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Study of transmetallation mechanisms of gadolinium complexes Doctoral Thesis Presentation, 16 December 2013 Vijetha MOGILIREDDY 1 16/12/2013 1 This work was funded by the Région Champagne Ardenne

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Page 1: PhD presentation-V.Mogilireddy

Study of transmetallation

mechanisms of gadolinium

complexes

Doctoral Thesis Presentation, 16 December 2013

Vijetha MOGILIREDDY

1

16/12/2013 1 This work was funded by the Région Champagne Ardenne

Page 2: PhD presentation-V.Mogilireddy

16/12/2013 2

Motivation

T1 image

H. William et. al, Clinical Radiol. 2000, 55, 825-831

Magnetic Resonance Imaging (MRI)

Anatomical imaging

Resolution Sensitivity

×

Page 3: PhD presentation-V.Mogilireddy

16/12/2013 3

Motivation

H. William et. al, Clinical Radiol. 2000, 55, 825-831

Magnetic Resonance Imaging (MRI)

Anatomical imaging

Resolution Sensitivity Contrast agents

T1 image

Paramagnetic systems

Reduced T1 values and increased brightness on T1 weighted images

T1 (cerebral lesion)without CA = 1000 ms

T1 (cerebral lesion)with CA = 330 ms

Page 4: PhD presentation-V.Mogilireddy

Constitution of Contrast agents

16/12/2013 4

Contributions

M

N

N

N

N

coo-

-OOC

-OOC

COO-

n

n = 0, 1M

Complex

Paramagnetic metal ions (Mn(II), Fe(III), Cu(II), Gd(III))

Gd3+ - 7 unpaired electrons

Free Gd3+ ion is toxic

Complexed with a cage like structure (multidentate chelate /

ligand)

J-C. G. Bunzli et al. Chem. Soc. Rev. 2005, 34, 1048–1077; A.E. Merbach, E. Toth (eds). The Chemistry of Contrast Agents in

Medical Magnetic Resonance Imaging. Wiley: New York, 2001

N N NCOO-

COO--OOC

-OOC

COO- N N

NNCOO--OOC

-OOC COO-

DTPA DOTA

Page 5: PhD presentation-V.Mogilireddy

16/12/2013 5

Contributions

M. Port et al. BioMetals. 2008, 21, 469–490

Ionic Non ionic Ionic Non ionic

Macrocyclic Linear

Gadolinium

Page 6: PhD presentation-V.Mogilireddy

16/12/2013 6

Contributions

Gd-DOTA Gd-HPDO3A Gd-DTPA

Gd-DTPA-BMA

Gadolinium

Gd-BTDO3A

Ionic Non ionic Ionic Non ionic

Macrocyclic Linear

2 2 2 2

CH2 CH2

O O

CH3 CH3

2 2

M. Port et al. BioMetals. 2008, 21, 469–490

Gd-DTPA-BMEA

Page 7: PhD presentation-V.Mogilireddy

First case in 1997

Damages internal organs sometimes leading to death

Patients with low glomerular filteration rate

16/12/2013 7

Nephrogenic Systemic Fibrosis (NSF) –

Problem definition

Peau d’orange appearance

Fibrosis of skin, joints, eyes

and internal organs

S. E. Cowper et al. The Lancet. 2000, 356, 1000–1001

Page 8: PhD presentation-V.Mogilireddy

Gd3+

N

N

N

N

coo-

-OOC

-OOC

COO-

n

n = 0, 1

A = PO43-, CO3

2-

B = Citrate, lactate, amino acids

M = Zn2+, Cu2+, Fe3+, Mg2+, Ca2+

16/12/2013 8

Link of NSF with contrast agents

T. Grobner, Nephrol. Dial. Transplant. 2006, 21, 1104–1108

P. Marckmann et al. J. Am. Soc. Nephrolog. 2006, 17, 2359–2362

E. Brücher. et al. Chem. Eur. J. 2000, 6, 719–724

GdL

L*GdL GdL* + L

+ L

GdLM + ML

Gd3+

Gd3+

GdHL

GdH2L+ H2LGd3+

+ HLGd3+

H+H+

L*

M

pH 3.6 – 5.2

Page 9: PhD presentation-V.Mogilireddy

16/12/2013 9

Link of NSF with contrast agents

Classification of European Medicine Agency

Safest cyclic structure

Intermediate ionic linear structure

least safest non ionic linear structure

Gd3+

N

N

N

N

coo-

-OOC

-OOC

COO-

n

n = 0, 1

A = PO43-, CO3

2-

B = Citrate, lactate, amino acids

M = Zn2+, Cu2+, Fe3+, Mg2+, Ca2+

T. Grobner, Nephrol. Dial. Transplant. 2006, 21, 1104–1108

P. Marckmann et al. J. Am. Soc. Nephrolog. 2006, 17, 2359–2362

Page 10: PhD presentation-V.Mogilireddy

16/12/2013 10

How to improve the Gd contrast agents

I. Lukes et al. Dalton Trans. 2008, 3027–3047

C. Alric et al. J. Am. Chem. Soc. 2008, 130, 5908–5915

Relaxivity enhancement

Rotational correlation time : R

Accelerate the exchange of H2O molecules : kex

Number of water molecules : q

Number of Gd(III) complexes : nGd

16/12/2013 10

Page 11: PhD presentation-V.Mogilireddy

16/12/2013 11

How to improve the Gd contrast agents

I. Lukes et al. Dalton Trans. 2008, 3027–3047

Relaxivity enhancement

Rotational correlation time : R

Accelerate the exchange of H2O molecules : kex

Number of water molecules : q

Number of Gd(III) complexes : nGd

16/12/2013 11

N

N

N

COOH

HOOC

O

NH

OHN S

S

HOOC

AuNP

N

N

NCOOH

COOH

NH

O

HNS

S

HOOC

N NN

COOHCOOH

O

NH

O

NH

S S

COOH

N N

NN

CO2H

HO2C

HO2C

N

HN

C. Alric et al. J. Am. Chem. Soc. 2008, 130, 5908–5915

Page 12: PhD presentation-V.Mogilireddy

16/12/2013 12

Contrast agents

Physico-chemical study of newly developped contrast agents

Safety Efficiency

Outline

Potentiometric study of ligands and metal complexes

Kinetic inertness evaluation of Gd complexes towards demetallation

Investigation of transmetallation mechanisms

Page 13: PhD presentation-V.Mogilireddy

16/12/2013 13

Macrocyclic ligands

N N

NN

CO2H

HO2C

HO2C

N

HN

L1H4

N N

NN

CO2H

HO2C

HO2C

N

N

O2N

L2H3

Dr. S. J. Archibald group, University of Hull, UK

Page 14: PhD presentation-V.Mogilireddy

16/12/2013 14

Species distribution diagrams

HYSS treatment

Potentiometric titrations of ligands L1H4

2 4 6 8 10 120

20

40

60

80

100

[L1]4-

L1H

3-L

1H

2

2-L1H

3

-

L1H

4

L1H

5

+L1H

6

2+

% o

f p

roto

na

ted

sp

ecie

s o

f L

1H

4

pH

N N

NN

CO2H

HO2C

HO2C

N

HN

[L] = 2×10-3 M, 25°C, NMe4Cl (0.1 M)

L1H4

log b log K

LH 12.5 12.5

LH2 22.4 9.9

LH3 30.7 8.3

LH4 35.4 4.7

LH5 39.5 4.1

LH6 42.1 2.6

Page 15: PhD presentation-V.Mogilireddy

N N

NN

CO2H

HO2C

HO2C

HN

HN

16/12/2013 15

200 300 4000,0

0,5

1,0

Ab

so

rba

nce

(nm)

UV NMR

Identification of the species

A. El Majzoub et al. Eur. J. Inorg. Chem. 2007, 5087–5097

BIMHn

Page 16: PhD presentation-V.Mogilireddy

16/12/2013 16

UV spectroscopic studies

Bathochromic and hypochromic shift

( = 274 and 280 nm) between pH 4.1 and 6

Evolution of spectra as a function of pH

N N

NN

CO2H

HO2C

HO2C

HN

HN

A. El Majzoub et al. Eur. J. Inorg. Chem. 2007, 5087–5097

HN

NH

HN

NBIMH2

+ BIMH

240 280 3200,0

0,5

1,0

Ab

sorb

ance

(nm)

pH = 2.3

pH = 3.4

pH = 4.1

pH = 6.0

Page 17: PhD presentation-V.Mogilireddy

16/12/2013 17

UV spectroscopic studies

Bathochromic and hypochromic shift

( = 274 and 280 nm) between pH 4.1 and 6

Evolution of spectra as a function of pH

N N

NN

CO2H

HO2C

HO2C

HN

HN

Hyperchromic shift beyond pH 11 HN

N

N

NBIM-BIMH

A. El Majzoub et al. Eur. J. Inorg. Chem. 2007, 5087–5097

HN

NH

HN

NBIMH2

+ BIMH

240 280 3200,0

0,5

1,0

Ab

sorb

ance

(nm)

pH = 2.3

pH = 3.4

pH = 4.1

pH = 6.0

240 280 3200,0

0,5

1,0

Ab

sorb

ance

(nm)

pH = 8.2

pH = 10.6

pH = 11.5

Page 18: PhD presentation-V.Mogilireddy

16/12/2013 18

NMR spectroscopic studies

7.8 7.6 7.4 7.2 4.4 4.2 4.0 3.8

Aromatic zone Aliphatic zoneppm

pH = 11.0

pH = 6.5

pH = 1.9

pH = 1.0

pH = 3.0

pH = 3.7

pH = 4.7

pH = 8.8

pH = 7.7

pH = 10.0

7.8 7.6 7.4 7.2 4.4 4.2 4.0 3.8

Aromatic zone Aliphatic zoneppm

7.8 7.6 7.4 7.2 4.4 4.2 4.0 3.8

Aromatic zone Aliphatic zoneppm

7.8 7.6 7.4 7.2 4.4 4.2 4.0 3.8

Aromatic zone Aliphatic zone

7.8 7.6 7.4 7.2 4.4 4.2 4.0 3.8

Aromatic zone Aliphatic zoneppm

pH = 11.0

pH = 6.5

pH = 1.9

pH = 1.0

pH = 3.0

pH = 3.7

pH = 4.7

pH = 8.8

pH = 7.7

pH = 10.0

7.8 7.6 7.4 7.2 4.4 4.2 4.0 3.8

Aromatic zone Aliphatic zoneppm

Upfield chemical shift between pH 3.7

and 6.5

Evolution of spectra as a function of pH, D2O, 300 MHz

N N

NN

CO2H

HO2C

HO2C

HN

HN*

Page 19: PhD presentation-V.Mogilireddy

16/12/2013 19

NMR spectroscopic studies

7.8 7.6 7.4 7.2 4.4 4.2 4.0 3.8

Aromatic zone Aliphatic zoneppm

pH = 11.0

pH = 6.5

pH = 1.9

pH = 1.0

pH = 3.0

pH = 3.7

pH = 4.7

pH = 8.8

pH = 7.7

pH = 10.0

7.8 7.6 7.4 7.2 4.4 4.2 4.0 3.8

Aromatic zone Aliphatic zoneppm

7.8 7.6 7.4 7.2 4.4 4.2 4.0 3.8

Aromatic zone Aliphatic zoneppm

7.8 7.6 7.4 7.2 4.4 4.2 4.0 3.8

Aromatic zone Aliphatic zone

7.8 7.6 7.4 7.2 4.4 4.2 4.0 3.8

Aromatic zone Aliphatic zoneppm

pH = 11.0

pH = 6.5

pH = 1.9

pH = 1.0

pH = 3.0

pH = 3.7

pH = 4.7

pH = 8.8

pH = 7.7

pH = 10.0

7.8 7.6 7.4 7.2 4.4 4.2 4.0 3.8

Aromatic zone Aliphatic zoneppm

Upfield chemical shift between pH 3.7

and 6.5

Evolution of spectra as a function of pH, D2O, 300 MHz

N N

NN

CO2H

HO2C

HO2C

HN

HN*

HN

NH

HN

NBIMH2

+ BIMH

4.7

Page 20: PhD presentation-V.Mogilireddy

16/12/2013 20

Protonation scheme

N N

NN

-OOC COO-

-OOCN

N

[L1]4-

N N

NN

-OOC COO-

-OOCN

HN

[L1H]3-

N N

NN

-OOC COO-

-OOCN

HN

[L1H2]2-

H+

N N

NN

-OOC COO-

-OOCN

HN

[L1H3]-

2H+

N N

NN

-OOC COO-

-OOCHN

HN

[L1H4]

2H+

N N

NN

-OOC COOH

-OOCHN

HN

[L1H5]+

2H+

N N

NN

-OOC COOH

HOOCHN

HN

[L1H6]2+

2H+

2.6

4.1 4.7

8.3

12.5 9.9

Page 21: PhD presentation-V.Mogilireddy

16/12/2013 21

Determination of stability constants

of the metal complexes - Methodology

Out-of-Cell Method

Storage at 37°C under argon for one month

Measurement of pH of each cell at 25°C

0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,72,0

2,5

3,0

3,5

4,0

4,5

pH

VOH

- /mL

J. Moreau et al. Chem. Eur. J. 2004, 10, 5218–5232

1 2

3 4

5 6

7 pH

[L] = [M] = 10-3 M, NMe4Cl (0.1 M)

Page 22: PhD presentation-V.Mogilireddy

16/12/2013 22

Determination of stability constants

Methodology

Out-of-Cell Method

Storage at 37°C under argon for one month

Measurement of pH of each cell at 25°C

Selection of a tube (appropriate pH) followed

by the titration with NMe4OH in conventionnal manner

0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,72,0

2,5

3,0

3,5

4,0

4,5

pH

VOH

- /mL

0,0 0,1 0,2 0,3 0,4 0,5 0,6

4

5

6

7

8

9

10

11

12

pH

VOH

- /mL

1 2

3 4

5 6

7 pH

J. Moreau et al. Chem. Eur. J. 2004, 10, 5218–5232

Page 23: PhD presentation-V.Mogilireddy

16/12/2013 23

Species distribution diagrams of Gd(III) and

Eu(III) complexes

2 4 6 8 10 120

20

40

60

80

100

L1H

4

L1H

5

+

L1H

7

3+

L1H

6

2+

[GdL1]-[GdL

1H]

[GdL1H

2]+

Gd3+

%G

d

pH 2 4 6 8 10 120

20

40

60

80

100

[EuL1]-[EuL

1H]

[EuL1H

2]+

L1H

5

+

L1H

6

2+

Eu3+

% E

u

pH

• Gd(III) • Eu(III)

[L] = [M] = 2×10-3 M, 25°C, NMe4Cl (0.1 M)

N N

NN

CO2H

HO2C

HO2C

N

HN

L1H4

Page 24: PhD presentation-V.Mogilireddy

2 4 6 8 10 120

20

40

60

80

100

LH5

+

LH6

2+

[GdL]-

[GdLH]

[GdLH2]+

Gd3+

%G

d

pH

7800

8000

8200

8400

8600

8800

9000

(

mo

l-1 L

cm

-1)

16/12/2013 24

UV spectroscopic studies

[L] = [M] = 2×10-3 M, 25°C, NMe4Cl (0.1 M)

2 4 6 8 10 120

20

40

60

80

100[EuL

1H]

[EuL1]-

[EuL1H

2]+

Eu3+

L1H

6

2+

L1H

5

+

%E

upH

8400

8600

8800

9000

9200

9400

(m

ol-1

L c

m-1)

M = Gd

M = Eu

N N

NN

CO2H

HO2C

HO2C

HN

HN

A. El Majzoub et al. Eur. J. Inorg. Chem. 2007, 5087–5097

log K Gd Eu

L1H4

ML1H2 ML1H 3.0 4.1 4.67 BIMH2+ BIMH

ML1H ML1 8.4 9.3 12.5 BIMH BIM-

• 278nm = f(pH)

Page 25: PhD presentation-V.Mogilireddy

2 4 6 8 10 120

20

40

60

80

100

LH5

+

LH6

2+

[GdL]-

[GdLH]

[GdLH2]+

Gd3+

%G

d

pH

7800

8000

8200

8400

8600

8800

9000

(

mo

l-1 L

cm

-1)

16/12/2013 25

UV spectroscopic studies

[L] = [M] = 2×10-3 M, 25°C, NMe4Cl (0.1 M)

2 4 6 8 10 120

20

40

60

80

100[EuL

1H]

[EuL1]-

[EuL1H

2]+

Eu3+

L1H

6

2+

L1H

5

+

%E

upH

8400

8600

8800

9000

9200

9400

(m

ol-1

L c

m-1)

M = Gd

M = Eu

N N

NN

CO2H

HO2C

HO2C

HN

HN

A. El Majzoub et al. Eur. J. Inorg. Chem. 2007, 5087–5097

log K Gd Eu

L1H4

ML1H2 ML1H 3.0 4.1 4.67 BIMH2+ BIMH

ML1H ML1 8.4 9.3 12.5 BIMH BIM-

• 278nm = f(pH)

Involvement of BIMH moiety in the Ln(III)

coordination sphere

Page 26: PhD presentation-V.Mogilireddy

16/12/2013 26

Gd

Eu

3.0

4.1

8.4

9.3

N N

N NCO2

--O2C

-O2C

HN

NH

M

[ML1H2]+

N N

N NCO2

--O2C

-O2C

HN

N

M

N N

N NCO2

--O2C

-O2C

N

N

M

OH2

[ML1H] [ML1]-

OH2H2O OH2

Gd

Eu

3.0

4.1

8.4

9.3

N N

N NCO2

--O2C

-O2C

HN

NH

M

[ML1H2]+

N N

N NCO2

--O2C

-O2C

HN

N

M

N N

N NCO2

--O2C

-O2C

N

N

M

OH2

[ML1H] [ML1]-

OH2H2O OH2

Gadolinium and Europium complexes

nH2O determined by fluorescence (S.J Archibald group)

Complexation Schemes

3 8.4

4 9.3

Page 27: PhD presentation-V.Mogilireddy

16/12/2013 27

Stability of Gd(III) complexes

L4H4 > L1H4 > L5H3 M = Gd(III)

[L] = [M] = 2×10-3 M, 25°C, NMe4Cl (0.1 M)

Log([Gd]free/[Gd]total) = f(pH)

N N

NN

CO2H

HO2C

HO2C

N

HN

L1H4

N N

NN

CO2H

CO2H

CO2H

L4H4

HO2CN N

NN

CO2H

HO2C

HO2C L5H3

HOOH

OH

-12

-10

-8

-6

-4

-2

0

L1H

4

L5H

3

L4H

4

log(

[Gd

] free

/[G

d] to

tal)

2 4 6 8 10 12pH

Page 28: PhD presentation-V.Mogilireddy

16/12/2013 28

Species distribution diagrams of transition

metal complexes (Cu(II) and Zn(II))

2 4 6 8 10 120

20

40

60

80

100

L1H

6

2+

Cu2+

% Cu

[CuL1]2-[CuL

1H]

-

[CuL1H

2]

[CuL1H

3]+

pH2 4 6 8 10 12

0

20

40

60

80

100

LH5

+

LH6

2+

Zn2+

[ZnL1H

4]2+

[ZnL1H

3]+

[ZnL1H

2]

[ZnL1H]

-

[ZnL1]2-

% Z

n

pH

[L] = [M] = 2×10-3 M, 25°C, NMe4Cl (0.1 M)

N N

N NCO2

--O2C

-O2CHN

NHM

[ML1H2]

N N

N NCO2

--O2C

-O2CHN

N

MN N

N NCO2

--O2C

-O2CN

N

M

[ML1H]- [ML1]2-

Cu

Zn

4.5 9.2

5.1 9.7

Page 29: PhD presentation-V.Mogilireddy

Gd>Eu>Cu>Zn

16/12/2013 29

Stability of metal complexes

Comparison of stability of all metal

complexes

[L] = [M] = 2×10-3 M, 25°C, NMe4Cl (0.1 M)

N N

NN

CO2H

HO2C

HO2C

N

HN

-10

-8

-6

-4

-2

0

log

([M

] free/[

M] to

tal)

2 4 6 8 10 12

Zn-L1H

4

Cu-L1H

4

Eu-L1H

4

pH

Gd-L1H

4

Page 30: PhD presentation-V.Mogilireddy

16/12/2013 30

At pH~7:

- [GdLH] > 95%

- [ZnLH] < 5%

[L] = [Gd] = [Zn] = 2×10-3 M

Stability of metal complexes

N N

NN

CO2H

HO2C

HO2C

N

HN

What happens for L1H4 in the presence of Gd(III) and Zn(II)?

From thermodynamic determinations

Transmetallation? 2 4 6 8 10 12

0

20

40

60

80

100

ZnLH2

ZnLHZnLH

3

GdLH2

LH6

ZnLH4

GdLGdLH

%L

pH

Page 31: PhD presentation-V.Mogilireddy

16/12/2013 31

Relaxometric measurements, phosphate buffer (pH~7.4)

R1(t)/R1(t=0) = f(t)

What is expected :

Gd release R1(t) < R1(t = 0) in the current conditions

S. Laurent et al. CMMI, 2010, 5, 305–308

Kinetic inertness

GdL ZnL ++ Zn Gd

Relaxation rate versus time, [GdL] =[Zn] = 1:1; T= 37°C, pH 7.4, in phosphate buffer

Page 32: PhD presentation-V.Mogilireddy

16/12/2013 32

Relaxometric measurements, phosphate buffer at pH~7.4

R1(t)/R1(t=0) = f(t)

Here no decrease in R1(t) values

S. Laurent et al. CMMI, 2010, 5, 305–308

Kinetic inertness

GdL ZnL ++ Zn Gd

No transmetallation was detected

0 1000 2000 3000 4000 50000,0

0,2

0,4

0,6

0,8

1,0

1,2

R1

t / R

10

t (min)

Gd-L1H

4

Gd-L4H

4

Relaxation rate versus time, [GdL] =[Zn] = 1:1; T= 37°C, pH 7.4, in phosphate buffer

Page 33: PhD presentation-V.Mogilireddy

16/12/2013 33

Linear ligands

N

N

N

COOH

HOOC

O

NH

OHN S

S

HOOC

AuNP

N

N

NCOOH

COOH

NH

O

HNS

S

HOOC

N NN

COOHCOOH

O

NH

O

NH

S S

COOH

L@2H3

Dr. S. Roux group, Université de Franche-Comté, France

N N N

COOHHOOC

COOH

OO

NHHN SHHS

L@1H5

Page 34: PhD presentation-V.Mogilireddy

16/12/2013 34

Protonation constants of ligands

Potentiometry, [L] = 2×10-3 M, OH- = 5×10-2 M, HCl = 1×10-2 M

10.37 (2) 9.77 (1) 8.96 (2) 4.79 (1) 3.43 (1) 2.34 (1)

C.F.G.C Geraldes et al. MRI 1995, 13, 401–420. G. Crisponi et al. Polyhedron 2002, 21, 1319–1327

9.4 4.4 3.1

NH NH HN

C

COO

COO

C

OOC

O

NH

CH3O

HN

H3C

L@1H5

DTPA – BMA or L@3H3

NH NH HN

C

COO

COO

C

OOC

O

NH SH

O

HNHS

Page 35: PhD presentation-V.Mogilireddy

16/12/2013 35

Protonation constants of ligands

Potentiometry, [L] = 2×10-3 M, OH- = 5×10-2 M, HCl = 1×10-2 M

L. J. Garces et al. J. Phys. Chem. B. 2009, 113, 15145–15155. C. David et al. J. Phys. Chem. B. 2007, 111, 10421–10430

NH NH HN

C

COO

COOH

C

OOC

O

NH SH

O

HNHS

L@1H5

L@2H3

N

N

N

COOH

HOOC

O

NH

OHN S

S

HOOC

AuNP

N

N

NCOOH

COOH

NH

O

HNS

S

HOOC

N NN

COOHCOOH

O

NH

O

NH

S S

COOH

Basicity increase for L@2H3

L@2H3 11.26(3) 10.12(2) 7.27(3) 5.75(2) 3.78(1)

L@1H5 10,37(2) 9.77(1) 8.86(2) 4.79(1) 3.43(1) 2.34(1)

Page 36: PhD presentation-V.Mogilireddy

16/12/2013 36

Protonation constants of ligands

Potentiometry, [L] = 2×10-3 M, OH- = 5×10-2 M, HCl = 1×10-2 M

L@1H5

• Ligand packing at the nanoparticle surface

• H bond network that stabilize added protons

L. Morrigi et al., JACS 2009, 131, 10828-–10829

L@2H3

NH NH HN

C

COO

COOH

C

OOC

O

NH SH

O

HNHS

L@2H3 11.26(3) 10.12(2) 7.27(3) 5.75(2) 3.78(1)

L@1H5 10,37(2) 9.77(1) 8.86(2) 4.79(1) 3.43(1) 2.34(1)

Basicity increase for L@2H3

Page 37: PhD presentation-V.Mogilireddy

16/12/2013 37

2 4 6 8 10 120

20

40

60

80

100

[GdL@

1]2-

[GdL@

1H]

-

[GdL@

1H

2]

Gd3+

L@

1H

5

%G

d

pH2 4 6 8 10 12

0

20

40

60

80

100

[GdL@

2]

[GdL@

2H]

[GdL@

2H

2]

Gd3+

% G

d

pH

Stability constants obtained through direct titrations

using potentiometry

Species distribution diagrams of Gd(III)

complexes

[L] = [M] = 2×10-3 M, 25°C, NMe4Cl (0.1 M)

N N N

COOHHOOC

COOH

OO

NHHN SHHS

N

N

N

COOH

HOOC

O

NH

OHN S

S

HOOC

AuNP

N

N

NCOOH

COOH

NH

O

HNS

S

HOOC

N NN

COOHCOOH

O

NH

O

NH

S S

COOH

Page 38: PhD presentation-V.Mogilireddy

16/12/2013 38

Stability of Gd(III) complexes

Log([Gd]free/[Gd]total)

[L] = [M] = 2×10-3 M, 25°C, NMe4Cl (0.1 M)

N N N

COOH

COOHHOOC

HOOC

COOH L@4H5

N

N

N

COOH

HOOC

O

NH

OHN S

S

HOOC

AuNP

N

N

NCOOH

COOH

NH

O

HNS

S

HOOC

N NN

COOHCOOH

O

NH

O

NH

S S

COOH

L@2H3

L@4H5 = L@

2H3 > L@1H5 M = Gd(III)

-10

-8

-6

-4

-2

0

L@

1H

5

L@

2H

3

pH

log (

[Gd] fr

ee/[

Gd] to

tal)

L@

4H

5

2 4 6 8 10 12

N N N

COOHHOOC

O

NH

O

HNHS SH

COOH

L@1H 5

Page 39: PhD presentation-V.Mogilireddy

16/12/2013 39

[L] = [M] = 2×10-3 M, 25°C, NMe4Cl (0.1 M)

2 4 6 8 10 120

20

40

60

80

100

[Cu2L

@

1(OH)

2]3-

[CuL@

1]3-[CuL

@

1H]

2-

[Cu2L

@

1]-

[CuL@

1H

2]-

[Cu2L

@

1H]

Cu2+

[CuL@

1H

3]%

Cu

pH

Species distribution diagrams of Cu(II) and

Zn(II) complexes (M/L = 1/1)

2 4 6 8 10 120

20

40

60

80

100

[ZnL@

1H]

-[ZnL

@

1]2-

[ZnL@

1H

2]

[ZnL@

1H

3]+

Zn2+

%Z

n

pH

N N N

C

COOH

COOH

C

HOOC

O

NH SH

O

HNHS

L@1H5

• M = Cu(II)

• M = Zn(II)

Dinuclear Cu(II) complexes even in

M/L = 1/1 conditions

Page 40: PhD presentation-V.Mogilireddy

16/12/2013 40

[L] = [M] = 2×10-3 M, 25°C, NMe4Cl (0.1 M)

2 4 6 8 10 120

20

40

60

80

100

[CuL@

2H

2]

[CuL@

2H] [CuL

@

2]

Cu2+

%C

u

pH

2 4 6 8 10 120

20

40

60

80

100

[ZnL@

2]

[ZnLH@

2]

[ZnL@

2H

2]

Zn2+

%Z

n

pH

• M = Cu(II)

• M = Zn(II)

L@2H3

Species distribution diagrams of Cu(II) and

Zn(II) complexes

N

N

N

COOH

HOOC

O

NH

OHN S

S

HOOC

AuNP

N

N

NCOOH

COOH

NH

O

HNS

S

HOOC

N NN

COOHCOOH

O

NH

O

NH

S S

COOH

Page 41: PhD presentation-V.Mogilireddy

16/12/2013 41

Stability of metal complexes

Cu>Gd>(Zn>Ca)

[L] = [M] = 2×10-3 M, 25°C, NMe4Cl (0.1 M)

Gd>(Cu>Zn>Ca)

N N N

COOHHOOC

COOH

OO

NHHN SHHS

L@1H5

-16

-14

-12

-10

-8

-6

-4

-2

0

Ca-L@

1H

5

Zn-L@

1H

5

Gd-L@

1H

5

pH

log([

M] fr

ee/[

M] to

tal)

Cu-L@

1H

5

2 4 6 8 10 12

-10

-8

-6

-4

-2

0

Ca-L@

2H

3

Gd-L@

2H

3

Cu-L@

2H

3

pH

log([

M] to

tal/[

M] fr

ee)

Zn-L@

2H

3

2 4 6 8 10 12

L@2H3

N

N

N

COOH

HOOC

O

NH

OHN S

S

HOOC

AuNP

N

N

NCOOH

COOH

NH

O

HNS

S

HOOC

N NN

COOHCOOH

O

NH

O

NH

S S

COOH

Page 42: PhD presentation-V.Mogilireddy

16/12/2013 42

Stability of metal complexes

• At pH = 7.4

N N N

COOHHOOC

COOH

OO

NHHN SHHS

L@1H5 L@

2H3

N

N

N

COOH

HOOC

O

NH

OHN S

S

HOOC

AuNP

N

N

NCOOH

COOH

NH

O

HNS

S

HOOC

N NN

COOHCOOH

O

NH

O

NH

S S

COOH

0

2

4

6

8

10

1

Gd Cu Zn Ca

Log([

M] f

ree/[M

] tota

l)

Cu > Gd > Zn > Ca 0

2

4

6

8

10

1

Gd Cu Zn Ca

Gd > Cu > Zn > Ca

Log([

M] f

ree/[M

] tota

l)

[L] = [M] = 2×10-3 M, 25°C, NMe4Cl (0.1 M)

Page 43: PhD presentation-V.Mogilireddy

16/12/2013 43

Stability of metal complexes

At pH~7

- [GdL@1H2] > 80%

- [ZnL@1H2] < 20%

0

20

40

60

80

100

2 4 6 8 10 12pH

% M

Gd3+

Zn2+

ZnL1@H3

ZnL1@H2

ZnL1@H ZnL

1@

ZnL1@(OH)

GdL1@H2

GdL1@H

GdL1@

[L] = [Gd] = [M] = 2×10-3 M

What happens for L@1H5 in the presence of Gd(III) and Zn(II)?

Transmetallation

N N N

COOHHOOC

COOH

OO

NHHN SHHS

From thermodynamic determinations

Page 44: PhD presentation-V.Mogilireddy

16/12/2013 44

Stability of metal complexes

[L] = [Gd] = [M] = 2×10-3 M

At pH~7

-[GdL@2] > 95%

-[ZnL@2H] < 5%

0

20

40

60

80

100

2 4 6 8 10 12

pH

% M

Zn2+Gd

3+

GdL2@

ZnL2@H2

ZnL2@H

What happens for L@2H3 in the presence of Gd(III) and Zn(II)?

Transmetallation

N

N

N

COOH

HOOC

O

NH

OHN S

S

HOOC

AuNP

N

N

NCOOH

COOH

NH

O

HNS

S

HOOC

N NN

COOHCOOH

O

NH

O

NH

S S

COOH

From thermodynamic determinations

Page 45: PhD presentation-V.Mogilireddy

16/12/2013 45

Kinetic inertness

With Zn(II) in excess

(mechanism)

M = competitive cations (Zn(II))

GdL ML ++ M Gd

Under stoichiometric conditions

between GdL and Zn(II)

Relaxometry UV-vis spectroscopy

L = L@1H5 and L@

2H3 L = L@1H5

Page 46: PhD presentation-V.Mogilireddy

16/12/2013 46

Stoichiometric conditions

Relaxation rates are measured as a function of time

Relaxation rate versus time, [GdL] =[Zn] = 1:1; T= 37°C, pH 7.4, in phosphate buffer

0 1000 2000 3000 4000 50000,0

0,2

0,4

0,6

0,8

1,0

R1(t

)/R

1(t

=0)

t (mins)

DTPA:Gd

L@

1H

5:Gd

L@

2H

3:Gd

Kinetic index

t80%: Time for R1(t)/ R1(t = 0) = 0.8 GdL ZnL ++ Zn Gd

Gd-L@1H5 Gd-L@

2H3 Gd-DTPA

t80% 108 min

≈ 2h 216 min

≈ 4h 275 min

≈ 5h

Kinetic stability order

Gd-DTPA > Gd-L@2H3 > Gd-L@

1H5

S. Laurent et al. CMMI, 2010, 5, 305–308

Page 47: PhD presentation-V.Mogilireddy

16/12/2013 47

Stoichiometric conditions

Relaxation rates are measured as a function of time

Relaxation rate versus time, [GdL] =[Zn] = 1:1; T= 37°C, pH 7.4, in phosphate buffer

0 1000 2000 3000 4000 50000,0

0,2

0,4

0,6

0,8

1,0

R1(t

)/R

1(t

=0)

t (mins)

DTPA:Gd

L@

1H

5:Gd

L@

2H

3:Gd

Kinetic index

t80%: Time for R1(t)/ R1(t = 0) = 0.8 GdL ZnL ++ Zn Gd

Thermodynamic index

% GdL 4320min = R1(t = 4320) / R1(t = 0)

Gd-L@1H5 Gd-L@

2H3 Gd-DTPA

t80% 108 min

≈ 2h 216 min

≈ 4h 275 min

≈ 5h

Gd-L@1H5 Gd-L@

2H3 Gd-DTPA

% GdL 4320min

10% 30% 42%

S. Laurent et al. CMMI, 2010, 5, 305–308

Page 48: PhD presentation-V.Mogilireddy

16/12/2013 48

Excess of competitive cation

In the presence of excess Zn(II) and at various pH conditions

4×10-3 M < [Zn2+] < 10×10-3 M

5.8 < pH < 6.5

A = f(t), [GdL] =5×10-4 M; [Zn] = 4×10-3 M; T= 25°C, pH 6.5, NMe4Cl (0.1M)

200 250 300 350 400

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

GdDTPA(0.5mM)+Cu(5mM) in NMe4Cl at 25°C

Ab

so

rba

nce

Wavelength(nm)

t kAA

AAln obs

e0

et

y = -0,001xR² = 0,991

-1,4

-1,2

-1

-0,8

-0,6

-0,4

-0,2

0

0 200 400 600 800 1000

t (mins)

Ln(A

t-A

e/A

0-A

e)

Pseudo first order

kobs

N N N

COOHHOOC

COOH

OO

NHHN SHHS

Page 49: PhD presentation-V.Mogilireddy

16/12/2013 49

4×10-3 M < [Zn2+] < 10×10-3 M, 5.8 < pH < 6.5, T= 25°C, NMe4Cl (0.1 M)

• kobs = f([Zn2+])

pH = 5.8 pH = 6.0

pH = 6.2 pH = 6.5

Investigation of transmetallation mechanism

• For a given [H+]: kobs ↗ when [Zn2+] ↗

0,004 0,005 0,006 0,007 0,008 0,009 0,01010

15

20

25

30

k ob

s (1

0-4

s-1

)

[Zn2+

] (mol L-1

)

Page 50: PhD presentation-V.Mogilireddy

16/12/2013 50

4×10-3 M < [Zn2+] < 10×10-3 M, 5.8 < pH < 6.5, T= 25°C, NMe4Cl (0.1 M)

• kobs = f([Zn2+])

pH = 5.8 pH = 6.0

pH = 6.2 pH = 6.5

Investigation of transmetallation mechanism

• For a given [Zn2+]: kobs ↗ when [H+] ↗

• For a given [H+]: kobs ↗ when [Zn2+] ↗

GdLZnHn complexes involved in the transmetallation mechanism

0,004 0,005 0,006 0,007 0,008 0,009 0,01010

15

20

25

30

k ob

s (1

0-4

s-1

)

[Zn2+

] (mol L-1

)

Page 51: PhD presentation-V.Mogilireddy

16/12/2013 51

Investigation of transmetallation mechanism

5.8 < pH < 6.5, T= 25°C, NMe4Cl (0.1 M)

E. Brücher. et al. Chem. Eur. J. 2000, 6, 719–724

GdLHn

Zn

Zn

GdLZnH + H

GdLZn + 2H

ZnL + Gd + 2H

Zn2L + Gd + 2H

ZnL + Gd + 2H

Zn2L + Gd + 2H

Zn

Zn

ZnZnxLHn + H

- spontaneous

- H assisted

transmet. pathways

Page 52: PhD presentation-V.Mogilireddy

16/12/2013 52

Investigation of transmetallation mechanism

2

54

22

3

2

21obs

ZnBB

ZnBZnBBk

76

2

5

43

2

2

3

1

obsPHPHP

PHPHPHPk

Versus [Zn2+] pH fixed

Versus [H+] [Zn2+] fixed

Tobsi i GdLkvv

GdLZnGdLZnHGdLHGdLHGdLGdL 2T

Page 53: PhD presentation-V.Mogilireddy

16/12/2013 53

Investigation of transmetallation mechanism

E. Brücher. et al. Chem. Eur. J. 2000, 6, 719–724

GdLHn

Zn

Zn

GdLZnH + H

GdLZn + 2H

ZnL + Gd + 2H

Zn2L + Gd + 2H

ZnL + Gd + 2H

Zn2L + Gd + 2H

Zn

Zn

k1

k2

k3

k4

ZnZnxLHn + H

- spontaneous

- H assisted

transmet. pathways

(1)

(2)

(3)

(4)

1 2 3 4

ki (104 M-1s-1) 38 0.08

ki (104 M-2s-1) 1007 654

Transmetallation is driven by Zn(II) attack on heteronuclear GdLZnH and GdLZn complexes

Page 54: PhD presentation-V.Mogilireddy

16/12/2013 54

Conclusion

Page 55: PhD presentation-V.Mogilireddy

16/12/2013 55

Conclusion

- half-life of the Gd complexes

- demetallation pathways in

competitive conditions

2 4 6 8 10 120

20

40

60

80

100

[GdL@

2]

[GdL@

2H]

[GdL@

2H

2]

Gd3+

% G

d

pH

Thermodynamic stability

- stability of Gd complexes

- identification of the species at

physiological pH

Kinetic inertness

From a methodological point of view

GdLHn

Zn

Zn

GdLZnH + H

GdLZn + 2H

ZnL + Gd + 2H

Zn2L + Gd + 2H

ZnL + Gd + 2H

Zn2L + Gd + 2H

Zn

Zn

ZnZnxLHn + H

- spontaneous

- H assisted

transmet. pathways

Page 56: PhD presentation-V.Mogilireddy

16/12/2013 56

Conclusion

No transmetallation

N N

NN

CO2H

HO2C

HO2C

N

HN

Gd-L@2H3 Gd-DTPA

t1/2 257 min 277 min

Thermodynamic stability

Kinetic inertness

Good candidates for MRI applications

0

1

2

3

4

5

6

7

8

1Gd- L4H4

Gd- L1H4

Gd- L5H3

Log(

[M] f

ree/

[M] t

ota

l)

0

1

2

3

4

5

6

7

8

1Gd- L@

4H5

Gd- L@

2H3

Log(

[M] f

ree/

[M] t

ota

l)

Page 57: PhD presentation-V.Mogilireddy