Inorganica Chimica Acta 288 (1999) 244–248

Note

Interaction of ATP with a Gd3+ complex employed asparamagnetic contrast agent in NMR imaging

Antonio Bianchi a,*, Luisella Calabi b,1, Massimo Foresti b, Pietro Losi b, Lino Paleari b,Agustin Rodriguez c, Barbara Valtancoli a

a Department of Chemistry, Uni6ersity of Florence, Via Maragliano 75/77, 50144 Florence, Italyb Bracco SpA, Milano Research Center, Via E. Folli 50, 20134 Milan, Italy

c Department of Inorganic Chemistry, Uni6ersity of La Laguna, La Laguna, Canary Islands, Spain

Received 19 November 1997; accepted 22 February 1999

Abstract

The interaction of adenosine 5%-triphosphate (ATP) and tripolyphosphate (TPP) with the complex GdBOPTA2−, a contrastagent for NMR imaging (international nonproprietary name: gadobenate), has been studied by means of potentiometric (pHmetric) titrations in 0.15 mol dm−3 NaCl at 298.1 K. Both ATP and TPP interact with GdBOPTA2− forming mono- topenta-protonated mixed-ligand complexes. The equilibrium constants for the formation of such mixed-ligand complexes, as wellas for the formation Gd3+ complexes with ATP and TPP have been determined. The results indicate that around physiologicalpH (7.4), under the experimental conditions employed ([Gd3+]= [BOPTA]= [ATP]=1×10−3 mol dm−3, 298.1 K), only a verysmall amount of GdBOPTA2− (B5%) is bound to the nucleotide. Relaxation experiments demonstrated that the interaction ofATP with GdBOPTA2− in vitro does not compromise the effectiveness of this complex as paramagnetic contrast agent for NMRimaging, nor cause degradation of the complex for a long time. © 1999 Elsevier Science S.A. All rights reserved.

Keywords: Nuclear magnetic resonance imaging; Gadolinium complexes; Nucleotide complexes

1. Introduction

In the last decade increasing interest has beendevoted to Gd3+ complexes of polyaminopolycarbo-xylic ligands, since they have proved useful as contrastagents for magnetic resonance imaging in clinical diag-nostics [1–3]. The 1H NMR image of a tissue is largelydetermined by the NMR signal of water protons and itsintensity is dependent on nuclear relaxation times.Paramagnetic contrast agents, such as Gd3+ com-plexes, enhance the 1H NMR image modifying therelaxation times of the neighboring nuclei. A principalrelaxation mechanism operating with this type of com-plex takes place via a water molecule directly coordi-

nated to the metal ion and exchanging with the bulksolvent [1]. In order to facilitate this mechanism, Gd3+

complexes are selected in which at least one coordina-tion site on the metal ion is not occupied by a liganddonor atom. This characteristic allows the binding ofwater molecules to the metal center but, at the sametime, exposes the complex to undesired coordination ofexogen ligands from the medium. These secondary in-teractions can modify at large extent the relaxationproperties of the complex.

The nucleotide adenosine 5%-triphosphate (ATP) con-tains a triphosphate chain which is a good binding sitefor hard metal cation such as Gd3+. Indeed EDTAcomplexes of trivalent lanthanide cations interact withtripolyphosphate P3O10

5− (TPP) and ATP giving riseto stable complexes in which both EDTA and thesecondary ligand seem to be directly bound to the metalcenters [4]. Recently it has been reported that similar

* Corresponding author. Tel.: +39-055-354 841; fax: +39-055-354 842.

E-mail address: bianchi@chimat.chim1.unifi.it (A. Bianchi)1 Also corresponding author.

0020-1693/99/$ - see front matter © 1999 Elsevier Science S.A. All rights reserved.

PII: S 0 0 2 0 -1693 (99 )00106 -1

A. Bianchi et al. / Inorganica Chimica Acta 288 (1999) 244–248 245

ATP complexes are also formed by Gd3+ complexeswith DTPA (diethylentrinitrilopentacetic acid) andDTPA derivatives [5].

Considering the large presence of ATP in tissues, wehave undertaken a study devoted to evidence to whatextent this nucleotide interacts with the complexGdBOPTA2−, a contrast agent for NMR imaging (in-ternational nonproprietary name: gadobenate) [6], andthe modifications produced by the interaction in therelaxation mechanism of this complex. The presentstudy is of particular interest considering that it hasbeen suggested recently [7] that GdBOPTA2− pene-trates into hepatocytes where ATP concentration rangeis several mmol dm−3. In order to better understandthe importance of the triphosphoric chain of ATP inthe binding to the metal center we have also anal-yzed the coordination properties of TPP towardsGdBOPTA2−.

2. Experimental

The interaction of ATP4− and P3O105− with the

Gd3+ complexes of BOPTA was studied by means ofpotentiometric (pH metric) titration in 0.15 mol dm−3

NaCl solutions. Although the ligands form weak Na+

complexes in solution, we chose this medium for study-ing the present complexation equilibria in order toreproduce the physiological conditions. As a conse-quence of this choice the equilibrium constants reportedin this paper are conditional stability constants andtheir significance is limited to solutions containingabout 0.15 mol dm−3 Na+.

2.1. Materials

ATP disodium salt (99%) and TPP pentasodium salt(99%) were obtained from Aldrich and Fluka, respec-

tively, and used without further purification. BOPTA(4-carboxy-5,8,11-tris(carboxymethyl)-1-phenyl-2-oxa-5,8,11-triazatridecan-13-oic acid) was furnished byBracco SpA (synthesis of this compound is described inRef. [3]). Gd3+ stock solutions were prepared in doublydistilled water and standardized by complexometrictitrations using EDTA.

2.2. Potentiometric measurements

All the pH metric measurements (pH= − log [H+])were carried out in degassed 0.15 mol dm−3 NaClsolutions, at 298.190.1 K, by using the equipment andthe methodology that has already been described [8].The combined Ingold 405 S7/120 electrode was cali-brated as a hydrogen concentration probe by titratingknown amounts of HCl with standardized CO2-freeNaOH solutions and determining the equivalent pointusing Gran’s method [9] which allows one to determinethe standard potential E° and the ionic product ofwater (pKw=13.73(1) at 298.1 K in 0.15 mol dm−3

NaCl).All equilibria involved in the studied systems were

determined, or redetermined, under the present experi-mental conditions in order to obtain a consistent set ofdata. The concentration of Gd3+ and BOPTA was1×10−3 mol dm−3 in all experiments, while the con-centration of ATP and TPP was varied in the range of(1–2)×10−3 mol dm−3. At least three measurements(about 100 data points each) were performed for eachsystem in the pH range of 2.5–10.5 and the relevante.m.f. data were treated by means of the computerprogram HYPERQUAD [10] which furnished the com-plexation constants listed in Tables 1 and 2. Backtitrations were performed on solutions containing ATPto check on possible nucleotide dephosphorylation. Theligand protonation constants used in calculation (Table3) were taken from Ref. [11] for ATP4− and redeter-mined under our experimental conditions for BOPTA

Table 1Logarithms of the equilibrium constants for the formation of Gd3+

complexes with ATP4−, and P3O105− determined in 0.15 mol dm−3

NaCl solution at 298.190.1 K

Reaction log K

Gd3++ATP4−=GdATP− 6.34(4)a

4.28(4)Gd3++HATP3−=GdHATP3.57(6)Gd3++H2ATP2−=GdH2ATP+

3.64(4)GdATP−+ATP4−=Gd(ATP)25−

8.37(2)Gd3++P3O105−=GdP3O10

2−

Gd3++HP3O104−=GdHP3O10

− 5.10(3)4.73(1)GdP3O10

2−+OH−=GdP3O10(OH)3−

5.02(5)GdP3O102−+P3O10

5−=Gd(P3O10)27−

a Values in parentheses are standard deviations on the last signifi-cant figure.

A. Bianchi et al. / Inorganica Chimica Acta 288 (1999) 244–248246

Table 2Logarithms of stability constants of Gd3+ complexes with BOPTA(H5L) and of the mixed-ligand complexes formed in the systemsGd3+/BOPTA/ATP4−, and Gd3+/BOPTA/P3O10

5−, determined in0.15 mol dm−3 NaCl solution at 298.190.1 K

Reaction log K

22.61(4)aGd3++L5−=GdL2−

24.33(3)Gd3++L5−+H+=GdHL−

31.76(2)Gd3++ATP4−+L5−+H+=H[Gd(ATP)L]5−

Gd3++ATP4−+L5−+2H+=H2[Gd(ATP)L]4− 35.43(4)38.1(1)Gd3++ATP4−+L5−+3H+=H3[Gd(ATP)L]3−

Gd3++ATP4−+L5−+4H+=H4[Gd(ATP)L]2− 40.8(1)43.23(9)Gd3++ATP4−+L5−+5H+=H5[Gd(ATP)L]−

32.34(4)Gd3++P3O105−+L5−+H+=H[Gd(P3O10)L]6−

37.90(4)Gd3++P3O105−+L5−+2H+=H2[Gd(P3O10)L]5−

40.88(6)Gd3++P3O105−+L5−+3H+=H3[Gd(P3O10)L]4−

43.71(4)Gd3++P3O105−+L5−+4H+=H4[Gd(P3O10)L]3−

Gd3++P3O105−+L5−+5H+=H5[Gd(P3O10)L]2− 46.25(5)

a Values in parentheses are standard deviations on the last signifi-cant figure.

An inversion recovery sequence using a threeparameters fitting [13] was employed to determine thelongitudinal relaxation times.

3. Results and discussion

3.1. Gd3+ complexes of ATP and TPP

The equilibrium constants for Gd3+ complexes withATP determined in the present work under our experi-mental conditions (Table 1) are in good agreement withvalues previously determined in 0.1 mol dm−3 KClsolution at the same temperature (298.1 K) [14]. In thecase of TPP complexes the unique values found in theliterature are in disagreement with our results, whichare on the other hand, consistent with data for Gd3+

complexation by ligands bearing triphosphate chains[14].

As can be seen from Table 1, Gd3+ shows a goodtendency to interact with fully deprotonated and partlyprotonated forms of the two ligands forming complexesin a wide range of pH. The Gd3+ complex with thefully deprotonated P3O10

5− anion (GdP3O102−) is more

stable by two logarithmic units than the analogouscomplex with ATP4−, indicating that in alkaline solu-tions TPP is more efficient than ATP in the binding ofthis metal ion, probably due to the greater negativecharge of the former anion. On the other hand, due tothe higher basicity of TPP and to the greater ability ofprotonated forms of ATP to bind Gd3+, the nucleotidebecomes a stronger ligand for the metal ion in acidicsolutions. The stability trend observed in the 1:1 metal-to-ligand complexes is also found for 1:2 species.

All Gd3+ complexes formed with ATP and TPP arelargely weaker than the very stable complexes formedby BOPTA (Table 2). As a consequence there is verysmall competition between the two ligands and BOPTAin the formation of single-ligand complexes and, ac-cordingly, no transmetallation reactions were observedin the potentiometric measurements on addition ofATP or TPP to solutions of BOPTA complexes.

3.2. ATP and TPP binding to Gd3+ complexes withBOPTA

Potentiometric (pH metric) titration performed onsolutions containing Gd3+, BOPTA and ATP, or TPPallowed to evidence the formation of several mixed-lig-and complexes and to determine their formation con-stants (Table 2). The distribution of the species formedas a function of pH calculated for equimolar concentra-tion of all reactants (1910−3 mol dm−3) in the sys-tems Gd3+/BOPTA/ATP and Gd3+/BOPTA/TPP arereported in Figs. 1 and 2, respectively.

and P3O105−. The values obtained for the last two

ligands are in good agreement with previous determina-tions [3,12].

2.3. Relaxometric study

Water relaxation times were determined by means ofa Bruker-Minispec spectrometer operating at 20 MHz.Samples of GdBOPTA2− (2×10−3 mol dm−3) and ofGdBOPTA2− (2×10−3 mol dm−3) containing differ-ent amounts of ATP (from 2×10−3 to 1×10−2 moldm−3) were prepared in 0.15 mol dm−3 NaCl at pH7.3. A solution of ATP in the absence of complex wasused as a control solution. The relaxation times weremeasured at 298 K after thermal equilibration of thesamples.

Table 3Logarithms of protonation constants for BOPTA (H5L), ATP4− andP3O10

5− determined in 0.15 mol dm−3 NaCl solution at 298.190.1 K

log KReaction

L5−+H+=HL4− 10.76(2)a

8.17(2)HL4−+H+=H2L3−

H2L3−+H+=H3L2− 4.31(2)2.69(2)H3L2−+H+=H4L−

H4L−+H+=H5L 2.18(2)6.24bATP4−+H+=HATP3−

HATP3−+H+=H2ATP2− 4.00b

H2ATP2−+H+=H3ATP− 1.76b

P3O105−+H+=HP3O10

4− 7.61(1)5.32(1)HP3O10

4−+H+=H2P3O103−

H2P3O103−+H+=H3P3O10

2− 1.3(1)

a Values in parentheses are standard deviations on the last signifi-cant figure.

b Taken from Ref. [11].

A. Bianchi et al. / Inorganica Chimica Acta 288 (1999) 244–248 247

Fig. 1. Distribution diagram of the complexed species formed as afunction of pH in the system Gd3+/BOPTA (L)/ATP. [Gd3+]=[BOPTA]= [ATP]=1×10−3 mol dm−3, 298.1 K.

dm−3) the mixed-ligand complexes formed by ATP areminor species over all the pH range considered (2.5–9),GdBOPTA2− remaining the principal complexed form.It is worth noting that around physiological pH (7.4),under the conditions employed, only a very small por-tion of GdBOPTA2− is bound to the nucleotide.

As shown in Table 2, TPP also forms, withGdBOPTA2−, only protonated species containing oneto five H+ ions. A comparison of the distributiondiagrams reported in Figs. 1 and 2 clearly evidences alower tendency of TPP with respect to ATP to bindGdBOPTA2−, in spite of the greater ability displayedby TPP in the coordination of free Gd3+. This featurecan be ascribed to the lower negative charge present onthe ATP anions and hence to a lower electrostaticrepulsion between the negatively charged ligands in theformation of the mixed-ligand complexes. Further, sta-bilization of such ATP complexes may arise from p–pstaking interactions between the adenine moiety of thenucleotide and the aromatic ring of BOPTA.

An interesting point in the formation of these com-plexes is the possible structure of such mixed-ligandspecies. The negative charges presented byGdBOPTA2− and the ATP or TPP anions, at least inthe lower protonation degrees, allow us to exclude theformation of second sphere complexes, and thus ATPand TPP are expected to bind directly to the metalcenter in the BOPTA complex. The molecular structureof the BOPTA complex, determined in the [GdBOPTA-(H2O)]Na2·1.5H2O solid compound, evidences themetal ion in a nine-coordinate geometry which com-prises the three amine nitrogens, five oxygens of theacetate residues, and a water molecule, defining a dis-torted tricapped trigonal prism around the metal ion[4]. The water molecule is responsible for the relaxationmechanism observed for this complex in solution. Inthe absence of crystallographic information on mixed-ligand complexes it is not possible to propose a struc-tural model for the simultaneous binding of BOPTAand ATP to the metal ion. Nevertheless, in view of theclinical application of the GdBOPTA2− complex, animportant point is to know whether the metal coordi-nated water molecule is retained in the mixed-ligandcomplexes of ATP, since the relaxation properties ofsuch complexes are very sensitive to the removal of thecoordinated water molecule. In other words it is ofgreat interest to verify whether GdBOPTA2− in themixed-ligand complex is still able to enhance the waterproton relaxation.

To this purpose we have determined the water relax-ation time of GdBOPTA2− sample solutions contain-ing increasing amounts of ATP at physiological pH.The longitudinal relaxation time of GdBOPTA2− (2×10−3 mol dm−3) in 0.15 mol dm−3 NaCl at 312 K(t1=87.90(3) ms) does not change significantly upon

As shown in Table 2 and depicted by the figures, thefully deprotonated ATP4− and P3O10

5− anions arecompletely unable to bind GdBOPTA2−, which re-mains the unique complexed species present in alkalinesolutions. On the other hand, on lowering the solutionpH many protonated mixed-ligand complexes areformed. Most likely protonation of the ligands favorsthe formation of such complexes by lowering the elec-trostatic repulsion between the two anionic ligands andreducing the number of donor atoms available in eachligand for the coordination to the metal ion.

A monoprotonated mixed-ligand complex with ATPstarts forming at about neutral pH, reaching the maxi-mum percentage of formation (ca. 30%) at about pH 5.In more acidic solutions also, polyprotonated speciescontaining up to five H+ ions are present but all ofthem are formed in less than 20%. On the whole, underthe experimental conditions employed to calculate thedistribution diagrams (all reactants 1×10−3 mol

Fig. 2. Distribution diagram of the complexed species formed as afunction of pH in the system Gd3+/BOPTA (L)/TPP. [Gd3+]=[BOPTA]= [TPP]=1×10−3 mol dm−3, 298.1 K.

A. Bianchi et al. / Inorganica Chimica Acta 288 (1999) 244–248248

addition of ATP (t1=87.14(3), 86.71(3) and 86.60(3)ms in the presence of ATP, respectively in 2×10−3,6×10−3 and 1×10−2 mol dm−3 concentrations, cor-responding to 10, 26 and 37% of GdBOPTA2− boundto ATP) indicating that the formation of the mixed-lig-and complexes with ATP does not give rise to apprecia-ble alterations of the relaxation properties ofGdBOPTA2−. Furthermore, such relaxation propertiesare maintained for a long time, both in absence and inpresence of ATP, as evidenced by checking the relax-ation times of the above samples over several months.In other words, the interaction of ATP withGdBOPTA2− in vitro does not compromise the effec-tiveness of this complex as paramagnetic contrast agentfor NMR imaging, nor causes degradation of the com-plex for a long time.

Acknowledgements

Financial support to A. Rodriguez from HumanCapital and Mobility, contract CHRX-CT94-0632, isgratefully acknowledged.

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