Precursor-Directed Biosynthesis of Azinomycin A and Related Metabolites by Streptomyces sahachiroi

Azinomycins A and B are bioactive compounds produced by Streptomyces species. These naturally occurring antibiotics exhibit potent in vitro cytotoxic activity, promising in vivo antitumor activity and exert their effect by disruption of DNA replication by the formation of interstrand cross-links. The electrophilic C-10 and C-21 carbons contained within the aziridine and epoxide moieties are known to be responsible for the interstrand cross-links through alkylation of N-7 atoms of guanine bases. The naphthoate moiety (3-methoxy-5-methyl-naphthoate) is believed to play a role in the azinomycins’ biological activity through hydrophobic interactions in the DNA major groove. In this study, precursor directed biosynthesis (PDB) has been investigated for the production of unnatural azinomycin analogues. A series of naphthoic acid analogues were fed to the azinomycin producing bacteria (Streptomyces sahachiroi ATCC 33158). LCMS analyses revealed that 1-naphthoic acid, 4-fluoro-1-naphthoic acid, 4-methyl-1-naphthoic acid and 3-methoxy-1-naphthoic acid were successfully incorporated into the azinomycin A biosynthesis pathway and resulted in the development of novel azinomycin A analogues. These novel products were isolated and purified using preparative HPLC and were characterised by diode array detection and electrospray tandem mass spectrometry. NMR characterisation and biological studies could not yet be conducted due to the low production levels (~100 μg in 500 mL fermentation broth) and the persistence of some impurities in these novel azinomycin A analogues. In the course of this work, additional metabolites were found in the fermentations. LCMS analysis revealed that a range of naphthoic acids were biotransformed into primary amides. Column chromatography has been carried out towards purification of all the produced amides. As consequence, 1-naphthoic amide and 4-fluoro-1-naphthoic amide were successfully purified and characterised by NMR and LCMS. Further amides were so far only characterised by LCMS, as impurities hampered NMR analysis. Antibacterial and antifungal tests were carried out to investigate the biological activity of the purified amide. The results revealed that they were inactive against the tested microorganisms