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Paramagnetic GdIIIFeIII heterobimetallic complexes of DTPA-bis-salicylamide
Spectrochinu'caActa,Vol. 49A, No. 9, pp. 1315-1322,1993 Printed in Great Britain
0584-8539/93$6.00+0.00 ~) 1993PergamonPressLtd
Paramagnetic Gdm-Fe m heterobimetallic complexes of DTPA-bis-salicylamide S. AIME,* M . BOTI'A, M . FASANO a n d E . TERRENO Dipartimento di Chimica Inorganica, Chimica Fisica e Chimica tel Materiali, Universiti~ di Torino, via P. Giuria 7, 10125 Torino, Italy
(Received 28 April 1992; accepted 8 June 1992) Almtract--The reaction between DTPA (diethylenetriaminepenta-acetic acid)-anhydride and p-aminosalicylic acid (PAS) affords a novel ligand, [DTPA(PAS)2], able to form stable beterobimetallic complexes with Gd 3+ and Fe 3+ ions. The lanthanide ion occupies an internal coordination cage formed by three nitrogen atoms, two carboxylate and two carboxoamido groups of the ligand, whereas the outer salicylic moieties form stable chelate rings with Fem ions. The stoichiometry of the resulting heterobimetallic complexes, established by measurements of water proton relaxation enhancement, is [(H20)-Gd--DTPA(PAS)2]2--Fe(H20)2 or [(H20)-Gd-DTPA(PAS)2b-Fe depending on the pH of the aqueous solution. The individual contributions t o the observed relaxation enhancement from Gd 3+ and Fe 3+ paramagnetic ions have been clearly distinguished and analysed.
INTRODUCTION
THE RAPID development of Magnetic Resonance Imaging (MRI) techniques has aroused new interest in paramagnetic gadolinium(III) complexes for their potential utility as contrast agents (CAs). In particular, polyaminocarboxylate chelates have undergone intense scrutiny for their favourable relaxation properties associated with high thermodynamic stabilities, which are expected to limit the toxicological problems arising from the interaction of both the lanthanide ions and the free ligands with biological substrates [1]. Current efforts are mainly devoted to the search for a higher specificity towards organs and tissues as well as for an increased relaxivity (i.e. the water proton relaxation enhancement per mM concentration of paramagnetic ion) of these CAs in order to achieve a maximum contrast/dose ratio. Actually, it has been shown that, at the typical magnetic fields employed by commercial imaging instruments, a marked increase of relaxivity of gadolinium complexes may be pursued through a lengthening of their molecular reorientation times, rR. This was accomplished either through the formation of protein-Gd-DTPA complex conjugates [2] or by exploiting the non-covalent interactions taking place between suitable functionalized complexes and slowly tumbling systems such as fl-cyclodextrin [3], micelles [4] or plasma proteins [5]. In this work we explore an additional way for enhancing the solvent proton relaxation rate upon the formation of heterobimetallic Gd-Fe paramagnetic complexes. Such species have been obtained through the synthesis of the novel ligand [DTPA(PAS)2] containing a DTPA-Iike coordination cage able to bind the Gd 3÷ ions. EXPERIMENTAL All reagents were purchased commercially and were used without further purification. ~H and
~ac N M R spectra were recorded on a Jeol EX-400 spectrometer. Nas[DTPA(PAS)2] A mixture of p-aminosalicylic acid (PAS) sodium salt dihydrate (475 rag, 2.25 mmol) and diethylenetriaminepenta-acetic acid ( D T P A ) dianhydride (357 mg, 1 mmol) in anhydrous D M S O * Author to whom correspondence should be addressed. 1315
1316
S. AIMEet
al.
(5 ml) was stirred under nitrogen at room temperature for 90 min. After centrifugation the resulting solution was neutralized by addition of three equivalents of NaOH dissolved in methanol. The ligand was precipitated as white crystals after addition of anhydrous ethanol. The solid, separated by centrifugation, was washed three times with ethanol and isolated after drying to constant weight (yield 55%). ~H NMR (020, pH 7, T--25°C, reference t-BuOH at 6 1.29 ppm), 6(ppm):3.21 (t, 4), 3.48 (s, 4), 3.50 (t, 4), 3.60 (s, 4), 3.91 (s, 2), 6.97 (dd, 2), 7.11 (d, 2), 7.73 (d, 2). t3C NMR (D20, pH 7, T=25°C, reference t-BuOH at 6 31.3 ppm), t5 (ppm):51.9, 54.5, 56.7, 60.2, 61.2, 109.4, 113.2, 116.2, 132.7, 143.0, 162.3, 172.2, 174.0, 176.6, 180.5.
Na2[LnDTPA(PAS)2] Gd m and Eu in complexes were prepared by mixing equimolar amounts from stock solutions of lanthanide chlorides and the ligand and by adjusting the pH to 8 with NaOH.
NMR spectral measurements The spin-lattice relaxation rates (1/T1) of the water protons were measured at 20 MHz and 25°C on a Stelar Spinmaster spectrometer by using the inversion-recovery pulse sequence. The 1/TI NMRD profiles of water protons were measured at 25°C over a continuous range of magnetic fields from 2.5 x 10-4 to 1.4 T (0.01-50 MHz proton Larmor frequencies) using the Koenig-Brown relaxometer installed at the Department of Chemistry of the University of Florence.
RESULTS
AND
DISCUSSION
Synthesis and characterization of [DTPA(PAS)2] ligand The synthesis of [DTPA(PAS)2] ligand was carried out by reacting D T P A dianhydride with PAS sodium salt in anhydrous DMSO in the stoichiometric ratio of 1 : 2 (Scheme 1). From the reaction mixture, [DTPA(PAS)2] was precipitated as the pentasodium salt and characterized by lH and 13C N M R spectroscopy (Figs 1 and 2). The assignment of the 1H and 13C resonances (Fig. 3) were made by comparison with the NMR spectra of the parent DTPA and PAS, as well as on the basis of homo- and heteronuclear 2D-COSY experiments. Attempts to synthesize the mono-substituted product were made by using a 1:1 stoichiometric ratio between the reactants, but although we had evidence from the NMR spectra of the formation of the desired product, we were unable to separate it from the bis-substituted product always present with some amounts of unreacted DTPA and PAS. [DTPA(PAS)2] complexes The G d 3+ complex has been synthesized by adding aliquots of a concentrated solution of gadolinium ions to an aqueous solution of [DTPA(PAS)2] sodium salt.
0
o.
o~ 0
OH
OH
.o
N~/~./~__/o.
.O/~o I ~ Od complex V I I = Bu complex
Scheme 1.
OH
0
Gdm-Fem heterobimetallic complexes
1317
E D
''''l''''l''' 7.1 7.0 PPM
,
M
C
JI I
'
8
~
'
'
'
lAB
Ill I
'
7
'
'
'
I
6
'
'
'
'
I
'
5
'
'
'
I
4
'
'
'
'
I
3
PPM Fig. 1. 1H NMR spectrum (400 MHz) of the ligand [DTPA(PAS)]2 (0.1 M) in D,O at pH 7 and 25°C. The water signal at 6 4.8 ppm has been suppressed by presaturation. The resonances have been labelled according to Fig. 3.
AB E D RM
IF
L
Jli 1
~ A .......... J ..... T
'
I
'
180
'
'
I
160
'
'
'
I
140
'
'
'
I
'
120 PPM
'
'
I
100
'
'
'
I
80
'
'
*
I
'
60
'
Fig. 2. 13C NMR spectrum (100.5 MHz) of the ligand [DTPA (PAS)2] (0.1 M) in D20 at pH7 and 25°C. The resonances have been labelled according to Fig. 3.
0
H
O
OH
~ p
OH
O
M L
HNL~
OH
Fig. 3. [DTPA(PAS)2]: labelled structure.
P
1318
S. A1ME et al. 5
4-
J_0
2~
0
I 0.2
I 0.4
I 0.6
0.8
1.0
[Gd 3+] (raM) Fig. 4. Plot of the water proton longitudinal relaxation rate (R~o~) of a I m M solution of [DTPA(PAS)2] as a function of Gd 3+ concentration, measured at 20 MHz, 25°C and p H 6.8.
The measurement of the solvent proton relaxation times following stepwise addition of Gd 3+ ions (Fig. 4) allows the determination of the relaxivity Rip (in m M - l s -l) of the Gd 3÷ complex according to the equation: 1 globs = T]obs= R~p[Gd-DTPA(PAS)2] + C where C is simply the water proton relaxation rate in the absence of the paramagnetic metal ion. The [Gd-DTPA(PAS)2] 2- complex has a relaxivity of 4.6 mM -1 s -~ at 25°C and 20MHz, which compares well with the value of 4.8 found for the parent [Gd-DTPA] 2- chelate [1]. This finding supports the view that [DTPA(PAS)2] 2- also acts as an octadentate ligand towards Gd 3+ through its three nitrogens, two carboxylate oxygens and two carboxoamido groups. The ninth coordination position is then occupied by a water molecule, which is involved in a fast exchange process with the bulk water and is responsible for the inner sphere contribution to the observed paramagnetic relaxation of solvent water protons. Further support to the proposed structure arises from the measurement of the 1/Tt NMRD (Nuclear Magnetic Relaxation Dispersion) profile [6] (Fig. 5) of the complex over a wide range of frequencies. This has been done on a field-cycling spectrometer, which allows quick and very accurate determination of water proton longitudinal relaxation times in the 0.01-50 MHz frequency range. Since the theory of paramagnetic relaxation of solutions containing small metal complexes has been described in detail in several papers [7], it will not be repeated here. We simply deal with the results of the fitting procedure of the experimental data to the I0 'm
m~B
I~B--t--B~i~im
m m"'m • •
u a~ 0 0
l 0.I
I 1.0
I 10 Proton Larmor Frequency (MHz)
l
100
Fig. 5. 1/T~ N M R D profile of a 1 m M a q u e o u s solution of [ G d - D T P A ( P A S ) 2 ] 2- m e a s u r e d at 25°C and p H 6.8. The lower curve represents the outer sphere contribution to the relaxivity.
1319
G d m - F e m heterobimetallic complexes Table 1. N M R parameters for [Gd--DTPA] 2- obtained from the fitting of the N M R D profile with relaxation theory rso (ps)
rv (ps)
ra (ps)
r (.~)
81 57
18 21
74 90
3.07 3.14
Gd-DTPA Gd-DTPA(PAS)2
established theory of both inner and outer sphere relaxation by using r, ra, rSO and rv as adjustable parameters and assuming one coordinated water molecule with arm (the residence lifetime at the paramagnetic centre) of 5 ns and the usual values of 3.6 A for the distance of closest approach of the water molecules in the outer coordination sphere of the complex, the relative diffusion coefficient being 2.6 x 10 -5 cm -2. The solid lines through the experimental data points in Fig. 4 are calculated from the parameters reported in Table 1. In this table the corresponding parameters obtained from the fitting procedure of G d - D T P A are also reported. On going from G d - D T P A to A only minor changes are detected, namely a lengthening of the molecular reorientation time ra as a consequence of the increased molecular size and a slight decrease of rso, which probably reflects a decreased symmetry in the coordination cage around the Gd 3+ ion upon substitution of two carboxylate groups with two carboxoamido functionalities. As was anticipated in the Introduction, the salicylic functionality was chosen in order to have a suitable group for the linkage of the Fe IH ions on the surface of this Gd m complex. It is in fact well established that salicylate ligands form three types of chelate with Fe 3+ ions, namely (PAS)Fe(H20)4 (B; violet), (PAS)EFe(H20)2 (C; red) and (PAS)3Fe (D; yellow) [8] (Fig. 6). Whereas species B is present only at very acidic pHs, complex C is the main species present at pH 5 and complex D is dominant at pH 9. We thus expect that complex A will also form, with Fe 3÷ ions, heterobimetallic complexes of the same stoichiometry at the same pH value. The formation of these heterobimetallic complexes may be conveniently followed by measuring the solvent proton longitudinal relaxation rates. Firstly, we measured the relaxitivites of complexes C and D, which were found to be 4.5 and 0.21 mM -1 s -l respectively. The very low R~p value found for the latter species is consistent with the
/\
H20 I~Ff,~O H20
--R
OH,
R
OH2
/ NH
O
o•NH O
\
O
R
~
O
II1 R = H I V R = - - C - - NH ~ ( G d ~ D T P A ~ P A S )
II O
V R=H Vl R = ~
C~
NH~(Od ~DTPA~PA$)
U O
Fig. 6. T h e three types of chelated structure formed by Fe 3÷ and salicylate moieties in a q u e o u s solution.
1320
S. AIIdE et al. 9
7
5 4 0
I
I
0.2
0.4
[Pe3+l (raM) Fig. 7. Plot of the water proton longitudinal relaxation rate (Rlo~) of a 1 mM solution of [Gd-DTPA(PAS)2] 2- as a function of Fe 3÷ concentration, measured at 20 MHz, 25°C and pH 5.
absence of inner sphere coordinated water, i.e. the observed relaxivity has to be ascribed only to the paramagnetic contribution of the water molecules diffusing in the proximity of the complex (outer sphere contribution). This is slightly lower than the value previously reported for the [Fem-DTPA] 2- complex [1], probably as a consequence of an increased average distance of minimum approach between the diffusing water molecules and the paramagnetic centre and of a difference in the electronic relaxation times for the two species. On the other hand, the good relaxivity of C is mainly determined by the two water molecules directly coordinated to the paramagnetic centre. This relaxivity value compares well with the value of 10.7 mM -1 s -~ reported, at the same magnetic field strength, for the hexa-aquo ion (at pH 2) [9], which corresponds to a contribution slightly lower than 2 mM -~ s -~ for each coordinated water molecule. The formation of the heterobimetallic (Gd-Fe) complex E was followed by measuring the water proton relaxation rate after successive addition of aliquots of Fe 3÷ ions to a 1 mM solution of 1 (Fig. 7) at pH 5. The observed R~ values are given by the sum of four terms: Rxo~ -- RIGd(A) + R1Gd(E) + RIFe(E) + R l d
where R~a and RIGd(A) a r e known (0.38 s -[ and 4.6 mM -~ s -~ respectively). Since R1Fe is independent of molecular size (as the short electronic relaxation time of Fe 3+ ions is the correlation time which modulates the magnetic interaction), we may assume that its value corresponds to that measured for C. The slight increase of R1Gd(E) with respect to
5
4~ A
.3~2 1-
0
I
0.2
I
0.4
I
0,6
I
0.8
I
1.0
I
1.2
I
1.4
I
1.6
1.8
lFe3÷l (raM) Fig. 8. Plot of the water proton longitudinal relaxation rate (Rto~) of a 1 mM solution of [DTPA(PAS)2] as a function of Fe 3+ concentration, measured at 20 MHz, 25°C and pH 5.
Gdm-Fem heterobimetalliccomplexes
1321
4.0 3.5
I
a.e2.5 I iw v m
g
2.01.5 1.00.5-
I 0.2
I 0.4
[Fe s+] (raM)
Fig. 9. Plot of the water proton longitudinalrelaxation rate (Riot) of a 1mM solution of [Eu-DTPA(PAS)2]2- as a functionof Fea+ concentration,measuredat 20 MHz, 25°Cand pH 5.
R1GdtA)(from 4.6 to 5.7 mM -~ s -l) may then be ascribed to the increased molecular size of the complex. Furthermore, this observation seems to indicate that the proximity of the two paramagnetic centres with quite different electronic relaxation times does not alter their individual relaxation properties, which remain unchanged upon the formation of a heterobimetallic complex, thus suggesting the absence of any detectable mutual interactions. The above discussions implicitly excluded the possibility of iron replacing the Gd 3+ ion in the DTPA-like coordination cage of the ligand. This appears to be a good assumption if we consider the high affinity of the salicylate function for Fe 3+ ions, but it needs to be proved. First, we investigated the coordination properties of the ligand towards Fe 3+ ions by measuring the water proton relaxation rate of a I mM aqueous solution of the ligand at pH 5 as a function of increasing concentrations of the metal ion. The results, shown in Fig. 8, provide clear evidence of the presence of two distinct coordination steps. Up to 1 mM iron concentration the metal ion enters the DTPA-like cavity, giving a complex which does not contain any water molecules in the inner coordination sphere, as indicated by the relaxivity value of 1.03 mM -~ s -1, typical for outer sphere coordination only. At higher iron concentration, a marked increase in the Rl values is observed, as expected for chelation by the salicylic moieties, to give the complex E. Then, in order to check whether iron replacement takes place or not, we prepared the [Eu-DTPA(PAS)2] 2- complex (G) which, due to the well known similarity of chemical properties among the lanthanide series, is expected to have the same coordination geometry and stability constant as the analogous Gd a+ complex I [10]. F u r t h e r m o r e , Eu m has a very short electronic relaxation time, of the order of 10-13 s, which results in very low relaxivity values of the aqueous solutions of its complexes [11]. Following the addition of Fe 3+ ions to a solution of compound G at pH 5, there are two possibilities: (a) iron replacement of the Eu 3+ ion in the internal cavity in the complex or (b) iron coordination to the salicylate moieties. In the first case we do not expect any increase in the water proton relaxation rate, while in the second case we should obtain a linear increase in R~ as function of iron concentration. Experimentally (Fig. 9) we observe the latter behaviour, which confirms the absence of replacement of the Gd a+ inside the DTPA-like coordination cage of the ligand and substantiates the above conclusions. In summary, we have shown that heterobimetallic Gd-Fe complexes may be easily synthesized through the introduction of suitable functionalities on the surface of the DTPA-like cage. This approach may be extended to other metal ions to provide a variety of building blocks able to modulate size, shape and relaxation properties of novel CAs for MRI.
1322
S. AIME et al. REFERENCES
[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]
R. B. Lauffer, Chem. Rev. 87, 901 (1987). R. B. Lauffer and T. J. Brady, Magn. Reson. Med. 3, 11 (1985). S. Aime, M. Botta, M. Panero, F. Uggeri and M. Grandi, Magn. Reson. Chem. 29, 923 (1991). S. Aime, L. Barbero and M. Botta, Magn. Reson. lmag. 9, 843 (1991). J.-H. Hwang, A. G. Webb, R. L. Belford and R. B. Clarkson, Abstract of the lOth SMRM, p. 509 (1991). S. H. Koenig and R. D. Brown, III, NMR Spectroscopy of Cells and Organisms, Vol. II (Edited by R. K. Gupta), p. 75. CRC Press, Boca Raton (1987). cf. S. H. Koenig and R. D. Brown III, Prog. NMR Spectrosc. 22, 487 (1990) for a review and extensive references. A. Agren, Acta Chem. Scand. 8, 1059 (1954). S. Aime (Unpublished data). G. R. Choppin, in Lanthanide Probes in Life, Chemical and Earth Sciences (Edited by J.-C. G. Biinzli and G. R. Choppin), Chap. 1. Elsevier, Amsterdam (1990). I. Bertini and C. Luchinat, NMR of Paramagnetic Molecules in Biological Systems. Benjamin/Cummings Publishing Company, Menlo Park (1986).
0584-8539/93$6.00+0.00 ~) 1993PergamonPressLtd
Paramagnetic Gdm-Fe m heterobimetallic complexes of DTPA-bis-salicylamide S. AIME,* M . BOTI'A, M . FASANO a n d E . TERRENO Dipartimento di Chimica Inorganica, Chimica Fisica e Chimica tel Materiali, Universiti~ di Torino, via P. Giuria 7, 10125 Torino, Italy
(Received 28 April 1992; accepted 8 June 1992) Almtract--The reaction between DTPA (diethylenetriaminepenta-acetic acid)-anhydride and p-aminosalicylic acid (PAS) affords a novel ligand, [DTPA(PAS)2], able to form stable beterobimetallic complexes with Gd 3+ and Fe 3+ ions. The lanthanide ion occupies an internal coordination cage formed by three nitrogen atoms, two carboxylate and two carboxoamido groups of the ligand, whereas the outer salicylic moieties form stable chelate rings with Fem ions. The stoichiometry of the resulting heterobimetallic complexes, established by measurements of water proton relaxation enhancement, is [(H20)-Gd--DTPA(PAS)2]2--Fe(H20)2 or [(H20)-Gd-DTPA(PAS)2b-Fe depending on the pH of the aqueous solution. The individual contributions t o the observed relaxation enhancement from Gd 3+ and Fe 3+ paramagnetic ions have been clearly distinguished and analysed.
INTRODUCTION
THE RAPID development of Magnetic Resonance Imaging (MRI) techniques has aroused new interest in paramagnetic gadolinium(III) complexes for their potential utility as contrast agents (CAs). In particular, polyaminocarboxylate chelates have undergone intense scrutiny for their favourable relaxation properties associated with high thermodynamic stabilities, which are expected to limit the toxicological problems arising from the interaction of both the lanthanide ions and the free ligands with biological substrates [1]. Current efforts are mainly devoted to the search for a higher specificity towards organs and tissues as well as for an increased relaxivity (i.e. the water proton relaxation enhancement per mM concentration of paramagnetic ion) of these CAs in order to achieve a maximum contrast/dose ratio. Actually, it has been shown that, at the typical magnetic fields employed by commercial imaging instruments, a marked increase of relaxivity of gadolinium complexes may be pursued through a lengthening of their molecular reorientation times, rR. This was accomplished either through the formation of protein-Gd-DTPA complex conjugates [2] or by exploiting the non-covalent interactions taking place between suitable functionalized complexes and slowly tumbling systems such as fl-cyclodextrin [3], micelles [4] or plasma proteins [5]. In this work we explore an additional way for enhancing the solvent proton relaxation rate upon the formation of heterobimetallic Gd-Fe paramagnetic complexes. Such species have been obtained through the synthesis of the novel ligand [DTPA(PAS)2] containing a DTPA-Iike coordination cage able to bind the Gd 3÷ ions. EXPERIMENTAL All reagents were purchased commercially and were used without further purification. ~H and
~ac N M R spectra were recorded on a Jeol EX-400 spectrometer. Nas[DTPA(PAS)2] A mixture of p-aminosalicylic acid (PAS) sodium salt dihydrate (475 rag, 2.25 mmol) and diethylenetriaminepenta-acetic acid ( D T P A ) dianhydride (357 mg, 1 mmol) in anhydrous D M S O * Author to whom correspondence should be addressed. 1315
1316
S. AIMEet
al.
(5 ml) was stirred under nitrogen at room temperature for 90 min. After centrifugation the resulting solution was neutralized by addition of three equivalents of NaOH dissolved in methanol. The ligand was precipitated as white crystals after addition of anhydrous ethanol. The solid, separated by centrifugation, was washed three times with ethanol and isolated after drying to constant weight (yield 55%). ~H NMR (020, pH 7, T--25°C, reference t-BuOH at 6 1.29 ppm), 6(ppm):3.21 (t, 4), 3.48 (s, 4), 3.50 (t, 4), 3.60 (s, 4), 3.91 (s, 2), 6.97 (dd, 2), 7.11 (d, 2), 7.73 (d, 2). t3C NMR (D20, pH 7, T=25°C, reference t-BuOH at 6 31.3 ppm), t5 (ppm):51.9, 54.5, 56.7, 60.2, 61.2, 109.4, 113.2, 116.2, 132.7, 143.0, 162.3, 172.2, 174.0, 176.6, 180.5.
Na2[LnDTPA(PAS)2] Gd m and Eu in complexes were prepared by mixing equimolar amounts from stock solutions of lanthanide chlorides and the ligand and by adjusting the pH to 8 with NaOH.
NMR spectral measurements The spin-lattice relaxation rates (1/T1) of the water protons were measured at 20 MHz and 25°C on a Stelar Spinmaster spectrometer by using the inversion-recovery pulse sequence. The 1/TI NMRD profiles of water protons were measured at 25°C over a continuous range of magnetic fields from 2.5 x 10-4 to 1.4 T (0.01-50 MHz proton Larmor frequencies) using the Koenig-Brown relaxometer installed at the Department of Chemistry of the University of Florence.
RESULTS
AND
DISCUSSION
Synthesis and characterization of [DTPA(PAS)2] ligand The synthesis of [DTPA(PAS)2] ligand was carried out by reacting D T P A dianhydride with PAS sodium salt in anhydrous DMSO in the stoichiometric ratio of 1 : 2 (Scheme 1). From the reaction mixture, [DTPA(PAS)2] was precipitated as the pentasodium salt and characterized by lH and 13C N M R spectroscopy (Figs 1 and 2). The assignment of the 1H and 13C resonances (Fig. 3) were made by comparison with the NMR spectra of the parent DTPA and PAS, as well as on the basis of homo- and heteronuclear 2D-COSY experiments. Attempts to synthesize the mono-substituted product were made by using a 1:1 stoichiometric ratio between the reactants, but although we had evidence from the NMR spectra of the formation of the desired product, we were unable to separate it from the bis-substituted product always present with some amounts of unreacted DTPA and PAS. [DTPA(PAS)2] complexes The G d 3+ complex has been synthesized by adding aliquots of a concentrated solution of gadolinium ions to an aqueous solution of [DTPA(PAS)2] sodium salt.
0
o.
o~ 0
OH
OH
.o
N~/~./~__/o.
.O/~o I ~ Od complex V I I = Bu complex
Scheme 1.
OH
0
Gdm-Fem heterobimetallic complexes
1317
E D
''''l''''l''' 7.1 7.0 PPM
,
M
C
JI I
'
8
~
'
'
'
lAB
Ill I
'
7
'
'
'
I
6
'
'
'
'
I
'
5
'
'
'
I
4
'
'
'
'
I
3
PPM Fig. 1. 1H NMR spectrum (400 MHz) of the ligand [DTPA(PAS)]2 (0.1 M) in D,O at pH 7 and 25°C. The water signal at 6 4.8 ppm has been suppressed by presaturation. The resonances have been labelled according to Fig. 3.
AB E D RM
IF
L
Jli 1
~ A .......... J ..... T
'
I
'
180
'
'
I
160
'
'
'
I
140
'
'
'
I
'
120 PPM
'
'
I
100
'
'
'
I
80
'
'
*
I
'
60
'
Fig. 2. 13C NMR spectrum (100.5 MHz) of the ligand [DTPA (PAS)2] (0.1 M) in D20 at pH7 and 25°C. The resonances have been labelled according to Fig. 3.
0
H
O
OH
~ p
OH
O
M L
HNL~
OH
Fig. 3. [DTPA(PAS)2]: labelled structure.
P
1318
S. A1ME et al. 5
4-
J_0
2~
0
I 0.2
I 0.4
I 0.6
0.8
1.0
[Gd 3+] (raM) Fig. 4. Plot of the water proton longitudinal relaxation rate (R~o~) of a I m M solution of [DTPA(PAS)2] as a function of Gd 3+ concentration, measured at 20 MHz, 25°C and p H 6.8.
The measurement of the solvent proton relaxation times following stepwise addition of Gd 3+ ions (Fig. 4) allows the determination of the relaxivity Rip (in m M - l s -l) of the Gd 3÷ complex according to the equation: 1 globs = T]obs= R~p[Gd-DTPA(PAS)2] + C where C is simply the water proton relaxation rate in the absence of the paramagnetic metal ion. The [Gd-DTPA(PAS)2] 2- complex has a relaxivity of 4.6 mM -1 s -~ at 25°C and 20MHz, which compares well with the value of 4.8 found for the parent [Gd-DTPA] 2- chelate [1]. This finding supports the view that [DTPA(PAS)2] 2- also acts as an octadentate ligand towards Gd 3+ through its three nitrogens, two carboxylate oxygens and two carboxoamido groups. The ninth coordination position is then occupied by a water molecule, which is involved in a fast exchange process with the bulk water and is responsible for the inner sphere contribution to the observed paramagnetic relaxation of solvent water protons. Further support to the proposed structure arises from the measurement of the 1/Tt NMRD (Nuclear Magnetic Relaxation Dispersion) profile [6] (Fig. 5) of the complex over a wide range of frequencies. This has been done on a field-cycling spectrometer, which allows quick and very accurate determination of water proton longitudinal relaxation times in the 0.01-50 MHz frequency range. Since the theory of paramagnetic relaxation of solutions containing small metal complexes has been described in detail in several papers [7], it will not be repeated here. We simply deal with the results of the fitting procedure of the experimental data to the I0 'm
m~B
I~B--t--B~i~im
m m"'m • •
u a~ 0 0
l 0.I
I 1.0
I 10 Proton Larmor Frequency (MHz)
l
100
Fig. 5. 1/T~ N M R D profile of a 1 m M a q u e o u s solution of [ G d - D T P A ( P A S ) 2 ] 2- m e a s u r e d at 25°C and p H 6.8. The lower curve represents the outer sphere contribution to the relaxivity.
1319
G d m - F e m heterobimetallic complexes Table 1. N M R parameters for [Gd--DTPA] 2- obtained from the fitting of the N M R D profile with relaxation theory rso (ps)
rv (ps)
ra (ps)
r (.~)
81 57
18 21
74 90
3.07 3.14
Gd-DTPA Gd-DTPA(PAS)2
established theory of both inner and outer sphere relaxation by using r, ra, rSO and rv as adjustable parameters and assuming one coordinated water molecule with arm (the residence lifetime at the paramagnetic centre) of 5 ns and the usual values of 3.6 A for the distance of closest approach of the water molecules in the outer coordination sphere of the complex, the relative diffusion coefficient being 2.6 x 10 -5 cm -2. The solid lines through the experimental data points in Fig. 4 are calculated from the parameters reported in Table 1. In this table the corresponding parameters obtained from the fitting procedure of G d - D T P A are also reported. On going from G d - D T P A to A only minor changes are detected, namely a lengthening of the molecular reorientation time ra as a consequence of the increased molecular size and a slight decrease of rso, which probably reflects a decreased symmetry in the coordination cage around the Gd 3+ ion upon substitution of two carboxylate groups with two carboxoamido functionalities. As was anticipated in the Introduction, the salicylic functionality was chosen in order to have a suitable group for the linkage of the Fe IH ions on the surface of this Gd m complex. It is in fact well established that salicylate ligands form three types of chelate with Fe 3+ ions, namely (PAS)Fe(H20)4 (B; violet), (PAS)EFe(H20)2 (C; red) and (PAS)3Fe (D; yellow) [8] (Fig. 6). Whereas species B is present only at very acidic pHs, complex C is the main species present at pH 5 and complex D is dominant at pH 9. We thus expect that complex A will also form, with Fe 3÷ ions, heterobimetallic complexes of the same stoichiometry at the same pH value. The formation of these heterobimetallic complexes may be conveniently followed by measuring the solvent proton longitudinal relaxation rates. Firstly, we measured the relaxitivites of complexes C and D, which were found to be 4.5 and 0.21 mM -1 s -l respectively. The very low R~p value found for the latter species is consistent with the
/\
H20 I~Ff,~O H20
--R
OH,
R
OH2
/ NH
O
o•NH O
\
O
R
~
O
II1 R = H I V R = - - C - - NH ~ ( G d ~ D T P A ~ P A S )
II O
V R=H Vl R = ~
C~
NH~(Od ~DTPA~PA$)
U O
Fig. 6. T h e three types of chelated structure formed by Fe 3÷ and salicylate moieties in a q u e o u s solution.
1320
S. AIIdE et al. 9
7
5 4 0
I
I
0.2
0.4
[Pe3+l (raM) Fig. 7. Plot of the water proton longitudinal relaxation rate (Rlo~) of a 1 mM solution of [Gd-DTPA(PAS)2] 2- as a function of Fe 3÷ concentration, measured at 20 MHz, 25°C and pH 5.
absence of inner sphere coordinated water, i.e. the observed relaxivity has to be ascribed only to the paramagnetic contribution of the water molecules diffusing in the proximity of the complex (outer sphere contribution). This is slightly lower than the value previously reported for the [Fem-DTPA] 2- complex [1], probably as a consequence of an increased average distance of minimum approach between the diffusing water molecules and the paramagnetic centre and of a difference in the electronic relaxation times for the two species. On the other hand, the good relaxivity of C is mainly determined by the two water molecules directly coordinated to the paramagnetic centre. This relaxivity value compares well with the value of 10.7 mM -1 s -~ reported, at the same magnetic field strength, for the hexa-aquo ion (at pH 2) [9], which corresponds to a contribution slightly lower than 2 mM -~ s -~ for each coordinated water molecule. The formation of the heterobimetallic (Gd-Fe) complex E was followed by measuring the water proton relaxation rate after successive addition of aliquots of Fe 3÷ ions to a 1 mM solution of 1 (Fig. 7) at pH 5. The observed R~ values are given by the sum of four terms: Rxo~ -- RIGd(A) + R1Gd(E) + RIFe(E) + R l d
where R~a and RIGd(A) a r e known (0.38 s -[ and 4.6 mM -~ s -~ respectively). Since R1Fe is independent of molecular size (as the short electronic relaxation time of Fe 3+ ions is the correlation time which modulates the magnetic interaction), we may assume that its value corresponds to that measured for C. The slight increase of R1Gd(E) with respect to
5
4~ A
.3~2 1-
0
I
0.2
I
0.4
I
0,6
I
0.8
I
1.0
I
1.2
I
1.4
I
1.6
1.8
lFe3÷l (raM) Fig. 8. Plot of the water proton longitudinal relaxation rate (Rto~) of a 1 mM solution of [DTPA(PAS)2] as a function of Fe 3+ concentration, measured at 20 MHz, 25°C and pH 5.
Gdm-Fem heterobimetalliccomplexes
1321
4.0 3.5
I
a.e2.5 I iw v m
g
2.01.5 1.00.5-
I 0.2
I 0.4
[Fe s+] (raM)
Fig. 9. Plot of the water proton longitudinalrelaxation rate (Riot) of a 1mM solution of [Eu-DTPA(PAS)2]2- as a functionof Fea+ concentration,measuredat 20 MHz, 25°Cand pH 5.
R1GdtA)(from 4.6 to 5.7 mM -~ s -l) may then be ascribed to the increased molecular size of the complex. Furthermore, this observation seems to indicate that the proximity of the two paramagnetic centres with quite different electronic relaxation times does not alter their individual relaxation properties, which remain unchanged upon the formation of a heterobimetallic complex, thus suggesting the absence of any detectable mutual interactions. The above discussions implicitly excluded the possibility of iron replacing the Gd 3+ ion in the DTPA-like coordination cage of the ligand. This appears to be a good assumption if we consider the high affinity of the salicylate function for Fe 3+ ions, but it needs to be proved. First, we investigated the coordination properties of the ligand towards Fe 3+ ions by measuring the water proton relaxation rate of a I mM aqueous solution of the ligand at pH 5 as a function of increasing concentrations of the metal ion. The results, shown in Fig. 8, provide clear evidence of the presence of two distinct coordination steps. Up to 1 mM iron concentration the metal ion enters the DTPA-like cavity, giving a complex which does not contain any water molecules in the inner coordination sphere, as indicated by the relaxivity value of 1.03 mM -~ s -1, typical for outer sphere coordination only. At higher iron concentration, a marked increase in the Rl values is observed, as expected for chelation by the salicylic moieties, to give the complex E. Then, in order to check whether iron replacement takes place or not, we prepared the [Eu-DTPA(PAS)2] 2- complex (G) which, due to the well known similarity of chemical properties among the lanthanide series, is expected to have the same coordination geometry and stability constant as the analogous Gd a+ complex I [10]. F u r t h e r m o r e , Eu m has a very short electronic relaxation time, of the order of 10-13 s, which results in very low relaxivity values of the aqueous solutions of its complexes [11]. Following the addition of Fe 3+ ions to a solution of compound G at pH 5, there are two possibilities: (a) iron replacement of the Eu 3+ ion in the internal cavity in the complex or (b) iron coordination to the salicylate moieties. In the first case we do not expect any increase in the water proton relaxation rate, while in the second case we should obtain a linear increase in R~ as function of iron concentration. Experimentally (Fig. 9) we observe the latter behaviour, which confirms the absence of replacement of the Gd a+ inside the DTPA-like coordination cage of the ligand and substantiates the above conclusions. In summary, we have shown that heterobimetallic Gd-Fe complexes may be easily synthesized through the introduction of suitable functionalities on the surface of the DTPA-like cage. This approach may be extended to other metal ions to provide a variety of building blocks able to modulate size, shape and relaxation properties of novel CAs for MRI.
1322
S. AIME et al. REFERENCES
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R. B. Lauffer, Chem. Rev. 87, 901 (1987). R. B. Lauffer and T. J. Brady, Magn. Reson. Med. 3, 11 (1985). S. Aime, M. Botta, M. Panero, F. Uggeri and M. Grandi, Magn. Reson. Chem. 29, 923 (1991). S. Aime, L. Barbero and M. Botta, Magn. Reson. lmag. 9, 843 (1991). J.-H. Hwang, A. G. Webb, R. L. Belford and R. B. Clarkson, Abstract of the lOth SMRM, p. 509 (1991). S. H. Koenig and R. D. Brown, III, NMR Spectroscopy of Cells and Organisms, Vol. II (Edited by R. K. Gupta), p. 75. CRC Press, Boca Raton (1987). cf. S. H. Koenig and R. D. Brown III, Prog. NMR Spectrosc. 22, 487 (1990) for a review and extensive references. A. Agren, Acta Chem. Scand. 8, 1059 (1954). S. Aime (Unpublished data). G. R. Choppin, in Lanthanide Probes in Life, Chemical and Earth Sciences (Edited by J.-C. G. Biinzli and G. R. Choppin), Chap. 1. Elsevier, Amsterdam (1990). I. Bertini and C. Luchinat, NMR of Paramagnetic Molecules in Biological Systems. Benjamin/Cummings Publishing Company, Menlo Park (1986).