Acetyl-CoA-Mediated Post-Biosynthetic Modification of Desferrioxamine B Generates N- and N-O-Acetylated Isomers Controlled by a pH Switch.

Biosynthesis of the hydroxamic acid siderophore desferrioxamine D1 (DFOD1, 6), which is the N-acetylated analogue of desferrioxamine B (DFOB, 5), has been delineated. Enzyme-independent Ac-CoA-mediated N-acetylation of 5 produced 6, in addition to three constitutional isomers containing an N-O-acetyl group installed at either one of the three hydroxamic acid groups of 5. The formation of N-Ac-DFOB (DFOD1, 6) and the composite of N-O-acetylated isomers N-O-Ac-DFOB[001] (6a), N-O-Ac-DFOB[010] (6b), and N-O-Ac-DFOB[100] (6c) (defined as the N-O-Ac motif positioned within the terminal amine, internal, or N-acetylated region of 5, respectively), was pH-dependent, with 6a-6c dominant at pH < 8.5 and 6 dominant at pH > 8.5. The trend in the pH dependence was consistent with the pKa values of the NH3+ (pKa ∼ 10) and N-OH (pKa ∼ 8.5-9) groups in 5. The N- and N-O-acetyl motifs can be conceived as a post-biosynthetic modification (PBM) of a nonproteinaceous secondary metabolite, akin to a post-translational modification (PTM) of a protein. The pH-labile N-O-acetyl group could act as a reversible switch to modulate the properties and functions of secondary metabolites, including hydroxamic acid siderophores. An alternative (most likely minor) biosynthetic pathway for 6 showed that the nonribosomal peptide synthetase-independent siderophore synthetase DesD was competent in condensing N'-acetyl-N-succinyl-N-hydroxy-1,5-diaminopentane (N'-Ac-SHDP, 7) with the dimeric hydroxamic acid precursor (AHDP-SHDP, 4) native to 5 biosynthesis to generate 6. The strategy of diversifying protein structure and function using PTMs could be paralleled in secondary metabolites with the use of PBMs.

Directing macrocyclic architecture using iron(III)-, gallium(III)-, or zirconium(IV)-assisted ring closure of linear dimeric endo-hydroxamic acid ligands.

Dimeric hydroxamic acid macrocycles are a subclass of bacterial siderophores produced for iron acquisition. Limited yields from natural sources provides the impetus to develop synthetic routes to improve access to these compounds, which have potential utility in metal ion binding applications in the environment and medicine. This work has examined the role of metal ions in forming pre-complexes with linear endo-hydroxamic acid (endo-HXA) ligands bearing terminal amine and carboxylic acid groups optimally configured for in situ ring closure reactions. The 1:1 reaction between Fe(III) and the dimeric endo-HXA ligand 5-((5-(5-((5-aminopentyl)(hydroxy)amino)-5-oxopentanamido)pentyl)(hydroxy)amino)-5-oxopentanoic acid (PPH-PPH) (1) formed the pre-complex (PC) [Fe(PP-PP)-PC]+ with in situ amide coupling generating the macrocycle (MC) [Fe(PP)2-MC]+ and, following Fe(III) removal, the apo-macrocycle 1,13-dihydroxy-1,7,13,19-tetraazacyclotetracosane-2,6,14,18-tetraone (PPH)2-MC (2). The 1:2 reaction system between Fe(III) and the monomeric endo-HXA ligand 5-((5-aminopentyl)(hydroxy)amino)-5-oxopentanoic acid (PPH) gave significantly less [Fe(PP)2-MC]+ than the former system, due to the requirement to form two rather than one amide bond(s). The 1:1 Ga(III):1 system yielded [Ga(PP-PP)-PC]+ and [Ga(PP)2-MC]+. Neither [Zr(PP-PP)-PC]2+ nor [Zr(PP)2-MC]2+ was detected in the 1:1 Zr(IV):1 system. Instead, the Zr(IV) system showed the formation of a 1:2 Zr(IV):1 pre-complex [Zr(PP-PP)2-PC], which following in situ amide bond forming chemistry, generated two Zr(IV) macrocyclic complexes with distinct architectures: a dimer-of-dimers complex [Zr((PP)2)2-MC] and an end-to-end macrocycle [Zr(PP)4-MC]. The formation of [Fe(PP)2-MC]+, [Ga(PP)2-MC]+ or [Zr((PP)2)2-MC] was confirmed from reconstitution experiments with 2. The work has shown that the choice of metal ion in metal-assisted ring closure reactions directs the assembly of macrocyclic complexes with distinct architectures.

endo-Hydroxamic Acid Monomers for the Assembly of a Suite of Non-native Dimeric Macrocyclic Siderophores Using Metal-Templated Synthesis.

An expedited synthesis of endo-hydroxamic acid aminocarboxylic acid (endo-HXA) compounds has been developed. These monomeric ligands are relevant to the synthesis of metal-macrocycle complexes using metal-templated synthesis (MTS), and the downstream production of apomacrocycles. Macrocycles can display useful drug properties and be used as ligands for radiometals in medical imaging applications, which supports methodological advances in accessing this class of molecule. Six endo-HXA ligands were prepared that contained methylene groups, ether atoms, or thioether atoms in different regions of the monomer (1-6). MTS using a 1:2 Fe(III)/ligand ratio furnished six dimeric hydroxamic acid macrocycles complexed with Fe(III) (1a-6a). The corresponding apomacrocycles (1b-6b) were produced upon treatment with diethylenetriaminepentaacetic acid (DTPA). Constitutional isomers of the apomacrocycles that contained one ether oxygen atom in the diamine-containing (2b) or dicarboxylic acid-containing (3b) region were well resolved by reverse-phase high-performance liquid chromatography (RP-HPLC). Density functional theory calculations were used to compute the structures and solvated molecular properties of 1b-6b and showed that the orientation of the amide bonds relative to the pseudo-C2 axis was close to parallel in 1b, 2b, and 4b-6b but tended toward perpendicular in 3b. This conformational constraint in 3b reduced the polarity compared with 2b, consistent with the experimental trend in polarity observed using RP-HPLC. The improved synthesis of endo-HXA ligands allows expanded structural diversity in MTS-derived macrocycles and the ability to modulate macrocycle properties.

The chemical biology and coordination chemistry of putrebactin, avaroferrin, bisucaberin, and alcaligin.

Dihydroxamic acid macrocyclic siderophores comprise four members: putrebactin (putH2), avaroferrin (avaH2), bisucaberin (bisH2), and alcaligin (alcH2). This mini-review collates studies of the chemical biology and coordination chemistry of these macrocycles, with an emphasis on putH2. These Fe(III)-binding macrocycles are produced by selected bacteria to acquire insoluble Fe(III) from the local environment. The macrocycles are optimally pre-configured for Fe(III) binding, as established from the X-ray crystal structure of dinuclear [Fe2(alc)3] at neutral pH. The dimeric macrocycles are biosynthetic products of two endo-hydroxamic acid ligands flanked by one amine group and one carboxylic acid group, which are assembled from 1,4-diaminobutane and/or 1,5-diaminopentane as initial substrates. The biosynthesis of alcH2 includes an additional diamine C-hydroxylation step. Knowledge of putH2 biosynthesis supported the use of precursor-directed biosynthesis to generate unsaturated putH2 analogues by culturing Shewanella putrefaciens in medium supplemented with unsaturated diamine substrates. The X-ray crystal structures of putH2, avaH2 and alcH2 show differences in the relative orientations of the amide and hydroxamic acid functional groups that could prescribe differences in solvation and other biological properties. Functional differences have been borne out in biological studies. Although evolved for Fe(III) acquisition, solution coordination complexes have been characterised between putH2 and oxido-V(IV/V), Mo(VI), or Cr(V). Retrosynthetic analysis of 1:1 complexes of [Fe(put)]+, [Fe(ava)]+, and [Fe(bis)]+ that dominate at pH < 5 led to a forward metal-templated synthesis approach to generate the Fe(III)-loaded macrocycles, with apo-macrocycles furnished upon incubation with EDTA. This mini-review aims to capture the rich chemistry and chemical biology of these seemingly simple compounds.