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 St DesD 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 St DesD 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 St DesD 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 St DesD active site to promote condensation. The major product using ( 1 ) 2 -L was instead the tetrahydroxamic acid macrocycle ( 1 ) 4 -MC desferrioxamine T 1 (DFOT 1 , 7 ) formed from the sufficiently flexible precursor ( 1 ) 4 -L desferrioxamine S 1 (DFOS 1 , 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. The chemoenzymatic synthesis of end-to-end dihydroxamic acid macrocycles using the recombinant siderophore synthetase from Salinispora tropica ( St DesD) and a linear dihydroxamic acid substrate was more efficient upon embedding a polyethylene glycol (PEG) unit between the two monohydroxamic acid termini. Apo-macrocycles containing PEG units with different multiplicities were functional Ga(III) chelators. • Polyethylene glycol (PEG) inserts flanked by two hydroxamic acid units used as substrates. • Macrocycles with variable cavity sizes were produced by chemoenzymatic synthesis. • Product concentrations showed a non-linear dependence on the PEG insert multiplicity. • Ga(III) complexes were preferentially formed with macrocyclic over linear chelators.