Thermodynamics of binding of iron(III) by brasilibactin AJames 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.

Fe3

+

OH2

OH2

OH2

OH2

H2

O

H2

O

Fe3

+OHOH2

OH2

OH2

H2

OH2

OH+ H+ H+

Ksp = 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

Al

Al

Ca

Microbial Cell

Fe

Fe

Fe

FeAl

Microbes produce small molecules called siderophores, to solubilize iron, return it to the cell, and facilitate transport into the cell.

Fe

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.

N

O

O

HN

OHN

OHO

NH

O

O

RO

NCHOHO

Brasilibactin A, R = C15H31

ObjectiveCharacterize 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 (NaClO4)

Problem: reversibility of protonation?

N

O

O

HN

OHN

OHO

NH

O

O

O

NCHOHO

OH-

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 (NaClO4) Using 1 proton model, pKa1 = 9.05 ± .08

NOHO

NH

O

OH

Fragment 3&4

Spectrophotometric titration of Fragment 1-2

pKa1 = 10.09±0.03, pKa2 = 8.18 ±0.09

pKa1 = 4.8 ±0.2, pKa2 = 2.9 ±0.1

pH 6.0-10.6 pH 2.7-6.0

N

O

O

HN

OH

HO

O

NCHOHO

Fragment 1&2

H

N

O

O

HN

OHN

OHO

NH

O

O

O

NCHOHO

BbtH

Fe-BbtH spectrophotometric titration

Conditions: [Fe3+] = 2.3 x 10-4 M, [BbtH] = 2.4 x 10-4 M, 25 °C, μ = 0.10 M (NaClO4)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: [Fe3+] = 2.1 x 10-4 M, [BbtH] = 2.1 x 10-4 M, 25 °C, μ = 0.10 M (NaClO4)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

NO

O

NH

O

NO

O

HN

O

OO

N

C

O

O

H

Fe3+

N

O

O

HN

OHN

OHO

NH

O

O

RO

NCHOHO

+ EDTA Fe(EDTA) +

Conditions: [Fe3+] = 2.5 x 10-4 M, [BbtH] = 2.6 x 10-

4 M, 25 °C, μ = 0.10 M (NaClO4).

N

O

O

HN

OHN

OHO

NH

O

O

O

NHO

O 12

Mycobactin S

Siderophore Log β110 pFea

BbtH 26.961 22.73Mycobactin S 26.62 N/A

Desferrioxamine B

30.63 26.6

Aerobactin 27.64 23.3Exochelin MN 39.125 31.1

Rhodotorulic acid 62.2b 21.9apFe 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.bThis stability constant is a log β230. Ref. 6.

ConclusionsoThe Brasilibactin A analog hydrolyzes at basic pH.oThe presence of Fe stabilizes the Brasilibactin A analog through at least pH 8 (complex dissociates irreversibly ab). oMolecule forms a stable complex with iron(III), but less stable than other hexadentate siderophores.oBbtH exhibits a slower rate of complex formation with iron than AHA does.

References:1 – This work2 – 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.

O

NH

N

OOH

NH3

HN

O

NH2+

N

OH

O

HN

O

HN

O

NH+HN

HO

+H3N

Exochelin MN

N

O OHNH

O

O

NHO

NH

O

O

NHO

+H3N

Desferrioxamine B

HN

NHO

O

NN

OHOOHO

Rhodotorulic acid

O

N

HN

HO

HO

O O

HO

COOH

NH

O

N OHO

OH

OAerobactin

OH

N

OHN

O

NHO

O

O

O

NH

N

O

OH

H

pKa = 10.09 pKa = 2.9

pKa = 8.18

pKa = 9.05

O

H

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 (NaClO4)

Comparison of stability constants

oBbt 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.