Studies towards the efficient isolation and purification of analogues of amphotericin B from Streptomyces nodosus
thesisposted on 08.11.2013, 13:40 by Odubunmi Olajumoke Ibrahim
Amphotericin B, produced by Streptomyces nodosus, is a medically important antifungal that is also active against Leishmania parasites. However, its use is severely limited by its toxicity. Treatment would be much more effective if non-toxic analogues of amphotericin B were available. Genetic manipulation of S. nodosus can provide access to such analogues potentially at clinically affordable cost. Amphotericin B has been shown to be biosynthesised by a modular polyketide synthase (PKS) and three post PKS enzymes. Genetic disruption of one of the post PKS genes encoding a cytochrome P450 produces 16-descarboxyl-16-methyl-amphotericin B, which has been shown to be less toxic yet retains antifungal activity. This thesis describes efforts to obtain improved characterisation and X-ray analysis of the PKS product, 8-deoxy-16-descarboxyl-16-methyl amphoteronolide B, by synthesising an octabenzoyl derivative. Results obtained show the derivatisation of the lactol gave the benzoyl ketone. Crystals were obtained but failed to diffract. This thesis also describes the in vivo production, isolation, purification and characterisation of 16-descarboxyl-16-methyl-amphotericin B, 8-deoxy-16-descarboxyl-16-methyl amphotericin A and 16-descarboxyl-16-methyl-19-(O)-perosaminyl amphoteronolide B (pHap2). Evidence is also presented that the yield of extracts from various mutants of amphotericin B can be significantly improved by strain selection and optimising the growth conditions. Improved purification protocols involving the intermediacy of less polar N-fluorenylmethoxycarboxyl (Fmoc) derivatives are also described. The use of Fmoc intermediates enabled partial purification by flash chromatography. However, derivatisation of very low purity samples (especially pHap2) resulted in low yields and formation of by-products. The methodology reported in this thesis showed promise for assisting with large scale purification of a variety of glycosylated analogues of amphotericin B for extensive biological assays and assist with investigations into the mode of action of these antibiotics.