Endo-hydroxamic acids: from synthons to substrates

Abstract

Desferrioxamine B is a linear trihydroxamate siderophore that forms hexadentate complexes of high affinity with Fe(III). It was discovered from the soil bacterium Streptomyces pilosus. Following the discovery of desferrioxamine B a number of siderophores sharing structural similarity to desferrioxamine B were discovered, isolated and characterised. These compounds shared a similar structure made up of consecutive N-(5-aminopentyl)-N-hydroxyacetamide (AHDP or A[C]) and 4-((5-aminopentyl)(hydroxy)amino)-4-oxobutanoic acid (SHDP or S[C]) monomeric units coupled together by amide bonds. The desferrioxamine-type siderophores are a class of metal chelator with proven and potential utility. Desferrioxamine-type siderophores are the current mainstay treatment for iron overload disease and find use in diagnostic oncology by binding 89Zr in PET imaging. This class of molecule may also have use in the treatment of neurodegenerative disorders, as antibiotics, as well as further use in oncology by binding frontier radiometals. The utility of known and new desferrioxamine-type siderophores warrants investigation but is currently limited by means of access. This thesis sought to investigate the utility of an enzymatic approach for exploring and expanding the chemical space around desferrioxamine-type siderophores. Desferrioxamine biosynthesis is modulated by the DesABCD enzyme cluster present in a number of Actinomycetes. The enzyme DesD is responsible for the peptide coupling of endo-hydroxamic acid monomers AHDP and SHDP in nature. For the purposes of this study DesD was available as a recombinant preparation from Salinispora tropica CND-440. The first part of this work was the development of a synthetic strategy to access AHDP and SHDP, the monomeric building blocks of desferrioxamine-type siderophores. This study aimed to develop a general methodology that was safer and higher yielding than present synthetic routes. The flexibility of the proposed synthetic route was tested to produce ether substituted AHDP and SHDP. The route gave higher yields than current methodologies used and successfully gave access to ether substituted analogues, N-(2-(2-aminoethoxy)ethyl)-N-hydroxyacetamide (A[O]) and 4-((2-(2-aminoethoxy)ethyl)(hydroxy)amino)-4-oxobutanoic acid (S[O]) . The second stage of this project used AHDP and SHDP in condensation reactions with DesD to optimise the conditions to produce a diverse product profile. The linear desferrioxamine-type siderophores produced were dimeric AHDP-SHDP and SHDP-SHDP, trimeric DFOB and DFOG1, tetrameric DFOB-(SHDP)1 and DFOG1-(SHDP)1 as well as pentameric DFOB-(SHDP)2 and DFOG1-(SHDP)2. The enzyme was not observed to produce hexameric products. The system also produced macrocyclic desferrioxamine-type siderophores in the form of DFOE, DFOT and DFOT-(SHDP)1. The third stage of this project used A[C], S[C], A[O] and S[O] in a mixed ligand incubation with DesD to investigate the substrate tolerability of DesD. The mixed ligand incubation produced a complex suite of products with a lower intensity than observed for native AHDP and SHDP. The enzyme incorporated ether substitution into linear dimers, trimers, tetramers and pentamers as well as macrocyclic dimers, trimers and tetramers. As the number of ether substitutions increased there was an associated reduction in HPLC retention time dependant on the region of ether oxygen atom substitution. The native oligomers and macrocycles were formed preferentially over ether substituted analogues. The position of substitution and relative quantities also allow for the elucidation of a biosynthetic mechanism. AHDP-SHDP oligomers and SHDP-SHDP oligomers are likely to share the same biosynthetic pathway due to identical products and product distributions observed between AHDP-SHDP and SHDP-SHDP as well as DFOB and DFOG1. The biosynthesis is largely dependant on oligomer size and preference for the activating (A) or condensing (C) site of DesD. The final stage of this thesis sought to further investigate the substrate tolerability of DesD by incubating the enzyme with synthetically derived, dimeric AHDP-SHDP and N-Boc-SHDP. The use of blunt-end SHDP was observed to direct the enzyme to synthesise N-Boc-DFOB. The successful production of N-Boc-DFOB demonstrated the directional capability of DesD in producing a single product In situ deprotection of N-Boc DFOB gave DFOB. This is the first report of using a synthetically protected native substrate to direct enzymatic synthesis.