New Benzoquinone Ansamycins From Streptomyces Hygroscopicus And The Exploration Of Secondary Metabolites From Streptomyces By LC-DAD-MS

Microbial secondary metabolites with various chemical structures and biological activities are important sources of new drugs. So far, most known microbial drugs come from actinomycetes, particularly from Streptomyces. New secondary metabolites from Streptomyces remain to be a significant origin for new drug development. This dissertation is divided into two parts. The first part describes the identification of three novel benzoquinone ansamycins from Streptomyces hygroscopicus17997or Streptomyces hygroscopicus N02Z-0421. The second part involves the exploration of novel secondary metabolites from strains of the genus Streptomyces.Geldanamycin (GDM) is a benzoquinone ansamycin produced by Streptomyces hygroscopicus. Although with exceptional potency against cancer cells, GDM acts only as a lead compound in anticancer drug development because of its severe hepatoxicity and poor water solubility. GDM analogues with increased water solubility and low hepatoxicity are promising candidates in anticancer drug development. Of the hundreds of GDM analogues obtained by semi-synthesis, two compounds,17-allylamino-17-demethoxygeldanamycin (17-AAG) and7-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), entered into clinical trials for the treatment of solid tumors.Secondary metabolites from actinomycetes are usually produced as groups of molecules with similar structures. This phenomenon is also known as multi-components of antibiotics. A number of novel GDM analogues from different GDM producers were reported recently. Some of these GDM analogues displayed higher water solubility or in vitro anti-tumor cell activity than GDM, showing the potential for further antitumor drug development.Streptomyces hygroscopicus17997was a soil isolate from Institute of Medicinal Biotechnology, China Academy Of Medical Sciences, and identified as a GDM producer. Its secondary metabolites were analyzed carefully by us. As a result of our detailed analysis or comparison of silica gel TLC and LC-MS profiles of GDM analogues produced by Streptomyces hygroscopicus17997, a novel natural GDM analogue was discovered. The structure of the analogue was determined to be19-[(1’S,4’R)-4’-hydroxy-1’-methoxy-2’-oxopentyl]-geldanamycin (compound713) by extensive spectroscopic data analysis, including2D NMR, modified Mosher’s method, and electronic circular dichroism (ECD). The4,5-dihydro form of the analogue,19-[(1’S,4’R)-4’-hydroxy-l’-methoxy-2’-oxopentyl]-4,5-dihydrogeldanamycin (compound715), was then discovered and identified in the gdmP gene disruption mutant of Streptomyces hygroscopicus17997. Compared to GDM, compounds713and715showed much increased water solubility but decreased cytotoxicity against HepG2cells.Streptomyces hygroscopicus N02Z-0421was a producer of herbimycin A, another benzoquinone ansamycin that shares a very similar structure to GDM. Herbimycin A possesses also severe cytotoxicity against cancer cells, and like GDM, is a lead compound in anticancer drug development. To obtain new herbimycin A analogue(s), we supplemented Met in the culture medium for S. hygroscopicus N02Z-0421. A red compound appeared in the silica gel TLC of secondary metabolites of the strain. The compound was then identified by us as a new herbimycin A analogue, whose structure was determined as17,19-dimethylthioherbimycin A, with methylthiol group(s) in the molecule as expected. Preliminary assays indicated that17,19-dimethylthioherbimycin A showed a potent cytotoxic activity against HepG2cancer cells, with an IC50value of18.7μM (for herbimycin A,14.0μM).The identification of17,19-dimethylthioherbimycin A from Streptomyces hygroscopicus N02Z-0421in culture medium supplemented with methionine (Met) provides an additional strong evidence to support the synthetic mechanism and the origin of methylthiol group in19-S-methylgeldanamycin, a new GDM analogue discovered earlier in our laboratory from Streptomyces hygroscopicus17997. The synthetic mechanism and the origin of methylthiol group in19-S-methylgeldanamycin was clarified recently in our laboratory as Met being first metabolized to methanethiol, and the latter molecule then acting as a nucleophilic agent in its attacking GDM to form19-S-methylgeldanamycin.Streptomyces hygroscopicus17997produced not only GDM and its analogues, but also some un-identified secondary metabolites with antibacterial activities. One compound with anti-G+bacterial activity was determined by us to be elaiophylin. Elaiophylin is a macrolide antibiotic with many biological activities such as antibacteria, antitumor and antiprotozoal, and often appears in the secondary metabolites of Streptomyces hygroscopicus. In our identification of elaiophylin, a fast procedure was established, which consisted of silica gel TLC followed by color reaction with NaOH and a simple and fast genetic analysis of biosynthetic potential of elaiophylin by PCR, etc. It is worthy to note that elaiophylin in silica gel TLC turned light red upon spraying NaOH, which is a feature of elaiophylin never reported before. This feature has been used successfully to fast identify elaiophylin producers several times in our laboratory.As mentioned in the beginning of the abstract, Streptomyces produces abundant secondary metabolites with various biological activities. To obtain new secondary metabolites from Streptomyces, we made an initial inspection of a dozen strains of Streptomyces from China Pharmaceutical Culture Collection (CPCC). Two strains with antibacterial activities and diversified patterns of secondary metabolites were chosen by us for further investigation of new compounds with biological activities.Streptomyces sp. CPCC200002is a soil isolate with very strong antibacterial activities collected from Guangdong Province, China. The EtOAc extracts of the culture of this strain was fractionated by silica gel TLC and then analyzed by LC-DAD-MS. A group of five similar compounds with antibacterial activity were identified as thiolutin, thiolutin oxide, aureothricin, butyryl-pyrrothine and isobutyryl-pyrrothine. All of them belonged to dithiolopyrrolone antibiotics. Dithiolopyrrolone antibiotics were known for their very strong antimicrobial activities, and new interests about these compounds aroused due to their anticancer and antitumor properties.The biosynthesis of dithiolopyrrolone antibiotics is closely related to cysteines, and each of dithiolopyrrolone antibiotics comes from the condensation of two cysteines. As a close analogue of cysteine, homocysteine was supplemented in the culture of Streptomyces sp. CPCC200002, with the expectation of new condensation product(s) of dithiolopyrrolone antibiotics produced. Higher production levels of the above identified five compounds were observed, but new compounds with homocysteines or homocysteine-cysteine hybrid were not detected by us.Streptomyces sp. CPCC203577is a soil isolate with significant antibacterial activities collected from Yunnan Province, China. The EtOAc extracts of this strain were analyzed by silica gel TLC and LC-DAD-MS, which led us to identifiy lavanducyanin. Lavanducyanin belongs to phenazines antibiotics, and natural and synthetic phenazines derivatives are promising candidates for drug development.A close analysis of minor components from Streptomyces sp. CPCC203577led us to discover four lavanducyanin analogues. Of them, one was deduced to be WS-9659B, which was reported by Nakayama O, the other three should be new lavanducyanin analogues after our search in SciFinder database. The separation, purification and structure determination of these new lavanducyanin analogues are still in progress.