This doctoral thesis presentation summarizes research on the transmetallation mechanisms of gadolinium complexes. The work studied newly developed contrast agents for magnetic resonance imaging to improve safety and efficiency. Physicochemical properties of macrocyclic ligands and their gadolinium complexes were characterized using potentiometric titrations, UV-Vis spectroscopy, and NMR spectroscopy to determine protonation states and kinetic inertness towards demetallation. The goal was to better understand transmetallation mechanisms in order to develop safer and more effective MRI contrast agents.

1. 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

2. 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
×

3. 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

4. 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 N
COO-
COO--OOC
-OOC
COO- N N
NN
COO--OOC
-OOC COO-
DTPA DOTA

5. 16/12/2013 5
Contributions
M. Port et al. BioMetals. 2008, 21, 469–490
Ionic Non ionic Ionic Non ionic
Macrocyclic Linear
Gadolinium

6. 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
22 22
CH2 CH2
O O
CH3 CH3
2 2
M. Port et al. BioMetals. 2008, 21, 469–490
Gd-DTPA-BMEA

7.  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

8. Gd3+
N
N
N
N
coo-
-OOC
-OOC
COO-
n
n = 0, 1
A = PO4
3-, 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

9. 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 = PO4
3-, 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

10. 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

11. 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
O
HN S
S
HOOC
AuNP
N
N
N
COOH
COOH
N
H
O
HN
S
S
HOOC
N N
N
COOH
COOH
O
NH
O
NH
S S
COOH
N N
NN
CO2H
HO2C
HO2C
N
H
N
C. Alric et al. J. Am. Chem. Soc. 2008, 130, 5908–5915

12. 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

13. 16/12/2013 13
Macrocyclic ligands
N N
NN
CO2H
HO2C
HO2C
N
H
N
L1H4
N N
NN
CO2H
HO2C
HO2C
N
N
O2N
L2H3
Dr. S. J. Archibald group, University of Hull, UK

14. 16/12/2013 14
Species distribution diagrams
HYSS treatment
 Potentiometric titrations of ligands L1H4
2 4 6 8 10 12
0
20
40
60
80
100
[L
1
]
4-
L
1
H
3-
L
1
H2
2-L
1
H3
-
L
1
H4
L
1
H5
+L
1
H6
2+
%ofprotonatedspeciesofL
1
H4
pH
N N
NN
CO2H
HO2C
HO2C
N
H
N
[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

15. N N
NN
CO2H
HO2C
HO2C
HN
H
N
16/12/2013 15
200 300 400
0,0
0,5
1,0
Absorbance
 (nm)
UV NMR
Identification of the species
A. El Majzoub et al. Eur. J. Inorg. Chem. 2007, 5087–5097
BIMHn

16. 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
H
N
A. El Majzoub et al. Eur. J. Inorg. Chem. 2007, 5087–5097
H
N
N
H
H
N
N
BIMH2
+
BIMH
240 280 320
0,0
0,5
1,0
Absorbance
 (nm)
pH = 2.3
pH = 3.4
pH = 4.1
pH = 6.0

17. 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
H
N
 Hyperchromic shift beyond pH 11
H
N
N
N
N
BIM-BIMH
A. El Majzoub et al. Eur. J. Inorg. Chem. 2007, 5087–5097
H
N
N
H
H
N
N
BIMH2
+
BIMH
240 280 320
0,0
0,5
1,0
Absorbance
 (nm)
pH = 2.3
pH = 3.4
pH = 4.1
pH = 6.0
240 280 320
0,0
0,5
1,0
Absorbance
 (nm)
pH = 8.2
pH = 10.6
pH = 11.5

18. 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 zone
ppm
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 zone
ppm







7.8 7.6 7.4 7.2 4.4 4.2 4.0 3.8
Aromatic zone Aliphatic zone
ppm
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 zone
ppm
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 zone
ppm







 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
H
N
*

19. 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 zone
ppm
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 zone
ppm







7.8 7.6 7.4 7.2 4.4 4.2 4.0 3.8
Aromatic zone Aliphatic zone
ppm
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 zone
ppm
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 zone
ppm







 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
H
N
*
H
N
N
H
H
N
N
BIMH2
+
BIMH
4.7

20. 16/12/2013 20
Protonation scheme
N N
NN
-
OOC COO-
-
OOC
N
N
[L1]4-
N N
NN
-
OOC COO-
-
OOC
N
H
N
[L1H]3-
N N
NN
-
OOC COO-
-
OOC
N
H
N
[L1H2]2-
H+
N N
NN
-
OOC COO-
-
OOC
N
H
N
[L1H3]-
2H+
N N
NN
-
OOC COO-
-
OOC
HN
H
N
[L1H4]
2H+
N N
NN
-
OOC COOH
-
OOC
HN
H
N
[L1H5]+
2H+
N N
NN
-
OOC COOH
HOOC
HN
H
N
[L1H6]2+
2H+
2.6
4.1 4.7
8.3
12.5 9.9

21. 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 0,7
2,0
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
7pH
[L] = [M] = 10-3 M, NMe4Cl (0.1 M)

22. 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 0,7
2,0
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
7pH
J. Moreau et al. Chem. Eur. J. 2004, 10, 5218–5232

23. 16/12/2013 23
Species distribution diagrams of Gd(III) and
Eu(III) complexes
2 4 6 8 10 12
0
20
40
60
80
100
L
1
H4
L
1
H5
+
L
1
H7
3+
L
1
H6
2+
[GdL
1
]
-[GdL
1
H]
[GdL
1
H2
]
+
Gd
3+
%Gd
pH 2 4 6 8 10 12
0
20
40
60
80
100
[EuL
1
]
-[EuL
1
H]
[EuL
1
H2
]
+
L
1
H5
+
L
1
H6
2+
Eu
3+
%Eu
pH
• Gd(III) • Eu(III)
[L] = [M] = 2×10-3 M, 25°C, NMe4Cl (0.1 M)
N N
NN
CO2H
HO2C
HO2C
N
H
N
L1H4

24. 2 4 6 8 10 12
0
20
40
60
80
100
LH5
+
LH6
2+
[GdL]
-
[GdLH]
[GdLH2
]
+
Gd
3+
%Gd
pH
7800
8000
8200
8400
8600
8800
9000

(mol
-1
Lcm
-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 12
0
20
40
60
80
100
[EuL
1
H] [EuL
1
]
-
[EuL
1
H2
]
+
Eu
3+
L
1
H6
2+
L
1
H5
+
%Eu
pH
8400
8600
8800
9000
9200
9400

(mol
-1
Lcm
-1
)
M = Gd
M = Eu
N N
NN
CO2H
HO2C
HO2C
HN
H
N
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)

25. 2 4 6 8 10 12
0
20
40
60
80
100
LH5
+
LH6
2+
[GdL]
-
[GdLH]
[GdLH2
]
+
Gd
3+
%Gd
pH
7800
8000
8200
8400
8600
8800
9000

(mol
-1
Lcm
-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 12
0
20
40
60
80
100
[EuL
1
H] [EuL
1
]
-
[EuL
1
H2
]
+
Eu
3+
L
1
H6
2+
L
1
H5
+
%Eu
pH
8400
8600
8800
9000
9200
9400

(mol
-1
Lcm
-1
)
M = Gd
M = Eu
N N
NN
CO2H
HO2C
HO2C
HN
H
N
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

26. 16/12/2013 26
Gd
Eu
3.0
4.1
8.4
9.3
N N
N N
CO2
--
O2C
-
O2C
HN
N
H
M
[ML1
H2]+
N N
N N
CO2
--
O2C
-
O2C
HN
N
M
N N
N N
CO2
--
O2C
-
O2C
N
N
M
OH2
[ML1
H] [ML1
]-
OH2
H2O OH2
Gd
Eu
3.0
4.1
8.4
9.3
N N
N N
CO2
--
O2C
-
O2C
HN
N
H
M
[ML1
H2]+
N N
N N
CO2
--
O2C
-
O2C
HN
N
M
N N
N N
CO2
--
O2C
-
O2C
N
N
M
OH2
[ML1
H] [ML1
]-
OH2
H2O OH2
Gadolinium and Europium complexes
 nH2O determined by fluorescence (S.J Archibald group)
Complexation Schemes
3 8.4
4 9.3

27. 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
H
N
L1H4
N N
NN
CO2H
CO2H
CO2H
L4H4
HO2C
N N
NN
CO2H
HO2C
HO2C L5H3
HO
OH
OH
-12
-10
-8
-6
-4
-2
0
L
1
H4
L
5
H3
L
4
H4
log([Gd]free
/[Gd]total
)
2 4 6 8 10 12pH

28. 16/12/2013 28
Species distribution diagrams of transition
metal complexes (Cu(II) and Zn(II))
2 4 6 8 10 12
0
20
40
60
80
100
L
1
H6
2+
Cu
2+
% Cu
[CuL
1
]
2-
[CuL
1
H]
-
[CuL
1
H2
]
[CuL
1
H3
]
+
pH
2 4 6 8 10 12
0
20
40
60
80
100
LH5
+
LH6
2+
Zn
2+
[ZnL
1
H4
]
2+
[ZnL
1
H3
]
+
[ZnL
1
H2
]
[ZnL
1
H]
-
[ZnL
1
]
2-
%Zn
pH
[L] = [M] = 2×10-3 M, 25°C, NMe4Cl (0.1 M)
N N
N N
CO2
--
O2C
-
O2C
HN
N
HM
[ML1
H2]
N N
N N
CO2
--
O2C
-
O2C
HN
N
M
N N
N N
CO2
--
O2C
-
O2C
N
N
M
[ML1
H]-
[ML1
]2-
Cu
Zn
4.5 9.2
5.1 9.7

29.  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
H
N
-10
-8
-6
-4
-2
0
log([M]free
/[M]total
)
2 4 6 8 10 12
Zn-L
1
H4
Cu-L
1
H4
Eu-L
1
H4
pH
Gd-L
1
H4

30. 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
H
N
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
ZnLH
ZnLH3
GdLH2
LH6
ZnLH4
GdLGdLH
%L
pH

31. 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

32. 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 5000
0,0
0,2
0,4
0,6
0,8
1,0
1,2
R1t
/R10
t (min)
Gd-L
1
H4
Gd-L
4
H4
Relaxation rate versus time, [GdL] =[Zn] = 1:1; T= 37°C, pH 7.4, in phosphate buffer

33. 16/12/2013 33
Linear ligands
N
N
N
COOH
HOOC
O
NH
O
HN S
S
HOOC
AuNP
N
N
N
COOH
COOH
N
H
O
HN
S
S
HOOC
N N
N
COOH
COOH
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

34. 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
CH3
O
HN
H3C
L@
1H5
DTPA – BMA or L@
3H3
NH NH HN
C
COO
COO
C
OOC
O
NH SH
O
HNHS

35. 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. Garcés 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
O
HN S
S
HOOC
AuNP
N
N
N
COOH
COOH
N
H
O
HN
S
S
HOOC
N N
N
COOH
COOH
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)

36. 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

37. 16/12/2013 37
2 4 6 8 10 12
0
20
40
60
80
100
[GdL@
1
]
2-
[GdL@
1
H]
-
[GdL@
1
H2
]
Gd
3+
L@
1
H5
%Gd
pH
2 4 6 8 10 12
0
20
40
60
80
100
[GdL@
2
]
[GdL@
2
H]
[GdL@
2
H2
]
Gd
3+
%Gd
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
O
HN S
S
HOOC
AuNP
N
N
N
COOH
COOH
N
H
O
HN
S
S
HOOC
N N
N
COOH
COOH
O
NH
O
NH
S S
COOH

38. 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
O
HN S
S
HOOC
AuNP
N
N
N
COOH
COOH
N
H
O
HN
S
S
HOOC
N N
N
COOH
COOH
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@
1
H5
L@
2
H3
pH
log([Gd]free
/[Gd]total
)
L@
4
H5
2 4 6 8 10 12
N N N
COOHHOOC
O
NH
O
HNHS SH
COOH
L@
1H 5

39. 16/12/2013 39
[L] = [M] = 2×10-3 M, 25°C, NMe4Cl (0.1 M)
2 4 6 8 10 12
0
20
40
60
80
100
[Cu2
L@
1
(OH)2
]
3-
[CuL@
1
]
3-[CuL@
1
H]
2-
[Cu2
L@
1
]
-
[CuL@
1
H2
]
-
[Cu2
L@
1
H]
Cu
2+
[CuL@
1
H3
]
%Cu
pH
Species distribution diagrams of Cu(II) and
Zn(II) complexes (M/L = 1/1)
2 4 6 8 10 12
0
20
40
60
80
100
[ZnL@
1
H]
-
[ZnL@
1
]
2-
[ZnL@
1
H2
]
[ZnL@
1
H3
]
+
Zn
2+
%Zn
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

40. 16/12/2013 40
[L] = [M] = 2×10-3 M, 25°C, NMe4Cl (0.1 M)
2 4 6 8 10 12
0
20
40
60
80
100
[CuL@
2
H2
]
[CuL@
2
H] [CuL@
2
]
Cu
2+
%Cu
pH
2 4 6 8 10 12
0
20
40
60
80
100
[ZnL@
2
]
[ZnLH@
2
]
[ZnL@
2
H2
]
Zn
2+
%Zn
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
O
HN S
S
HOOC
AuNP
N
N
N
COOH
COOH
N
H
O
HN
S
S
HOOC
N N
N
COOH
COOH
O
NH
O
NH
S S
COOH

41. 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@
1
H5
Zn-L@
1
H5
Gd-L@
1
H5
pH
log([M]free
/[M]total
)
Cu-L@
1
H5
2 4 6 8 10 12
-10
-8
-6
-4
-2
0
Ca-L@
2
H3
Gd-L@
2
H3
Cu-L@
2
H3
pH
log([M]total
/[M]free
)
Zn-L@
2
H3
2 4 6 8 10 12
L@
2H3
N
N
N
COOH
HOOC
O
NH
O
HN S
S
HOOC
AuNP
N
N
N
COOH
COOH
N
H
O
HN
S
S
HOOC
N N
N
COOH
COOH
O
NH
O
NH
S S
COOH

42. 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
O
HN S
S
HOOC
AuNP
N
N
N
COOH
COOH
N
H
O
HN
S
S
HOOC
N N
N
COOH
COOH
O
NH
O
NH
S S
COOH
0
2
4
6
8
10
1
Gd Cu Zn Ca
Log([M]free/[M]total)
Cu > Gd > Zn > Ca
0
2
4
6
8
10
1
Gd Cu Zn Ca
Gd > Cu > Zn > Ca
Log([M]free/[M]total)
[L] = [M] = 2×10-3 M, 25°C, NMe4Cl (0.1 M)

43. 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 ZnL1
@
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

44. 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+Gd3+
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
O
HN S
S
HOOC
AuNP
N
N
N
COOH
COOH
N
H
O
HN
S
S
HOOC
N N
N
COOH
COOH
O
NH
O
NH
S S
COOH
From thermodynamic determinations

45. 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

46. 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 5000
0,0
0,2
0,4
0,6
0,8
1,0
R1
(t)/R1
(t=0)
t (mins)
DTPA:Gd
L@
1
H5
:Gd
L@
2
H3
: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

47. 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 5000
0,0
0,2
0,4
0,6
0,8
1,0
R1
(t)/R1
(t=0)
t (mins)
DTPA:Gd
L@
1
H5
:Gd
L@
2
H3
: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

48. 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 NMe4
Cl at 25°C
Absorbance
Wavelength(nm)
tk
AA
AA
ln obs
e0
et








y =-0,001x
R² =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(At-Ae/A0-Ae)
Pseudo first order
kobs
N N N
COOHHOOC
COOH
OO
NHHN SHHS

49. 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 0,010
10
15
20
25
30
kobs
(10
-4
s
-1
)
[Zn
2+
] (mol L
-1
)

50. 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 0,010
10
15
20
25
30
kobs
(10
-4
s
-1
)
[Zn
2+
] (mol L
-1
)

51. 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
Zn
ZnxLHn + H
- spontaneous
- H assisted
transmet. pathways

52. 16/12/2013 52
Investigation of transmetallation mechanism
   
 



 2
54
22
3
2
21
obs
ZnBB
ZnBZnBB
k
     
    76
2
5
43
2
2
3
1
obs
PHPHP
PHPHPHP
k



Versus [Zn2+]
pH fixed
Versus [H+]
[Zn2+] fixed
 Tobsi i GdLkvv  
           GdLZnGdLZnHGdLHGdLHGdLGdL 2T 

53. 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
Zn
ZnxLHn + 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

54. 16/12/2013 54
Conclusion

55. 16/12/2013 55
Conclusion
- half-life of the Gd complexes
- demetallation pathways in
competitive conditions
2 4 6 8 10 12
0
20
40
60
80
100
[GdL@
2
]
[GdL@
2
H]
[GdL@
2
H2
]
Gd
3+
%Gd
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
Zn
ZnxLHn + H
- spontaneous
- H assisted
transmet. pathways

56. 16/12/2013 56
Conclusion
No transmetallation
N N
NN
CO2H
HO2C
HO2C
N
H
N
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
1Gd-
L4H4
Gd-
L1H4
Gd-
L5H3
Log([M]free/[M]total)
0
1
2
3
4
5
6
7
8
1Gd-
L@
4H5
Gd-
L@
2H3
Log([M]free/[M]total)