Study On Structural Diversity Of Capsimycins From Streptomyces Xiamenensis 318 And Post-Modification In Biosynthesis

Actinomycetes from mangroves have a great potential to produce novel secondary metabolites with a wide range of bioactivities in living environments adapted to high moisture,salinity and hypoxia tolerance.These active natural products have been considered as an important source of new drugs development.In this study,PTMs?polycyclic tetramate macrolactams?from mangrove-derived Streptomyces xiamenensis 318were analyzed,and in vitro pharmacological activity was evaluated.Also,the role of a novel cytochrome P450 monooxygenase in post-modification of PTMs was emphasized.In this study,six compounds were isolated and identified from S.xiamenensis 318,including three known compounds--ikarugamycin?1?,capsimycin?2?and capsimycin B?3?,as well as three new compounds--capsimycin C?4?,capsimycin D?5?and capsimycin G?3'?.The stereochemistry of trans tert-tert diol in capsimycin C?4?was determined by NMR?Nuclear Magnetic Resonance?,CD?Circular Dichroism?,Mo₂?OAc?₄ method and chemical semi-synthesis.The chemical stability of capsimycin?2?and capsimycin B?3?both with epoxy structure was investigated by adding acid and heating under different conditions.Their methoxy derivatives--capsimycin E?6?and capsimycin F?7?,respectively,were obtained.Based on MS/MS fragmentation of compound 1-7,a rapid and preliminary method for determining of 5-6-5 ring PTMs and of the numbers of substituents in its carbocyclic ring based on MS/MS data was proposed.A putative cytochrome P450 superfamily gene,SXIM₄0690?ikaD?,which was proximally localized to the ikarugamycin biosynthetic pathway,was characterized.In vivo gene inactivation and complementation experiment and in vitro IkaD catalysis experiment confirmed that IkaD catalysed the C-13/14 epoxide-ring formation to form capsimycin B?3?and further hydroxylation of C-30 ethyl side chain to form capsimycin G?3'?.Binding affinities and kinetic parameters for the interactions between ikarugamycin?1?and capsimycin B?3?with IkaD were measured with Surface Plasmon Resonance.Compounds 1-3 were evaluated for anti-pancreatic cancer cell proliferation activity and toxicity using CCK-8 and LDH?Lactate DeHydrogenase?methods.The results showed that compounds 1-3 had a good anti-pancreatic cancer activity,IC₅₀ range from 1.30 to 3.37?M.In this study,we studied PTM compounds from S.xiamenensis 318,and identified three new PTM compounds.The anti-tumor activity of compounds was screened,and three of them had anti-pancreatic cancer activity.The preliminary structure-activity relationship analysis and toxicity evaluation were carried out.Further studies on the post-modification mechanism of PTMs were carried out,and a new class of cytochrome P450 monooxygenase IkaD catalyzed epoxidation and hydroxylation of ikarugamycin was described via heterologous expression and in vitro biochemical experiments.