phd presentation-v.mogilireddy
TRANSCRIPT
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
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
×
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
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
16/12/2013 5
Contributions
M. Port et al. BioMetals. 2008, 21, 469–490
Ionic Non ionic Ionic Non ionic
Macrocyclic Linear
Gadolinium
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
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
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
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
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
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
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
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
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
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
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
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
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*
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
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
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)
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
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
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)
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
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
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
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
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
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
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
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
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
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
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)
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
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
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
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
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
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
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)
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
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
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
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
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
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
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
)
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
)
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
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
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
16/12/2013 54
Conclusion
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
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)