JBIC (1997) 2 :470–479 Q SBIC 1997

ORIGINAL ARTICLE

Silvio Aime 7 Mauro Botta 7 Simonetta Geninatti CrichGiovanni B. Giovenzana 7 Roberto PagliarinMaurizio Piccinini 7 Massimo Sisti 7 Enzo Terreno

Towards MRI contrast agents of improved efficacy. NMR relaxometric

investigations of the binding interaction to HSA of a novel

heptadentate macrocyclic triphosphonate Gd(III)-complex

Received: 27 January 1997 / Accepted: 12 May 1997

Dedicated to the memory of Prof. Giancarlo Jommi

S. Aime (Y) 7 M. Botta 7 S. Geninatti Crich 7 E. TerrenoDipartimento di Chimica I.F.M., Università di Torino,Via P. Giuria 7, I-10125 Torino, ItalyTel.: c3911-6707520; Fax: c3911-6707524;e-mail: aime@silver.ch.unito.it

G. B. Giovenzana 7 R. Pagliarin 7 M. Piccinini 7 M. SistiDipartimento di Chimica Organica ed Industriale,Università di Milano, Viale Venezian 21, I-20133 Milano, Italy

Abstract A novel heptacoordinating ligand consistingof a thirteen-membered tetraazamacrocycle containingthe pyridine ring and bearing three methylenephospho-nate groups (PCTP-[13]) has been synthesized. ItsGd(III) complex displays a remarkably high longitudi-nal water proton relaxivity (7.7 mMP1 sP1 at 25 7C,20 MHz and pH 7.5) which has been accounted for interms of contributions arising from (1) one water mole-cule bound to the metal ion, (2) hydrogen-bonded wa-ter molecules in the second coordination sphere, or (3)water molecules diffusing near the paramagnetic che-late. Variable-temperature 17O-NMR transverse relax-ation data indicate that the residence lifetime of themetal-bound water molecule is very short (8.0 ns at25 7C) with respect to the Gd(III) complexes currentlyconsidered as contrast agents for magnetic resonanceimaging. Furthermore, GdPCTP-[13] interacts with hu-man serum albumin (HSA), likely through electrostaticforces. By comparing water proton relaxivity data forthe GdPCTP-[13]-HSA adduct, measured as a functionof temperature and magnetic field strength, with thosefor the analogous adduct with GdDOTP (a twelve-membered tetraaza macrocyclic tetramethylenephos-phonate complex lacking a metal-bound water mole-cule), it has been possible to propose a general pictureaccounting for the main determinants of the relaxationenhancement observed when a paramagnetic Gd(III)complex is bound to HSA. Basically, the relaxation en-hancement in these systems arises from (1) water mole-cules in the hydration shell of the macromolecule andprotein exchangeable protons which lie close to the in-

teraction site of the paramagnetic complex and (2) themetal bound water molecule(s). As far as the lattercontribution is concerned, the interaction with the pro-tein causes an elongation of the residence lifetime ofthe metal-bound water molecule, which limits, to someextent, the potential relaxivity enhancement expectedupon the binding of the paramagnetic complex toHSA.

Key words Gd(III) complex 7 MRI contrast agent 7Water exchange rate 7 Human serum albumin 7Relaxometry

Introduction

Contrast agents for magnetic resonance imaging (MRI)are mostly represented by stable complexes of Gd(III)ions, whose seven unpaired electrons provide an effi-cient source for the paramagnetic relaxation of the wa-ter protons in the tissues where they distribute [1, 2].The large majority of complexes so far considered forthis purpose are based on octadentate ligands to ensurea high thermodynamic (and possibly kinetic) stability.This choice has implied that only one water moleculemay enter the inner coordination sphere of the Gd(III)ion, which is characterized by a coordination number ofnine. As the relaxivity of a Gd(III) complex is directlyproportional to the number (q) of inner coordination-sphere water molecules, complexes involving hepta-coordinating ligands and possibly two water moleculeshave also been considered. It has been shown that aroute to obtaining heptadentate ligands is provided bythe inclusion of a pyridine moiety in a triazamacrocyclewhose three amine nitrogens are further used as linkingsites for three acetate arms [3, 4]. Gd(III) complexes ofthese PCTA (Pyridine-Containing Triaza MacrocyclicTriAcetate) ligands have been obtained with a macro-cycle size ranging from twelve to fourteen members. Inthe case of GdPCTA-[12] and -[13], the relaxometricdata support the occurrence of two water molecules in

471

Scheme 1

the inner coordination sphere of the paramagnetic ion.In spite of the decreased denticity of the ligands, thelatter complexes display a quite good thermodynamicstability [4–6].

An additional route to enhancing the relaxivity of aGd(III) complex may be pursued through the exploita-tion of the paramagnetic relaxation of water moleculesin the second coordination sphere, i.e. water moleculesstrongly interacting at the surface of the complex. Thiscontribution differs from the ordinary outer-spherecontribution owing to water molecules diffusing in theproximity of the complex, whose magnitude is fairlyconstant for complexes of similar size. In the case of thesecond coordination sphere [7–10], we are dealing withmolecules which lie on the complex for a time signifi-cantly longer than that associated with diffusion mo-tions. Their contributions to the overall relaxivity maybe treated in strict analogy to water molecules in theinner coordination sphere, though the average Gd(III)-H distance is much longer. The contribution from thesecond coordination sphere is related to the overall re-sidual charge and to the nature of the functionalities atthe surface of the complex. We have found that phos-phonate moieties are particularly able to tightly bindwater molecules at the second coordination sphere. Infact, GdDOTP (Scheme 1), which lacks a metal-coordi-nated water molecule [9, 11], is endowed with a relaxiv-ity typical of complexes with qp1. Thus, its good relax-ivity may be fully accounted for in terms of the sum oftwo contributions arising from the second coordinationsphere and from the water molecules diffusing aroundthe chelate, respectively.

On the basis of these considerations, we thoughtthat a good candidate for a contrast agent of improvedefficacy may be a complex where the inner-sphere

mechanism is added to the previous ones. This complexmight be a heptacoordinating ligand bearing phospho-nate groups able to capture water molecules in its sec-ond coordination sphere, as GdDOTP does.

In this paper we report the synthesis of a novel li-gand derived from PCTA-[13] following the substitu-tion of the three acetic with three methylenephos-phonic groups. In analogy with the triacetic derivativewe shall indicate this thirteen-membered macrocyclic li-gand with the acronym PCTP-[13] (Pyridine-Contain-ing macrocyclic TriPhosphonate) (Scheme 1). Further-more, we expect that GdPCTP-[13], endowed withthree negative charges at physiological pH, shouldshow, although to a lesser extent, binding affinity withthe serum protein analogous to that of GdDOTP (un-published results).

In fact, the reversible formation of adducts withHSA represents one of the major routes to further re-laxation enhancements by Gd(III) complexes as a re-sult of the lengthening of the molecular reorientationaltime tR upon formation of a macromolecular adduct[12]. However, enough evidence has now been col-lected to show that the large relaxation enhancement ofthe inner-sphere term (Ris

1p) associated with long tR

values, is partly “quenched” by the occurrence of a re-latively long exchange lifetime (tM) of the coordinatedwater [13].

According to Eq. 1 [14], as tM becomes longer thanthe relaxation time of the coordinated water protons(T1M), the contribution to the overall relaxivity fromthe water molecules in the inner coordination sphereRis

1p, is controlled by tM:

Ris1p p

q7[C]55.67(T1MctM)

(1)

where [C] represents the molar concentration of the pa-ramagnetic complex. Actually, rather slow exchangerates have been observed in some Gd(III) complexescurrently used as contrast agents for MRI [15], namelyGdBMA-DTPA (tMp2.2 ms at 25 7C), GdDTPA(tMp0.3 ms at 25 7C), GdDOTA (tMp0.2 ms at 25 7C).On the other hand, complexes containing substitutedDOTA ligands showed much shorter tM values [16],and the Gd(III) complex with the octacoordinatingEGTA shows an optimum tM value of 20 ns at roomtemperature (unpublished results). According to Mer-bach and coworkers [15], in complexes with qp1, thecause of the occurrence of long exchange lifetimes liesin the dissociative mechanism operating the exchangebetween coordinated and “bulk” water. On going fromocta- to heptadentate ligands, we expect that a differentexchange mechanism for the coordinated water(s) inthe corresponding Gd(III) complexes may possiblylead to an increased exchange rate. In turn, this behav-ior would contribute to the elimination, or at least re-duction, of the “quenching” effect observed upon theinteraction with the protein.

These considerations on the relationship betweenthe complex structure and its hydration (in term of q,

472

second sphere, and tM) prompted us to design thePCTP ligand and to investigate in detail its relaxomet-ric properties in the presence and absence of HSA.

Materials and methods

HSA (crystallized and lyophilized) was purchased from Sigma(St. Louis, Mo., USA) and was used without any further purifica-tion. The molecular weight was assumed to be 69 kDa [17].

High resolution 1H- 13C and 31P-NMR spectra were obtainedon a Bruker AC 200 (200, 50.2 and 80.9 MHz, respectively) spec-trometer.

Mass spectra were recorded with a VG 7070 EQ spectrometer(at 70 eV, m-nitrobenzyl alcohol or glycerol as matrix in theFABc ionization, isobutane in chemical ionization technique).Elemental analyses were performed with a Perkin-Elmer 240 ap-paratus. Melting points were determined with a Buchi 520 appa-ratus.

1,4,8-Tritosyl-1,4,8-triazaoctane was prepared according to thereported procedure [18] (63.0% yield after crystallization). 2,6-Bis(chloromethyl)pyridine was purchased from Fluka.

Synthesis of the ligands

3,6,10-Tritosyl-3,6,10,16-tetraazabicyclo[10.3.1]hexadeca-1(16),12,14-triene

A solution of 2,6-bis(chloromethyl)pyridine (2.07 g, 11.8 mmol) inanhydrous acetonitrile (15 ml) was added dropwise, over a periodof one hour, to a suspension of 1,4,7-tritosyl-1,4,7-triazaoctane(6.9 g, 12.2 mmol) and Na2CO3 (5.8 g, 41.9 mmol) in anhydrousrefluxing acetonitrile under nitrogen atmosphere. The reactionwas refluxed overnight. The solvent was evaporated and the resi-due extracted with aqueous sodium hydroxide-methylene chlo-ride; the organic phase was dried over sodium sulfate and concen-trated to give a solid which was recrystallized from acetone togive pure 3,6,10-tritosyl-3,6,10,16-tetrazabicyclo[10.3.1]hexadeca-1(16),12,14-triene (yield: 90%).

3,6,10,16-Tetraazabicyclo[10.3.1]hexadeca-1(16),12,14-triene,hydrobromide74HBr

3,6,10-Tritosyl-3,6,10,16-tetrazabicyclo[10.3.1]hexadeca-1(16),12,14-triene (1.93 g, 2.83 mmol) was refluxed for 12 h with a48% aqueous hydrobromic acid solution (89.4 g, 530 mmol), gla-cial acetic acid (26.2 g, 436 mmol) and phenol (2.76 g, 29.4 mmol).The solution was then diluted with water and extracted with me-thylene chloride (3!60 ml). The aqueous phase was evaporatedunder reduced pressure and the residue was treated with thesame quantity of reactants; the resulting solution was refluxed forfurther 12 h. Then, after the separation of the water and methy-lene chloride, the aqueous phase was concentrated (3 ml) and di-ethyl ether (50 ml) was added. The white-yellow crystals obtainedafter filtration were washed with diethyl ether and dried undervacuum to give 3,6,10,16-tetraazabicyclo[10.3.1]hexadeca-1(16),12, 14-triene, hydrobromide (1.15 g, 73%). Mp: 122–123 7C.1H-NMR (D2O): d (ppm) 2.11 (m, 2H), 3.11 (m, 4H), 3.40 (m,4H), 4.34 (s, 2H), 4.40 (s, 2H), 7.32 (d, Jp7.7 Hz, 1H), 7.37 (d,Jp7.7Hz, 1H), 7.79 (t, Jp7.7 Hz, 1H). 13C-NMR (D2O): d (ppm)22.76, 43.41, 44.33, 44.84, 45.82, 51.45, 51.96, 127.14, 127.44,142.47, 152.58, 152.78.

3,6,10,16-Tetraazabicyclo[10.3.1]hexadeca-1(16),12,14-triene 1

A solution of potassium hydroxide (5 g, 89.1 mmol) in water(5 ml) was added under stirring to a flask containing 3,6,10,16-tetraazabicyclo[10.3.1]hexadeca-1(16),12,14-triene, hydrobromide(1.06 g, 1.90 mmol) pre-cooled to 0 7C. After 15 min the solution

was extracted with methylene chloride (6!10 ml) and the organiclayers were dried over sodium sulfate and evaporated to give anoily residue which later solidified to the white, waxy compound 1.98.5% yield (0.410 g) was obtained. 1H-NMR (CDCl3): d (ppm)1.66 (q, Jp6 Hz, 2H), 2.56 (t, Jp6 Hz, 2H), 2.66 (t, Jp6 Hz, 2H),2.68 (m, 4H), 3.88 (s, 2H), 3.90 (s, 2H), 7.02 (d, Jp7.7 Hz, 2H),7.56 (t, Jp7.7 Hz, 1H). FT-IR: 3365, 1595, 1577, 1456, 1363, 1161,1001, 747 cmP1. Mass spectrum: found CI m/e 221 (MHc); calcd.for C12, H20, N4: 220.

3,6,10,16-tetraazabicyclo[10.3.1]hexadecane-3,6,10-tris(methane-phosphonic) acid 2

Compound 1 (197 mg, 0.89 mmol) was dissolved in HCl 37%(2 ml) and water (2 ml). To this solution was added phosphorousacid (429 mg, 5.23 mmol) and the mixture was heated to reflux.Paraformaldehyde (287 mg, 9.53 mmol) was added dropwise inca. 1 h, and the reflux was continued for further 10 h. The reac-tion mixture was concentrated in vacuo and isopropanol was ad-ded slowly, under continuous stirring. The solid product was sep-arated by filtration. The purification of the product was carriedout by its dissolution in HCl (37%) followed by reprecipitationwith dioxane. This work-up was repeated three times. The prod-uct was dried by prolonged standing in vacuo.

1H-NMR (D2O): d (ppm) 7.90(t, Jp7.8 Hz, 1H), 7.50(d,Jp7.8 Hz, 1H), 7.46(d, Jp7.8 Hz, 1H), 4.58(s, 4H), 3.42(d,Jp4.4 Hz, 2H), 3.36(d, Jp4.2 Hz, 2H), 3.30(d, Jp4.4 Hz, 2H),3.15(t, Jp6.3 Hz, 2H), 3.09(s, 2H), 3.03(m, 4H), 2.15(m, 2H).

31P-NMR (D2O): d (ppm) 12.09(s, 1P), 10.33(s, 1P), 9.88(s,1P).

CHN: Anal. calc. for C15H29N4O9P374HCl C 27.80% H 5.13%N 8.64%. Found Cp27.73% Hp5.25% Np8.51%.

FAB-MS (Gly): Calc. for C15H29N4O9P3p502.34 uma.Foundp503 (MHc), 525 (MNac), 541 (MKc), 422 (M-PO3H2).

1,4,7,10-Tetraazacyclododecane-N,Nb,Nn,Nm-tetrakis(methylenephosphonic acid (DOTP)

The ligand DOTP and its Gd(III) complex were synthesized fol-lowing the procedure reported in the literature [8].

Complexation procedure

The ligand 2 (0.2 mmol) was dissolved in H2O (5 ml) and the pHof the solution was adjusted to 7.5 with NaOH 1N. To this solu-tion, 3 ml of an aqueous solution of gadolinium trichloride(0.2 mmol) was added dropwise by maintaining the pH at 7.5again with NaOH 1N. At room temperature the complex forma-tion is instantaneous. The pH of the solution was then increasedto 8–9 by adding NaOH 1N in order to precipitate the eventualexcess of uncomplexed Gd(III) ions. The solution was then evap-orated under reduced pressure and the residue dried at 70 7Covernight.

NMR relaxometric measurements

The variable-temperature longitudinal solvent proton relaxationrates were obtained on a Stelar Spinmaster spectrometer [Stelar,Mede (PV), Italy] operating at 20 MHz by means of the standardinversion recovery technique (16 experiments, 4 scans). A typical907 pulse width was 3.5 ms and the reproducibility of T1 data wasB0.4%. The 1/T1 NMRD profiles were acquired on the Field Cy-cling Koenig-Brown relaxometer (University of Florence, Italy)which operates over a continuum of magnetic field strength from2.5!10P4 to 1.4 T (corresponding to 0.01–50 MHz proton Lar-mor frequencies). Details of the instrument and of the data acqui-sition procedure are given elsewhere [19].

473

Scheme 2

Results and discussion

Synthesis of GdPCTP-[13]

The synthesis of PCTP-[13] ligand requires severalsteps, the most difficult one being the formation of themacrocyclic ring. The synthesis of 3,6,10,16-tetraazabi-cyclo[10.3.1]hexadeca-1(16),12,14-triene, (1) has beencarried out by reacting 2,6-bis(chloromethyl)pyridineand the disodium salt of 1,4,8-tritosyl-1,4,8-triazaoctanein anhydrous acetonitrile as solvent and potassium car-bonate as base in heterogeneous conditions [4, 5]. Hy-drolysis of the tritosylates with hydrobromic acid fol-lowed by alkalinization gave 1 pure enough for the fol-lowing step. As depicted in Scheme 2, the introductionof the three methylenephosphonate moieties has beencarried out by reacting 1 with paraformaldehyde andphosphorous acid. 2 has been fully characterized by el-emental analysis (C,H,N), 1H- and 31P-NMR spectra,and mass spectrometry. Finally, the complexation ofGd(III) ion to PCTA-[13] has been carried out by ad-ding stoichiometric amounts of Gd(III) chloride to theaqueous solution of the ligand at pH 7.5 and at ambienttemperature.

Proton relaxation studies

At 25 7C and 20 MHz, GdPCTP-[13] displays a relaxivi-ty of 7.7 mMP1 sP1. This value is markedly higher thanthe values reported for systems with qp1 such asGdDTPA (R1pp4.7 mMP1 sP1) [2], GdDOTA(R1pp4.7 mMP1 sP1) [2], and GdBMA-DTPA(R1pp4.4 mMP1 sP1) [20]. Furthermore, this relaxivityvalue appears significantly larger than that observed forthe related GdPCTA-[13] complex (R1pp 6.3 mMP1

sP1) [5] to which two water molecules in the innercoordination sphere were assigned. On the other hand,if the comparison is carried out with GdDOTP(R1pp4.7 mMP1 sP1 at 25 7C) [8], the observed relaxiv-ity of GdPCTP-[13] may be accounted for in terms of alarge contribution from the second coordination spherein addition to an inner-sphere contribution.

Fig. 1a, b 1/T1 NMRD profiles for GdPCTP-[13] measured at25 7C and pH 7.5: a observed profile and b inner sphere contribu-tion calculated as described in the text and fitted with qp1(straight line) and qp2 (dotted line)

The simple comparison between the relaxivitiesmeasured at a single magnetic field does not allow oneto propose a hypothesis about the number of watermolecules directly coordinated to the metal ion inGdPCTP-[13].

In order to get more insight into the parameters in-volved in the paramagnetic relaxation pathway, wemeasured the 1/T1 NMRD profiles over an extendedrange of observation frequencies at 25 7C (Fig. 1a). Ateach frequency, the observed relaxation rate may beconsidered as the sum of four contributions:

Robs1 pR1dcRis

1pcR2nd1p cRos

1p (2)

where R1d represents the relaxation rate of water pro-tons in the presence of a 1 mM solution of a relateddiamagnetic analog, and it may be assumed to be equalto the relaxation rate of pure water (i.e. 0.38 sP1 at25 7C); Ris

1p arises from the exchange of water mole-cule(s) in the inner coordination sphere of the para-magnetic metal ion; R2nd

1p is the contribution from wa-ter molecules in the second coordination sphere and R-os1p deals with the contribution from water moleculeswhich diffuse in the proximity of the paramagneticcomplex.

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Table 1 Best-fitting parameters obtained from the analysis of theNMRD profiles for GdPCTP-[13] at 25 7C and pH 7.5 on the basisof two models differing in the number of water molecules (q)coordinated to the paramagnetic centre

q D2 (sP2)/1019 tv (ps) tM (ns) tR (ps) r (Å)

12

3.02.5

31.032.0

8.04.0

102.0120.0

3.03.4

The latter contribution has been experimentally de-termined for systems with qp0, and it has been evalu-ated by means of the Freed’s [21] equation developedfor stable organic radicals dissolved in organic solvents.For small sized Gd(III) complexes, similar to GdPCTP-[13], it accounts for ca. 2.3–2.5 sP1 mMP1. As far as Ris1p and R2nd

1p are concerned, they may be evaluated onthe basis of the Solomon-Bloembergen-Morgan [22, 23]equations (Eqs. 1 and 3–5):

TP11M a

Dis

r6 f (tci, vI, vs) (3)

tP1ci pTP1

iE ctP1M ctP1

R (4)

TP1iE aKi D

2 f (tV, vs) (5)

where r is the water proton-Gd distance, tci (ip1,2) arethe overall correlation times for the dipolar nucleus-electron interaction, tR is the reorientational correla-tion time of the H-Gd position vector, TiE are the relax-ation times of the Gd(III) unpaired electrons, Dis andKi are constant terms related to the nuclear and elec-tron relaxation mechanisms, D2 is the trace of thesquare of the transient zero field splitting (ZFS) tensor,tV is the correlation time for the processes modulatingthe ZFS hamiltonian and vS and vI are the electronand proton Larmor frequencies, respectively.

The best-fitting procedure of the experimental datawas carried out by considering two models differing inthe number of water molecules present in the innercoordination sphere of the metal ion (qp1 and qp2,respectively). In these simulations we have assumedthat the contribution arising from the outer-sphere wa-ter molecules for GdPCTP-[13] was similar to that forGdDOTP. As far as the second sphere contribution isconcerned, we have to consider the different number ofphosphonate groups between the two chelates, and wehave estimated this contribution for GdPCTP-[13] bymultipling that measured for GdDOTP by 3/4.

Finally, by subtracting the outer and the secondsphere contributions to the observed profile forGdPCTP-[13], we simply obtained an estimation of the1/T1 NMRD profile for the inner sphere term only (Fig.1). Both models yield a reasonably good fitting of theexperimental data, and the obtained parameters are re-ported in Table 1. However, from these data it is imme-diatelly evident that the Gd-H distance (r) found forthe model with qp2 is exceedingly long. On the basisof a number of Gd(III) chelates containing one or more

water molecules in the inner coordination sphere, thisdistance invariably lies in the range 2.8–3.1 Å. There-fore, this result strongly suggests that the model withqp1 is more suitable to represent the hydration shell ofGdPCTP-[13]. In both models the fitting procedure israther insensitive to changes in tM, indicating that itsvalue is significantly longer than tR and smaller thanT1M.

Determination of the exchange rate of theinner-sphere water molecule by 17O-NMR

The actual value of tM has been obtained by analyzingthe temperature dependence of the solvent water 17Otransverse relaxation rate. According to Swift and Con-nick [24] theory, the paramagnetic contribution Ro

2p tothe observed relaxation rate, Ro

2obs, (Ro2ppRo

2obsPRo2d,

the latter contribution being the diamagnetic term) isgiven by the following equation:

Ro2ppPM7toP1

M 7Ro

2M2ctoP1

M 7Ro2McDvo2

M

(Ro2MctoP1

M )2cDvo2

M

(6)

where PM is the molar fraction of the coordinated wa-ter, Ro

2M represents its 17O transverse relaxation rate, toM

its residence lifetime in the metal site and DvoM the

chemical shift difference between the coordinated andbulk water 17O-NMR resonances. At the magnetic fieldstrength used in this work (2.1 T) and for rapidly re-orienting Gd(III) chelates, Ro

2M is dominated by thescalar term, Rosc

2M:

Rosc2M p

13

71Ak 2

2

7S(Sc1)1tE1ctE2

1cv2s7t2

E22 (7)

where S is the electronic spin quantum number (7/2 forGd(III)). A/k is the Gd-17O scalar coupling constant,which has been set equal to P3.8!106 rad sP1 asfound in closely related systems [15]; tEi (ip1,2) repre-sents the correlation times of the processes modulatingthe scalar interaction. This modulation may occurthrough both the longitudinal and the transverse elec-tronic relaxation times (T1E and T2E) and the mean res-idence lifetime (to

M) of the water molecule at the para-magnetic site, i.e.

tP1IE ptoP1

M cTP1iE . (7)

As described in the previous section (see Eq. 5), TiE aredetermined by the modulation of the zero field splitting(ZFS) of the electronic spin states and tV representsthe correlation time related to this dynamic process.

Therefore, the temperature dependence of Ro2M is

determined by the temperature effect on toM, tv, and

DvoM according to the following equations:

tP1j p

(tP1j )298.157T

298.15exp3DHJ

R 1 1298.15

P1T24 (8)

DvoMp

ge7mB7S(Sc1)7B3kB T

7Ak

(9)

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Table 2 Best-fitting parameters obtained from the analysis of the temperature dependence of R2p of water 17O-NMR for GdPCTP-[13]

q D2 (sP2)/1019 tV (ps) tM (ns) tR (ps) DHV (kJ molP1) DHM (kJ molP1) DHR (kJ molP1)

12

3.916.0

10.018.0

8.04.0

102.0120.0

0.40.9

58.059.0

20.020.0

Fig. 2 Temperature dependence of the transverse water 17O re-laxation rate (Ro

2p) for a 25 mM solution of GdPCTP-[13], meas-ured at 2.1 T and pH 7.5

Fig. 3 Temperature dependence of the water proton longitudinalrelaxation rate (R1p) for 1 mM GdPCTP-[13] solution, measuredat 20 MHz and pH 7.5

where the subscript j refers to the different correlationtimes (jpM,v), DHj is the activation enthalpy for thecorresponding dynamic process, B is the magnetic fieldstrength, and kB is the Boltzmann constant.

As shown in Fig. 2, the transverse relaxation rate ofwater 17O-NMR resonance increases as the tempera-ture decreases to indicate the typical behavior expectedwhen the fast exchange conditions are met, i.e. toP1

M `Ro

2M. Thus, at any temperature, tM results to be signifi-cantly shorter than in systems like GdDTPA or GdDO-TA. In spite of the concerns inherent to the evalua-tion of short to

M, the value obtained at 25 7C forGdPCTP-[13] (8 ns) is similar to that reported forGdPDTA, which has two coordinated water molecules[25]. On comparing the results from the fitting of 17Odata using qp1 or qp2 (Table 2), it is evident that onlyin the former case we found a value for ZFS that iscomparable with those values until now reported forwater molecules coordinated to the Gd(III) ion [15].Thus, the 17O-NMR data are also in agreement withthe view that the hydration sphere in GdPCTP-[13] isbetter described by a model made up of one water mol-ecule in the inner coordination sphere and a secondsphere similar to that found in the case of GdDOTP.

The occurrence of such a very short exchange life-time causes a monoexponential dependence of protonR1p (measured at 20 MHz) over the range of tempera-ture 0–60 7C (Fig. 3). It is, then, likely that the structureadopted by GdPCTP-[13] is analogous to the antipris-matic one shown by TmDOTP[11], where one of thecoordination sites of the phosphonate groups has been

replaced by one water molecule. Thus, in this complexthe Gd(III) ion appears octacoordinated and, proba-bly, the exchange of the coordinated water occursthrough an associative mechanism involving a nona-coordinated activated state.

Binding of GdPCTP-[13] to HSA

The determination of the interaction strength betweenthe complex and HSA has been carried out by measur-ing the longitudinal water proton relaxation time, at thefixed frequency of 20 MHz, of a 0.166 mM solution ofthe complex in the presence of an increasing concentra-tion of the added protein [13, 26] (Fig. 4). Each value inthis titration is given by the sum of three contribu-tions:

R1obspRF1p[GdPCTP-[13]]

cRB1p[GdPCTP-[13]-HSA]cR1d (10)

where RF1p and RB

1p are the relaxivity of the complexand of the paramagnetic macromolecular [HSA-GdPCTP-[13]] adduct at 25 7C respectively, and R1d isthe diamagnetic contribution. For the equilibrium:

GdPCTPcHSAvGdPCTPPHSA

it is possible to obtain the association constant, KA, (ac-tually the product KA7n, where n is the number of in-dependent sites characterized by the same KA value)

476

Fig. 4 Plot of the longitudinal water proton relaxation rate of a0.166 mM GdPCTP-[13] solution as a function of HSA concentra-tion (20 MHz, pH 7.5 and 25 7C)

and RB1p (relaxivity of the adduct) by combining Eq. 10

with the expression for the association constant KA

KAp[GdPCTPPHSA]

[GdPCTP]7[n7HSA](11)

The interaction of GdPCTP-[13] with HSA is ratherweak (KA7np6!102 MP1), whereas the relaxivity ofthe paramagnetic macromolecular [HSA/GdPCTP-[13]] adduct is 45 mMP1 sP1 at 20 MHz and 25 7C. Thebinding parameters have been measured in a solutionbuffered with Hepes (4-(2-hyydroxyethyl)-1-piperazi-neethanesulfonic acid) 50 mM. The interaction strengthwas fairly independent of the experimental tempera-ture as KA values measured at 10 7C (KAp5.7!102

MP1), and 39 7C (KAp6.4!102 MP1) were similar tothe value measured at 25 7C.

The binding strength should result essentially fromelectrostatic forces and is intermediate between thoseobserved for the neutral GdPCTA-[13] (almost nobinding) and the tetranegatively charged GdDOTP(KAp3.1!103 MP1 and np1, at 25 7C and in 50 mMNaCl solution, unpublished results). In order to assessthe possible “quenching” effect of slow water exchangerate on the relaxation enhancement for the HSA-GdPCTP-[13] adduct, we measured the temperaturedependence of the solvent relaxation rates of a solutioncontaining 0.175 mM of GdPCTP-[13] and 2.90 mM ofHSA. The knowledge of KA, RF

1p and R1d at the varioustemperatures, allows us to evaluate the contributionsfrom each of the three terms of Eq. 10 to the observedrelaxation rates. From this evaluation we found that therelaxivity of the HSA-adduct (RB

1p) decreases as thetemperature increases, as shown in Fig. 5a. Althoughthe exponential decay of RB

1p vs T is somewhatsmoothed with respect to that observed for the samecomplex in the absence of albumin (Fig. 3), it differsmarkedly from those found in the case of the HSA ad-

Fig. 5 Temperature dependence of the water proton relaxivity ofa GdPCTP-[13]-HSA adduct and b GdDOTP-HSA adduct calcu-lated as described in the text (20 MHz and pH 7.5)

ducts with Gd(III)-complexes bearing hydrophobicsubstituents, which are characterized by an increase ofR1p with the temperature [13]. On the other hand, thetemperature dependence of the relaxivity of the adductbetween HSA and GdDOTP (qp0) (Fig. 5b) behavesanalogously to the GdPCTP-[13] adduct, although thedecrease of R1p with the increase of the temperature isdefinitely more marked. On the assumption that the re-laxivity value of the HSA-GdPCTP-[13] adduct is madeup of the sum of an inner and a second-outer-spherecontribution and that the latter terms may have a mag-nitude similar to the overall relaxivity value shown byHSA-GdDOTP adduct, we subtract the latter contribu-tion to those ones reported in trace b of Fig. 5. Theresult of this subtraction should provide the tempera-ture-dependent profile of the contribution to the ob-served relaxivity arising from water in the inner-coordi-nation sphere of the HSA-GdPCTP-[13] adduct. Tosome extent, as shown in Fig. 6, the resulting profiledisplays a similarity to those ones found in the case ofHSA adduct with Gd complexes bearing hydrophobicsubstituents, i.e. there is a small but very significant in-crease of the relaxation rates as the temperature in-creases. Thus, it appears that, also in the case of theHSA-GdPCTP-[13] adduct, a “quenching” effect on therelaxation enhancement from a water molecule in theinner coordination sphere is present, despite the veryshort tM value measured for the free complex.

Further insights into the understanding of the deter-minants of the observed relaxivities have been gainedby measuring the 1/T1 NMRD profile of GdPCTP-[13]in the presence of HSA. From the knowledge of KA,R1d, RF

1p, and RB1p at 25 7C, we have calculated the re-

laxivity profile of the HSA-GdPCTP-[13] adduct (Fig.7a). We have then proceeded in an analogous way tothat described above in the case of 20 MHz relaxationdata at variable temperature, i.e. the 1/T1 NMRD pro-file of the HSA-GdDOTP adduct has been subtracted

477

Fig. 6 Temperature dependence of the inner-sphere contributionto the relaxivity for the GdPCTP-[13]-HSA adduct, calculated at20 MHz and pH 7.5, as described in the text

Fig. 7a, b 1/T1 NMRD profiles, calculated, as described in thetext, at 25 7C and pH 7.5 for the GdPCTP-[13]-HSA adduct: a R1p

contribution, and b Ris1p contribution

Table 3 Best-fitting parameters obtained from the analysis of theNMRD profiles for GdPCTP-[13]-HSA adduct at 25 7C andpH 7.5

q D2 (sP2)/1019 tV (ps) tM (ns) tR (ns) r (Å)

1 2.2 12.0 290.0 30.0 3.0

from the NMRD profile of HSA-GdPCTP-[13] adduct(Fig. 7b). Again, the difference should correspond basi-cally to the profile of the inner sphere term, and as suchit has been fitted to the theoretical values computedthrough Eqs. 3 and 4. A reasonably good fit has beenobtained by using, for the various parameters, valuesvery close to those obtained from the analysis of theNMRD profile of GdPCTP-[13] in the absence of HSA,but allowing tR and tM to vary (Table 3). The obtained

tR is in good agreement with values usually reportedfor the serum protein [17]. Very important is the resultthat the inner sphere profile can be fitted only with re-latively long exchange lifetime tM, i.e. it is apparentthat, in the adduct, the inner-sphere water exchangerate is ca. two orders of magnitude slower than in thefree complex.

Concluding remarks

In summary, the following conclusions can be drawn:(a) GdPCTP-[13] shows a very good relaxivity, much

higher than those reported for other Gd(III) complexescurrently used as CA for MRI. Such high relaxivity isconsistent with the view that, in addition to the ordina-ry contributions from inner (qp1) and outer coordina-tion spheres, there is a large second sphere contributionbrought about by the phosphonic groups. Moreover,the exchange rate of the water molecule directly coor-dinated to the Gd(III) ion with the “bulk” is much fas-ter than the values commonly encountered for octa-coordinated chelates.

(b) GdPCTP-[13] interacts weakly with HSA. Therelaxivity of the adduct is rather high (45.0 mMP1 sP1

at 25 7C) and slightly decreases when the temperature isincreased. Apparently, this behavior suggests that suchan adduct should not suffer the limiting factor of theslow exchange between the bound and the bulk watershown by other similar adducts (Eq. 1). Actually, wefound that as far as the contribution from the watermolecule coordinated to the metallic center is con-cerned, its exchange rate is ca. two orders of magnitudeslower than that found for the free complex. Thus, theslight decrease in R1p of the HSA adduct with the in-crease in temperature is the result of a balance betweenthe increase of the R1p inner-sphere contribution and adecrease of the relaxivity contribution arising from wa-ter molecules in both the second and outer coordina-tion spheres.

(c) The 1/T1 NMRD profile of the HSA-GdPCTP-[13] adduct is similar to those found for a number ofother adducts (either covalent or non-covalent) be-tween HSA and Gd(III) complexes [27, 28]. They showincreased relaxivity values at low fields and smoothedrelaxivity peaks in the 20–30 MHz frequency range. Itmay be instructive to recall that, on the contrary, metal-lo-proteins show lower relaxation rates at low fieldsand sharper relaxivity peaks at high fields. The NMRDprofiles of a paramagnetic metalloprotein [29] closelyresembles the theoretical profile calculated for a gener-ic Gd(III) complex using the same parameters foundfor its aqueous solutions in the absence of a protein butintroducing a tR value increased by two to three ordersof magnitude. There is, then, a basic difference be-tween a paramagnetic metalloprotein and paramagneticadducts between HSA and Gd(III) complexes. In theformer case the paramagnetic ion is kept tightly in theprotein framework (highly hydrophobic) and is in close

478

Fig. 8 Schematic representation of the non-covalent interactionbetween a negatively charged paramagnetic chelate and a pro-tein

contact only with the water molecule(s) coordinated toit. In the latter case the paramagnetic complex interactsdipolarly with a number of H2O molecules includingthose in the first and second coordination sphere and inthe various layers of the hydration shell of the protein,in addition to possible interactions with X-H groupscontaining exchangeable protons. It follows that, in ageneric HSA-Gd(III) complex adduct, the observed re-laxation enhancement may be envisaged as the sum oftwo contributions as sketched in Fig. 8: (a) one arisingfrom water molecules surrounding the complex, whichbelong to the hydration layers of the protein and fromexchangeable protons on the protein which are in theproximity of the site of interaction of the paramagneticcomplex. This contribution is present also in systemscontaining qp0 [30, 31] and deals with water moleculesand protons which are involved in a relatively fast ex-change with the bulk water. Thus, this contribution de-creases with the increase of the temperature. The mag-nitude of this contribution depends chiefly on the char-acteristic features of the binding interaction (i.e.GdPCTP-[13] may be compared with GdDOTP be-cause in both cases the interaction with HSA is essen-tially electrostatic) and the electronic relaxation timeTiE, and (b) one arising from water molecule(s) directlycoordinated to the paramagnetic center. This contribu-tion may be evaluated by the SBM theory by assuminga value of D2 similar to that determined for the freecomplex. A common feature of all the systems so farconsidered is that this contribution appears“quenched” by the occurrence of a slow exchange rate.Thus, it is reasonable to assume that the interaction ofthe complex with the surface of the protein results in anoverall elongation of the residence lifetime of the coor-

dinated water. This behavior appears as a consequenceof the fact that the interaction with the protein involvesthe side containing the coordinated water, i.e. the nega-tively charged oxygens of phosphate (or carboxylatemoieties in related systems) play an active part in estab-lishing the binding with the protein through the forma-tion of hydrogen bonds with positively charged groups(Lys or His residues). This may introduce minor struc-tural and electronic changes in the coordination schemearound the Gd(III) ion which, as a net effect, lead tothe remarkable slowing down of the exchange rate ofthe coordinated water molecule.

This is also likely to occur when the driving force ofthe interaction is provided by hydrophobic substituentson the surface of the complex, owing to the high flexi-bility of the protein structure which may rearrange sothat these interactions will be more easily set up.

Acknowledgements Support from Bracco S.p.A. (Milan, Italy) isgratefully acknowledged. This work has been carried out underthe EU-BIOMED II-MACE Project. We thank the EU-Largescale PARABIO Facility at the University of Firenze (Italy) forthe use of the FC-Relaxometer.

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