagenesis Of Penicillium Purpurogenum G59

The secondary microbial metabolites play important roles in natural product research,which provided many leading compounds for new drug developement.In the screening of microbial strains for bioactive compounds,the majority of pure strains were inactive to produce bioactive metabolites in a given bioassay,which were not suitable for further chemical investigations of bioactive metabolites in the same bioassay.It is meaningful to make full use of these inactive strains to avoid waste of microbial resources.Recent progress in microbial genome studies has revealed that major gene clusters for secondary metabolite biosynthesis in microorganisms remain silent under general culture conditions.Thus above mentioned inactive microbial strains indeed possess potentials for the production of bioactive metabolites.Various chemical and physical factors may activate the silent gene clusters,leading to the production of cryptic secondary metabolites.On the basis of above research viewpoint,our group has undertaken a research project to develop new approaches for activating the silent gene clusters in inactive microbial strains to obtain bioactive secondary metabolites.Many bioactive mutants were obtained from inactive wild type strains and series of bioactive metabolites have been isolated from the bioactive mutants.Two mutants used in.the present study,BD-1-6 and BD-1-3,are fungal mutants obtained by another member of the same research group through a random diethyl sulfate mutagenesis of Penicillium purpurogenum G59.P.purpurogenum G59 is a marine-derived wild-type fungal strain and the EtOAc extract from its fermentation did not show any inhibitory effect on K562 cells at 100 ?g/ml or even at 1000 ?g/ml.However,EtOAc extracts from the two mutants significantly inhibited K562 cells at 100 ?g/ml.In the present study,the EtOAc extracts from the mutants BD-1-6 and BD-1-3 were separated by tracing antitumor metabolites newly produced by the mutants,through a bioassay-guided fractionation coupled with TLC/HPLC monitoring by direct comparison with the parent strain G59 sample.Eventually,twenty seven secondary metabolites were isolated from the mutants,including thirteen(1-13)from BD-1-6 and fourteen(14-27)from BD-1-3,all being produced newly by the mutants compared to their parent G59.Among them,structures of six new compounds,1,2 and 14-17,were elucidated by morden spectroscopic methods(Mb,UV,IR,ID and 2D NMR,,CD and other known compounds,3-13 and 18-27,were identified on the basis of their physicochemical and spectral data.Further,absolute configurations of new compounds 1 and 2 could be determined by their theoretically ECD calculations.The absolute configuration deduced by the empirical CD rules for 1 coincided with that from the theoretically ECD calculations.Compounds 1 and 2 belong to the meroterpenoids possessing a drimane skeleton and cyclohexenone moiety.Among them,1 provided a new substructural skeleton for the same class meroterpenoids,while 2 provided a new member of one subclass compounds rare in nature.Compound 14 is a new member of aranorosin-type compounds very rare in nature.Compound 15 and 16 provided a new class of chemical structures with dimerized fructigenine A and aranorosin skeletons.In HPLC and LC-MS analysis using 1-27 as reference,1-13 and 14-27 were detected in the EtOAc extracts of BD-1-6 and BD-1-3,respectively,while all 1-27 were not detected in the EtOAc extract of G59.This has thus reconfirmed that 1-27 are surely produced newly by the mutants compared to their parent G59,indicating that the production of some silent secondary metabolites in parent G59 has been activated by the DES-induced mutation(s)in mutants BD-1-6 and BD-1-3.In MTT assay,new compounds 1,2 and 14-16 showed strong inhibitory effects on human cancer K562,HL-60,HeLa,MCF-7 and BGC-823 cells,while new one 17 weakly inhibited only the K562 and MCF-7 cells of tested five cancer cells.Except 19,25 and 27,all other known compounds also showed inhibitory effect on K562 cells to a certain extent.The present study has demonstred that the production of some silent secondary metabolites in P.purpurogenum G59 has been activated by the DES-induced mutation(s)in BD-1-6 and BD-1-3.And thus,the DES mutagenesis strategy for activating cryptic secondary metabolite in G59 could be anticipated to be useful and practical in other fungal metabolite studies.