Iron(III) sequestration by synthetic hydroxypyridinone siderophores and exchange with desferrioxamine B

J. M. Harrington,1 S. Dhungana,1 S. Chittamuru,2 H. K. Jacobs,2 A. S. Gopalan,2 and A.L. Crumbliss1

1Department of Chemistry, Duke University, Durham, NC 27708-0346 and 2Department of Chemistry and

Biochemistry, New Mexico State University, Las Cruces, NM, 88003-8001

The Iron Paradox Precipitation of Fe(OH)3 (Fe2O3, etc.) Redox chemistry

O2.-O2

H2O2 OH HO-

Fe2+

Fe3+

+

Haber-Weiss Cycle

Able to Participate inHaber-Weiss Cycle

Synthetic Siderophores

N

OH

O

X

3-hydroxy-2-pyridinone

N NN N

O OHOHO

N2(LH)2

N N

N

N

OHO

N

O OH

N O

OH

N3(LH)3

N2(LH)2 synthesisNN NN

OH

O O

OH

N2(LH)2

N

O

BnO +

MeSO3-

N

O

N N N

O

ORRO

1. Piperazine, Et3N, CH3CN 55 °C

2. Conc. HBr/glacial acetic acid (1:1), rt

R = Bn (93%)R = H (93%)

• 2HBr

Lambert, T. N.; Chittamuru, S.; Jacobs, H. K.; Gopalan, A. S. Tetrahedron Lett., 2002, 43/41, 7379

N

OH

HO OEt

O

N

O

HOOEt

O

CsF, CH3CN, reflux, 74%

N

O

BnOOH

1. PhCH2Br, K2CO3CH3CN, reflux

2. BH3•THF, rt90% (both steps)

N

O

BnOOMs

1.(CH3SO2)2O, Et3N,CH2Cl2, 0 °C to rt

2.ChCl3, rt, 92%N+

BnO

O

MeSO4-

N2(LH)2 ThermodynamicsNN NN

OH

O O

OH

N2(LH)2

N NN N

O OHOHOH H

pKa1 pKa4

pKa3pKa2

pKa1 = 3.8 ± .1 pKa2 = 5.91

± .09 pKa3 = 7.94

± .05 pKa4 = 9.21

± .02

Fe-N2(LH)2 Competition

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

350 450 550 650 750

Wavelength (nm)

Ab

s

[EDTA] = 0 M

[EDTA] = 1.96 x 10-3 M

+ 2 EDTA

2 [Fe(EDTA)] + 3

[Fe3+] = 2.47 x 10-4 M, [N2(LH)2] = 3.70 x 10-4 M, T = 25 °C, μ = 0.10.

NN NNOH

O O

OH

N2(LH)2

232

22

232

2

]][)([

][])([

EDTALNFe

FeEDTALHNK

N

N

Fe

O O

O

O

N N

N

N

Fe

O

OO

NN

O

NN NNO

O O

O

N NN N

O OHOHO

Fe-N2(LH)2 spectrophotometric titration

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

350 450 550 650 750

Wavelength (nm)

Ab

s

pH 3.5

pH 7.5

553 nm

[Fe3+] = 2.0 x 10-4 M, [N2(LH)2] = 3.0 x 10-4 M, T = 25 °C, μ = 0.10.

NN NNOH

O O

OH

N2(LH)2

2 + + 2 OH-

]][)([][

])([

222

22

322

2

OHLHNLFeN

LNFeK

N

N

Fe

O O

O

O

N N

N

N

Fe

O

OO

NN

O

NN NNO

O O

O

N

N

Fe

O

OO

NN

O

OH2OH2

N NN N

O OHOHO

Log βFeLH of Fe-N2(LH)2

log β230 = 60.46 ± .02

log β110 = 20.39 ± .02

log β111 = 21.3 ± .1

NN NNOH

O O

OH

N2(LH)2

2 Fe3+ + 3 N2(LH)2

Fe3+ + N2(LH)2

Fe3+ + N2(LH)2 + H+

N

N

Fe

O O

O

O

N N

N

N

Fe

O

OO

NN

O

NN NNO

O O

O

N

N

Fe

O

OO

NN

O

OH2OH2

N

N

Fe

O

O

HO

NN

O

OH2OH2

OH2OH2

3 5 7 9 11pH

0

20

40

60

80

100

% fo

rma

tion

rela

tive to

Fe

Speciation for Fe-N2(LH)2 system

Fe(N2L2

)

Fe2(N2L2)

3

NN NNOH

O O

OH

N2(LH)2

Fe2(N2L2)3Fe(N2L2)

Fe3+

Fe(OH)4-

[Fe3+] = 2 x 10-4 M, [N2(LH)2] = 3 x 10-4 M, T = 25 °C, μ = 0.10.

N

N

Fe

O O

O

O

N N

N

N

Fe

O

OO

NN

O

NN NNO

O O

O

N

N

Fe

O

OO

NN

O

OH2OH2

Fe(OH)2+

N3(LH)3 synthesis

N

O

BnO +

RO

NO

RO

N N

N

N

OOR

N

O

MeSO3-

1. 1, 4,7-Triazacyclononane, Et3N, CH3CN, rt

R = Bn (83%)R = H (87%)

2. Conc. HBr/glacial acetic acid (1:1), rt

• 3HBr

N N

N

N

OHO

N

O OH

N O

OH

N3(LH)3

N3(LH)3 Thermodynamics

N N

N

N

OHO

N

O OH

N O

OH

H

H

pKa1

pKa2

pKa3

pKa4

pKa5

pKa1 = 3.97 ± .07

pKa2 = 5.1 ± .1 pKa3 = 7.50

± .02 pKa4 = 8.84

± .03 pKa5 = 10.40

± .04

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

210 230 250 270 290 310 330 350 370 390

Wavelength (nm)

Ab

s

N N

N

N

OHO

N

O OH

N O

OH

N3(LH)3

Fe(N3(LH)3)-EDTA Competition

[Fe+3] = [N3(LH)3] = 4 x 10-4 M, [EDTA] = 0-10:1 equivalents, T = 25 °C, μ =0.10.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

350 450 550 650 750

Wavelength (nm)

Ab

s

[EDTA] = 0 M

[EDTA] = 4.0 x 10-3

+ EDTA

Fe(EDTA) +

EDTALHNFe

LHNFeEDTAKeff ))((

)(

33

33

FeO

OO

O

OON

N N

N

N

N

N

N N

N

NN O

OH

O

HO

O

HO

N N

N

N

OHO

N

O OH

N O

OH

N3(LH)3

Fe-N3(LH)3 spectrophotometric titration

0

0.2

0.4

0.6

0.8

1

1.2

350 450 550 650 750

Wavelength (nm)

Ab

s

551 nm

pH 2.9

pH 8.02

pKa = 3.10 pKa2 = 13.22

0

0.2

0.4

0.6

0.8

1

1.2

350 450 550 650 750

Wavelength (nm)

Ab

s

397 nm pH 10.44

pH 8.0

1K

OH

•[Fe3+] = [N3(LH)3] = 4.4 x 10-4 M, T = 25 °C, μ =0.10

2K

OH Fe

OOH

OO

OO

N N N

N

N

N

H2O

H2O

FeO

OO

O

OON

N N

N

N

N

H2O

FeO

OO

O

OON

N N

N

N

N

HO

N N

N

N

OHO

N

O OH

N O

OH

N3(LH)3

log βFeLH of N3(LH)3

log β110 = 27.34 ± .04

log β111 = 30.44 ± .08

log β11-1 = 17.66 ± .09

Fe3+ + N3(LH)3 + H+

Fe3+ + N3(LH)3

Fe3+ + N3(LH)3 + OH-

Fe

OOH

OO

OO

N N N

N

N

N

H2O

H2O

FeO

OO

O

OON

N N

N

N

N

H2O

FeO

OO

O

OON

N N

N

N

N

HO

N N

N

N

OHO

N

O OH

N O

OH

N3(LH)3

4 8 12pH

0

20

40

60

80

100

% form

ation relative to

Fe

Speciation for Fe-N3L3 system

Fe

OOH

OO

OO

N N N

N

N

N

H2O

H2O

FeO

OO

O

OON

N N

N

N

N Fe(N3L3)H

Fe(N3L3

)Fe(N3L3)H

[Fe3+] = 1 x 10-4 M, [N3(LH)3] = 1 x 10-4 M, T = 25 °C, μ = 0.10.

Fe(N3L3) Fe(N3L3)OH

Fe3+

Fe(OH)4-

Fe(OH)2+

FeO

OO

O

OON

N N

N

N

N

HO

Fe(N3L3)OH-

N N

N

N

OHO

N

O OH

N O

OH

N3(LH)3

pFe valuespFe = -log[Fe3+]free

Ligand pFe1

Deferiprone 19.42

Rhodotorulic Acid 21.903

N2(LH)2 22.074

N3(LH)3 23.494

Deferasirox 23.55

Deferrioxamine B 26.63

Enterobactin 35.63

1 – [Fe+3] = 10-6, [L] = 10-5, pH = 7.42 – Liu, et al, J. Med. Chem., 1999, 42, 48143 – Harris, et al, JACS, 1979, 101, 2722

4 - This work 5 - Steinhauser, et al, Eur. J. Inorg. Chem., 2004, 2004, 4177

N

N

N

HO

OH

OH

O

Deferasirox

N

CH3

CH3

OH

O

Deferiprone

N

O OH NHO

O

NHO

NH

O

O

NHO

H3NDeferrioxamine B

HN

NH

O

O

NN

OHOOHO

Rhodotorulic acid

Enterobactin

O

O

O

O

O

O

HN

NH

HN

O

O

OOH

HO

HO

HO

HO

HO

N N

N

N

OHO

N

O OH

N O

OH

N3(LH)3

N NN N

O OHOHO

N2(LH)2

Host-Guest complex formation

Batinic-Haberle, I.; Spasojevic, I.; Crumbliss, A. L.; Inorg. Chem.; 1996, 35(8), 2352-2359.

Dhungana, S.; White, P. S.; Crumbliss, A. L.; JACS; 2003, 125(48), 14760-14767.

HN N

N

H HN

+

Host-Guest Complex

0

0.5

1

1.5

2

2.5

3

3.5

300 400 500 600 700

Wavelength (nm)

Ab

s

435 nm

0

0.5

1

1.5

2

2.5

3

3.5

300 400 500 600 700

Wavelength (nm)

Abs

420 nm 522 nm

EtOH/MeOH

EtOH/MeOH

00.20.40.60.8

11.21.41.6

350 450 550 650 750

Wavelength (nm)

Ab

s

428 nm

N N

N

FeO

OO

O

OON

N N

N

N

N

+ HH

H

NN

OO

Fe

HN

OO

O

NH

O

O

O

+ HH

H

NN

OO

Fe

HN

OO

O

NH

O

O

O

NN

Proposed Host-Guest complex

DFB: N3(LH)3 = 50:1 ESI-MS peak: Observed m/z = 1121.5 Proposed H2O adduct

+

N

N

NHN N

N

H H

NO

HO

OOH

O

HO

N

OO

Fe

HN

O

O

O

NH

O

O

O

Exchange kinetics of [FeN3L3] with Desferrioxamine B

Abs vs Wavelength (nm)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

400 450 500 550 600 650

Wavelength (nm)

Ab

s

Fe(N3L3)

FeHDFB+

Abs 510 nm vs Time (min)

0.3

0.4

0.5

0.6

0.7

0.80.9

1

1.1

1.2

1.3

0 200 400 600 800 1000 1200

Time (min)

Ab

s 5

10

nm

Fit to single exponential decay kobs = 8.8 x 10-5 sec-1, k2nd, app = 0.0242 M-1 sec-1.

+ +N

N N

N

NN O

OH

O

HO

O

HO

N

O OH NHO

O

NHO

NH

O

O

NHO

H3N

FeO

OO

O

OON

N N

N

N

N

+ HH

H

NN

OO

Fe

HN

OO

O

NH

O

O

O

Proposed exchange mechanism

+

+

…N

N N

N

NN O

OH

O

HO

O

HO

N

O OH NHO

O

NHO

NH

O

O

NHO

H3N

FeO

OO

O

OON

N N

N

N

N

FeO

OO

O

OON

N N

N

N

N

N

OHOHN

O

O

N OH

NH

O

O

N

OH

NH3+

FeO

OO

O

OON

N N

N

N

N

N

OHOHN

O

O

N OH

NH

O

O

N

OH

NH3+

+

HH H

N

N

OO

Fe

HN

OO

O

NH

O

O

O

Conclusions N2(LH)2 is a stable chelator of iron, and could

provide insight into development of more effective chelation therapy treatments for iron overload.

We also characterized the complexation reactions of N3(LH)3 with iron, showing that it can bind iron effectively.

An exchange reaction can be observed between N3(LH)3 and deferrioxamine B, but not N2(LH)2, suggesting that host-guest interaction may be involved in exchange mechanism.

Acknowledgements Thanks: Dr. Al Crumbliss Esther Tristani The Crumbliss Lab Group Duke University Center for Biomolecular and Tissue

Engineering NIH NSF