Studies On Active Metabolites Of Streptomyces Marinus IMB094 And Saccharomyces Cerevisiae 7-98-1

The rise in resistance of bacteria,particularly the Gram-negative to multi-drug is becoming a major threat to the global human health care.In contrast to the continuing increase of the antibiotic-resistannce of bacteria,the discovery rate of antibiotics that possess novel skeleton structures and new antibacterial mechanism has dramatically decreased in the last twenty years.Developing novel antibiotics are urgently necessary to treat the infections caused by the antibiotic-resistant pathogens.Actinomycetes have been an important resources of the microbial drug discovery,which accounted for 40%natural antibiotics produces by microbes.Marine microorganisms,due to their special living environment,may have developed different secondary metabolic biosynthetic pathways from their terrestrial couterparts and therefore,might produce bioactive compounds with different structures,which has become a research hotspot in the microbial natural products discovery in recent years.In this dissertation,we carried out the isolation and purification,structure elucidation,and bioassay of the antibacterial constituents of of the fermentation broth from the marine sediment-derived actinomycete strains Streptomyces sp.IMB094 and Saccharomonospora sp.7-98-1.The crude extract of Streptomyces sp.IMB094 and Saccharomonospora sp.7-98-1 showed significant antibacterial activities against methicillin-resistant Staphylococcus aureus?MRSA?and muti-drug resistant Gram-negative pathogens.respectively.Extensive investigation of the secondary metabolite composition of the IMB094 strain resulted in the isolation of a novel actinomycin chromophoric analog,neo-actinomycin A?1.Figure 1?,a new natural product,neo-actinomycin B?2?,and two known actinomycins D and X₂?3 and 4?guided by the bioassay..Their structures were characterized by spectroscopic data analysis,including UV,1D-NMR,2D-NMR and MS spectra.Structurally,the chromophore of compound 1 and compound 2 contain a fourth oxazole ring fused with the actinocin moiety,forming a tetracyclic 5H-oxazolo[4,5-b]phenoxazine ring,which has never previously been found in naturally occurring actinomycins.The NMR data of compound 2 was first reported in this dissertation.The structures of 3 and 4 were identified as actinomycin D and X2,respectively.The biosynthesis of compounds 1 and 2 was investigated.During the precursor-directed in situ synthesis experiments,it was found that the yields of compound 1 and 2 were increased 6-12-fold by adding ?-ketoglutarate and pyruvate into the cultures,respectively.In addition,we further explored the possibility of in vitro transformation of the precursors in a variety of solvents,including the fermentation M8 media,H2O,and MeOH.After incubation of 3 with ?-ketoglutaric acid,we observed approximately 10%conversion of 3 to 1 in H2O and in M8 media,but no production of 1 in MeOH.Incubation with pyruvic acid under the same conditions lead to 50%conversion of 3 to 2 in H2O and M8 media,and only 5%conversion in MeOH.In addition,?-ketoglutarate and pyruvate were determined to be present in the cultures in the fermtation broth cultured in galactose-glutamate-mineral salts?GGM?medium with a high concentration.Therefore,the chromophore ring system of 1 and 2 was proposed to form in a nonenzymatic condensation of actinomycin D with ?-ketoglutarate and pyruvate,respectively.Compounds 1 and 2 were evaluated for their in vitro antibacterial activity against a series of Gram-positive and Gram-negative drug-resistant pathogens.Compound 1 showed moderate antibacterial activities against methicillin-resistant Staphylococcus aureus?MRSA?and vancomycin-resistant Enterococci?VRE?strains with MIC values of 16-64?g/mL,64-256-fold less active than 3.No antibacterial effects were observed for 2?MIC>128 ?g/mL?.Compounds 3 and 4 showed potent antibacterial activities against methicillin-resistant Staphylococcus epidermidis?MRSE?,MRSA and VRE pathogens?MIC:0.06-0.25?g/mL?.Neo-actinomycin A?1?exhibited potent cytotoxicities against HCT116,A549 and HepG2 cancer cell lines at nanomolar concentrations?IC₅0:38.7,65.8,43.0 nM?,approximately 800-fold decrease in activity relative to 3.By analysis of the structure-activity relationship of these compounds,it is concluded that the 2-amino group and the planarity of the chromophore is crucial for the antibacterial and cytotoxicity of actinomycins.The chemical constituents of the extracts of the marine-derived Saccharomonospora sp.7-98-1 were investigated by using a combination of various chromotographic techniques.Four compounds?5-8?were isolated from the watersolube active extracts.There structures were identified as uridine?5?,deoxyuridine?6?,guanosine?7?,and adenosine?8?,respectively byspectroscopic data analysis.The antibacterial compounds were not characterized from the crude extracts due to their strong water-solublity although we tried various chromatographic methods.