Hydroxamic acid chelator profiles and Ga(III) complexes from siderophore synthetase-mediated synthesis using length-modulated substrates.

The recombinant Salinispora tropica CNB-440 siderophore synthetase StDesD catalysed the assembly of pools of hydroxamic acid-containing chelators from a set of linear, non-native substrates containing internal polyethylene glycol (PEG)n spacer units. The internal (PEG)n units were capped at each end by an equivalent of the native StDesD substrate N-hydroxy-N-succinyl-cadaverine (HSC, 1) to give the length-modulated substrate set 1-(PEG)n-1-L (n = 1-6) (12.1-12.6). Chemoenzymatic reactions analysed by liquid chromatography-mass spectrometry showed StDesD catalysed end-to-end condensation reactions of 12.1-12.6 producing the cognate dihydroxamic acid macrocycles 1-(PEG)n-1-MC (13.1-13.6) as major products. Minor amounts of the macrocycle using a (1)2-L substrate with no PEG insert was observed, showing the flexible PEG linker in 12.1-12.6 improved positioning of the reactive termini in the StDesD active site to promote condensation. The major product using (1)2-L was instead the tetrahydroxamic acid macrocycle (1)4-MC desferrioxamine T1 (DFOT1, 7) formed from the sufficiently flexible precursor (1)4-L desferrioxamine S1 (DFOS1, 4). Reaction mixtures incubated with Ga(III) produced 1:1 Ga(III)-13.1-13.6 complexes. The work demonstrates chemoenzymatic synthesis as a facile approach to assemble structurally diverse and functional chelators alongside providing mechanistic insight of siderophore synthetases.