1. Thermodynamics of binding of iron(III) by brasilibactin A James Harrington, Heekwang Park, Yongcheng Ying, Jiyong Hong, and Alvin L. Crumbliss, Department of Chemistry, Duke University, Durham, NC, 27708-0346 Iron is necessary but problematic. Fe 3+ OH 2 H2OH2O H2OH2O Fe 3+ OHOH OH 2 H2OH2O H2OH2O H+H+ H+H+ H+H+ K sp = 10 -39 However, iron(III) easily hydrolyzes and forms insoluble hydroxide and oxide salts, resulting in low aqueous concentrations at physiological conditions. Iron can also take part in redox reactions that produce reactive oxygen species and can harm organisms Microbial Iron Acquisition Fe Environment Na Ca SYNTHESIS SOLUBILIZATION TRANSPORT EXCHANGE AND UPTAKE RELEASE Mg Al Ca Microbial Cell Fe Al Microbes produce small molecules called siderophores, to solubilize iron, return it to the cell, and facilitate transport into the cell. Brasilibactin A is a membrane-bound siderophore produced by Nocardia brasiliensis, which has been found to be cytotoxic at low concentrations (~ 50 nM). It is hypothesized that this is due to iron binding, as iron(III) inhibits caspase 3, an enzyme in the apoptosis pathway. Objective Characterize the pKa’s and thermodynamics of interaction of iron(III) with brasilibactin A by spectrophotometric/potentiometric titrations Ligand Spectrophotometric titration Conditions: [L] = 1.4 x 10 -4 M, 25 °C, μ = 0.10 M (NaClO 4 ) Problem: reversibility of protonation? The irreversible spectral transition suggests chemical reaction, possibly hydrolysis. Ester moiety may be susceptible to hydrolysis at high pH. Similar behavior has been observed in other siderophores, such as enterobactin, fusarinines, and fusigens. Fragment Potentiometric titration Conditions: [L] = 5.8 x 10 -4 M, 25 °C, μ = 0.10 M (NaClO 4 ) Using 1 proton model, pK a1 = 9.05 ±.08 Spectrophotometric titration of Fragment 1-2 pK a1 = 10.09±0.03, pK a2 = 8.18 ±0.09 pK a1 = 4.8 ±0.2, pK a2 = 2.9 ±0.1 pH 6.0-10.6pH 2.7-6.0 Fe-BbtH spectrophotometric titration Conditions: [Fe 3+ ] = 2.3 x 10 -4 M, [BbtH] = 2.4 x 10 -4 M, 25 °C, μ = 0.10 M (NaClO 4 ) The transition at high pH is not reversible. Likely dissociation of the complex, then hydrolysis of the ligand. Low pH spectrophotometric titration of the Fe(III)-BbtH system Conditions: [Fe 3+ ] = 2.1 x 10-4 M, [BbtH] = 2.1 x 10-4 M, 25 °C, μ = 0.10 M (NaClO 4 ) At low pH, the spectrum slowly decreases to baseline. This shift is reversible by returning the pH to its original value. Indicates reversible dissociation of the Fe(III)-BbtH complex. Competition of Fe(III)-BbtH complex with EDTA was performed to determine the thermodynamic stability constant of the Fe(III)- BbtH complex. [Fe(BbtH)] stability constant + EDTA  Fe(EDTA) + Conditions: [Fe 3+ ] = 2.5 x 10 -4 M, [BbtH] = 2.6 x 10 -4 M, 25 °C, μ = 0.10 M (NaClO 4 ). Siderophore Log β 110 pFe a BbtH26.96 1 22.73 Mycobactin S26.6 2 N/A Desferrioxamine B30.6 3 26.6 Aerobactin27.6 4 23.3 Exochelin MN39.12 5 31.1 Rhodotorulic acid62.2 b 21.9 a pFe is the concentration of free aqueous iron(III) in solution at set conditions of [M] = 10 -6 M, [L] = 10 -5 M, and pH = 7.4. b This stability constant is a log β 230. Ref. 6. Conclusions o The Brasilibactin A analog hydrolyzes at basic pH. o The presence of Fe stabilizes the Brasilibactin A analog through at least pH 8 (complex dissociates irreversibly ab). o Molecule forms a stable complex with iron(III), but less stable than other hexadentate siderophores. o BbtH exhibits a slower rate of complex formation with iron than AHA does. References: 1 – This work 2 – MacCordick, Schleiffer, and Duplatre, Radiochim. Acta 1985, 38, 43. 3 – Schwarzenbach and Schwartzenbach, Helv. Chim. Acta 1963, 46, 1390. 4 – Kupper, Carrano, Kuhn, and Butler, Inorg. Chem. 2006, 45, 6028. 5 – Dhungana, Miller, Dong, Ratledge, Crumbliss, J. Am. Chem. Soc. 2003, 125, 7654. 6 – Spasojevic, Armstrong, Brickman, Crumbliss, Inorg. Chem. 1999, 38, 449. Acknowledgements: We thank Duke University, the Center for Biomolecular and Tissue Engineering, the NIH, NSF Grants CHE 0418006 and CHE 0809466, and the rest of the Crumbliss and Hong labs. Iron is necessary for a variety of cellular processes, i.e. small molecule transport, electron transport. Microbes require an effective concentration of at least 10 -5 M for survival Introduction Conditions: [L] = 1.7 x 10 -4 M, 25 °C, μ = 0.10 M (NaClO 4 ) Comparison of stability constants o Bbt complex exhibits slow formation kinetics (relative to AHA). Addition of iron(III) to solution of BbtH at low pH (~2) resulted in complex formation over 3 times as long as complex formation was observed with AHA as evidenced by change in solution color.