The Discovery And Exploration Of (4S/R)-4,5-dihydro-4-hydroxygeldanamycin And Other New Secondary Metabolites From Streptomyces

Microbial secondary metabolites with various chemical structures and biological activities are important sources of new microbial drugs which play a large role in clinic. These years, the need to find new microbial drugs is becoming more and more urgent. So far, most of the known microbial drugs come from Streptomyces belonging to actinomycetes. Therefore, to strengthen the exploration of secondary metabolites from Streptomyces remains to be a significant way of new drug development.This dissertation is divided into two parts. The first part describes the identification of two novel geldanamycin analogues from Streptomyces hygroscopicus 17997. The second part involves the exploration of novel secondary metabolites from two Streptomyces strains, CPCC203471 and CPCC 200466.Geldanamycin (GDM) is a benzoquinone ansamycin produced by Streptomyces hygroscopicus. It binds to heat shock protein 90 and inhibits its function. 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. So, to explore new geldanamycin analogues with low hepatoxicity and increased water solubility is an important task. Hundreds of GDM analogues have been obtained by semi-synthesis, and some of them show excellent in vivo activities, for example, 17-allylamino-17-demethoxygeldanamycin (17-AAG) has entered into clinical trials for the treatment of several tumors. Besides, a number of natural GDM analogues with higher water solubility and better activity against cancer cells are isolated from Streptomyces, which indicate that natural GDM analogues are also promising candidates in anticancer drug development.Understandings of GDM biosynthesis help us obtain novel biosynthetic GDM analogues. GDM biosynthesis can be divided into two stages:proGDM formation and post-PKS tailoring modifications. The second stage includes modifications of C-17 hydroxylation, C-17-O-methylation, C-21 hydroxylation, C-7 carbamoylation and C-4,5 oxidation. Among them, the C-4,5 oxidation (desaturation) is catalyzed by GdmP, a cytochrome P450 oxidase. Most cytochrome P450 oxidases for secondary metabolite biosynthesis in actinomycetes function as hydroxylases/monooxygenases. Cytochrome P450 oxidases acting as desaturases are rare. In the few known desaturation reactions catalyzed by cytochrome P450 oxidases, substrate hydroxylation (and/or epoxidation) may also take place. Therefore, GdmP may also catalyze C-4/5 hydroxylation in GDM biosynthesis.Streptomyces hygroscopicus 17997 is a GDM producer from Institute of Medicinal Biotechnology, China Academy of Medical Sciences. Its secondary metabolites were analyzed by silica gel TLC and LC-MS in detail and compared carefully. Two novel natural GDM analogues were discovered. The structures of the two analogues were determined to be (4S)-4,5-dihydro-4-hydroxygeldanamycin (1) and (4R)-4,5-dihydro-4-hydroxygeldanamycin (2) by extensive spectroscopic data analysis, including HRESIMS, IR, NMR, and modified Mosher’s method. Both 1 and 2 were not bioconverted to GDM by GDM-pks, suggesting they are probably not normal intermediates of GDM biosynthesis but shunt products of GdmP-catalyzed C-4,5 oxidation. The discovery of the two new analogues deepen our understandings of C-4,5 oxidation of GDM biosynthesis. Compounds 1 and 2 showed decreased cytotoxicity against HepG2 cells.The secondary part of the dissertation describes the exploration of new secondary metabolites from some Streptomyces strains. We made an initial inspection of secondary metabolite profiles of 26 Streptomyces strains from China Pharmaceutical Culture Collection. Two strains (CPCC 203471, CPCC 200466) with antibacterial activities and diversified patterns of secondary metabolites were chosen for further investigation of new compounds with biological activities.Streptomyces sp. CPCC 203471 is a soil isolate with strong antibacterial activities from western Yunnan, China. The EtOAc extracts of the culture of this strain was analyzed by silica gel TLC, LC-MS and HRMS to get the essential data (UV, molecular mass, molecular formula, etc) of the secondary metabolites. These data helped us to identify seven compounds from secondary metabolites of CPCC 203471 by searching natural products database and SciFinder database. The major product with antibacterial activity was identified as novobiocin, a representative antibiotic of coumarins. Six other compounds were also identified as novobiocin analogues. LC-MS data show their molecular mass as follows:555 (b),642 (d, NOV-642),555 (e),569 (f),569 (g),598 (i). Among them, compound b was speculated to be 4"-O-desmethyl-3"-O-descarbamoylnovobiocin, one of compounds f, g was probably 3"-O-descarbamoylnovobiocin (the other one was possibly a new novobiocin analogue with low yield), compound i was identified as 4"-O-desmethylnovobiocin, and compound d (NOV-642) was proved to be a new novobiocin analogue after our searching SciFinder database. The structure of NOV-642 was determined to be 5-methoxylnovobiocin by NMR data analysis.Novobiocin shows strong activities against Gram-positive bacteria. It used to be a clinical drug for MRSA infections. What’s more, as the first Hsp90 C-terminal inhibitor discovered, novobiocin can inhibit tumor growth. We tested and compared the antibacterial activities (MIC) of novobiocin and NOV-642, and found out that NOV-642 showed 3-7 times lower antimicrobial activity than novobiocin.A macrolactam antibiotic, BE-14106, was also identified from Streptomyces sp. CPCC 203471. BE-14106 was reported to have cytotoxic activity against cancer cells.Streptomyces sp. CPCC 200466 showed strong antifungal activities. It was determined to be an oxazolomycin producer by silica gel TLC, LC-MS and HRMS analysis. Six oxazolomycin analogues with the same molecular mass (655) were identified from Streptomyces sp. CPCC 200466 (3,4,5,6,7,8). After searching SciFinder database, we found out that there were only three known oxazolomycins with molecular mass of 65, which were oxazolomycin A (4’Z,6’Z,8’E), oxazolomycin B (4’E,6’E,8’E) and oxazolomycin C (4’Z,6’E,8’E). Consequently, there should be at least three new isomers of oxazolomycins among the six compounds.Oxazolomycin contains a y-lactam/β-lactone, an oxazole and a triene (Z, Z, E) in its molecule. The unique y-lactam/βactone is similar to the pharmacophore in salinosporamide A, which is a 20S proteases inhibitor. Oxazolomycin shows antitumor, antiviral and antibacterial activities, and it has the potential as a lead compound for drug development.We studied the separation and purification of the six oxazolomycin analogues (mainly compound 5), and found these isomers very difficult to separate well because their structures are too similar. More efforts are needed to separate these isomers.