An elastic siderophore synthetase and rubbery substrates assemble multimeric linear and macrocyclic hydroxamic acid metal chelators

Abstract

The trihydroxamic acid bacterial siderophore desferrioxamine B (DFOB, 1) produced by the DesABCD biosynthetic cluster coordinates metals beyond Fe(III), which identifies potential to modify this chelator type to broaden metal sequestration and/or delivery applications. Rather than producing discrete chelators by total chemical synthesis from native monomers including N-hydroxy-N-succinyl-cadaverine (HSC, 2), the recombinant siderophore synthetase from Salinispora tropica CNB-440 (StDesD) was used with different substrate combinations to produce biocombinatorial mixtures of hydroxamic acid chelators. The mixtures were screened with Ga(III) or Zr(IV) as surrogates of immunological positron emission tomography (PET) imaging radiometals ⁶⁸Ga(III) or ⁸⁹Zr(IV) to inform known or new coordination chemistry. The last-in-line enzyme DesD forms amide bonds between two equivalents of 2 and N-hydroxy-N-acetyl-cadaverine to produce trimeric 1. Although hexadentate 1 is the terminal product evolved for Fe(III) complexation, it was conceived amine-containing 1 might remain a viable DesD substrate for further iteration with 2 to generate higher-order hydroxamic acid multimers. Incubation of StDesD, cofactors ATP and Mg(II), and 1 and 2, generated the octadentate hydroxamic acid DFOB-HSC (3) (previously characterised and named DFO*), decadentate DFOB-(HSC)₂ (4), dodecadentate DFOB-(HSC)₃ (5) and tetradecadentate DFOB-(HSC)₄ (6). The system with StDesD and 2 alone generated a set of linear multimers containing flanking amine and carboxylic acid groups (HSC)_x-L (x = 2 (7), x = 3 (8), x = 4 (9), x = 5 (10)) and a subset of the cognate ring-closed macrocycles (HSC)_x-MC (x = 3 (12), x = 4 (13), x = 5 (14), with x = 2 (11) not detected). Liquid chromatography-mass spectrometry metal screening experiments detected 1 : 1 complexes of Ga(III) or Zr(IV) and 1, 3–5, 8–10, and 12–14. Complexes of 2 : 1 stoichiometry were formed between Ga(III) and the high-denticity, high-cavity-volume chelators 4–6, and 14. A processive intra-cavity assembly mechanism has been posited for this flexible siderophore synthetase in delivering a large set of multimeric chelators.