Bioconjugate Chem. 2004, 15, 881−889
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A Gadolinium(III) Complex of a Carboxylic-Phosphorus Acid Derivative of Diethylenetriamine Covalently Bound to Inulin, a Potential Macromolecular MRI Contrast Agent Petra Lebdusˇkova´,†,‡ Jan Kotek,†,‡ Petr Hermann,‡ Luce Vander Elst,§ Robert N. Muller,§ Ivan Lukesˇ,*,‡ and Joop A. Peters*,† Laboratory of Applied Organic Chemistry and Catalysis, Delft University of Technology, Julianalaan 136, 2628 BL Delft, The Netherlands, Department of Inorganic Chemistry, Charles University, Hlavova 2030, 12840, Prague, Czech Republic, and Department of Organic Chemistry, University of Mons-Hainaut, B-7000, Mons, Belgium. Received January 30, 2004; Revised Manuscript Received April 21, 2004
A novel conjugate of a polysaccharide and a Gd(III) chelate with potential as contrast agent for magnetic resonance imaging (MRI) was synthesized. The structure of the chelate was derived from H5DTPA by replacing the central pendant arm by a phosphinic acid functional group, which was covalently bound to the polysaccharide inulin. On the average, each monosaccharide unit of the inulin was attached to approximately one (0.9) chelate moiety. The average molecular weight is 23110 and the average number of Gd3+ ions per molecule is 24. The ligand binds the Gd3+ ion in an octadentate fashion via three nitrogen atoms, four carboxylate oxygen atoms, and one P-O oxygen atom, and its first coordination sphere is completed by a water molecule. This compound shows promising properties for application as a contrast agent for MRI thanks to a favorable residence lifetime of this water molecule (170 ns at 298 K), a relatively long rotational correlation time (866 ps at 298 K), and the presence of two water molecules in the second coordination sphere of the Gd3+ ion. Furthermore, its stability toward transmetalation with Zn(II) is as high as that of the clinically used [Gd(DTPA)(H2O)]2-.
INTRODUCTION
Paramagnetic metal complexes are widely used in clinical diagnostics as contrast agents for magnetic resonance imaging (MRI). They are used to enhance the contrast between normal and diseased tissue or to indicate blood flow and/or organ function (1, 2). Nowadays, more than 30% of the examinations are performed after administration of a contrast agent. Paramagnetic contrast agents may improve the image contrast by enhancing the relaxation rate of water proton nuclei in their surroundings. The most commonly used paramagnetic metal ion in these contrast agents is Gd3+, which is very efficient in enhancing relaxation rates by virtue of its seven unpaired electrons and its relatively long electronic relaxation time. The efficiency of an MRI contrast agent usually is expressed as the relaxivity, r1, which is the enhancement of the water proton relaxation rate in s-1 mM-1 per Gd3+ ion. The relaxivity is governed mainly by the residence lifetime of water molecules coordinated in the first coordination sphere of the central metal ion, τM, by the complex reorientation time, τR, and by the longitudinal and transversal electronic relaxation times, τs1, τs2 (1, 3, 4). Currently applied low molecular weight complexes, such as Gd3+ complexes of 1,1,4,7,7-diethylenetriaminepentaacetic acid (H5DTPA) or 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (H4DOTA), have their relaxivity limited by fast rotation * Corresponding authors. J.A.P.: telephone: (+31)15 27 85892; fax: (+31)15 27 84289; e-mail:
[email protected]. I.L.: telephone: (+420)221952357; fax: (+420)221952378; e-mail:
[email protected]. † Delft University of Technology. ‡ Charles University. § University of Mons-Hainaut.
and, therefore, a short rotational correlation time, τR. New generations of contrast agents with improved relaxivity and specificity are desired. There are possibilities to enhance the relaxivity by increasing the τR value. This can be achieved by, for example, covalent or noncovalent binding of a low molecular weight chelate to macromolecules (3) or by self-assembly of monomeric complexes into micelles or particulates (5). The residence lifetime of the inner-sphere water molecule, τM, is a very important parameter in relation to the relaxivity of high molecular weight MRI contrast agents. Theory predicts that its optimal value is in the range of 20-50 ns at 298 K (3). The currently used contrast agents, such as the above-mentioned [Gd(DTPA)(H2O)]2- and [Gd(DOTA)(H2O)]-, have τM values of 303 and 244 ns at 298 K, respectively, which is far from the optimum (6). Upon conjugation of these complexes to a macromolecule, the relaxivities may therefore be limited by this parameter. The water exchange rate can be enhanced by, for example, introducing negative charge (7) in the neighborhood of the Gd3+ bound water or by increasing its steric strain, which favors the dissociative mechanism of the water exchange (8-12). Previously, we reported on the low-molecular weight complex [Gd(DT4APPh)(H2O)]2- (see Chart 1), which was designed on the basis of these ideas (11). It appeared to have a τM value of 92 ns, which is three times smaller than the currently used contrast agent [Gd(DTPA)(H2O)]2-. Furthermore, this complex has two second-sphere water molecules, which are contributing to the overall relaxivity. Here, we report on the synthesis of an analogous chelate (8) and its conjugate with the polysaccharide inulin (L, Chart 1). The relaxivity of both the parent chelate (8) and its conjugate and the parameters governing it are evaluated.
10.1021/bc049966g CCC: $27.50 © 2004 American Chemical Society Published on Web 05/27/2004
882 Bioconjugate Chem., Vol. 15, No. 4, 2004 Chart 1. Molecular Structures of Ligands Discussed
EXPERIMENTAL PROCEDURES
Materials. Commercially available diethylenetriamine, phthalanhydride, ethyl chloroformate, and ethyl bromoacetate had synthetic purity and were used as received. Arsenazo III used for free-gadolinium tests was purchased from Aldrich Chemical Co. (Milwaukee, WI). The 10% Pd/C-catalyst for the hydrogenation reactions was obtained from Acros (Geel, Belgium). Inulin was obtained from Sensus Coo¨peratie Cosun U.A. (Roosendaal, The Netherlands) and had an average degree of polymerization (dp) of 25. This “long chain” fraction was obtained by recrystalization of inulin from water, thereby selectively removing the low-molecular-weight components. Paraformaldehyde was obtained by filtration of aged aqueous formaldehyde solutions and was dried in a desiccator over concentrated sulfuric acid. p-Nitrobenzyl phosphinic acid was prepared by alkylation of P(CH(OEt)2)(OSiMe3)(OEt) with p-nitrobenzyl bromide followed by hydrolysis in aqueous HCl according to a procedure published elsewhere (13). NMR Spectroscopy. 1H (300 MHz), 13C (75.5 MHz), 17 O (40.7 MHz), and 31P NMR spectra (121.5 MHz) were recorded on a Varian INOVA-300 spectrometer using 5 mm sample tubes. Unless stated otherwise, NMR experiments were performed at 25 °C. Chemical shifts δ are given in ppm. For measurements in D2O, tert-butyl alcohol was used as an internal standard with the methyl signal referenced to 1.2 ppm (1H) or 31.2 ppm (13C). Deuterium oxide (100%) was used as an external chemical shift reference for 17O resonances. The 31P chemical shifts were measured with respect to 1% H3PO4 in D2O as an external standard (substitution method). The pH values of the samples were measured at ambient temperature using a Corning 125 pH meter with a calibrated microcombination electrode purchased from Aldrich Chemical Co. The pH values of the solutions were adjusted using dilute aqueous solutions of NaOH and HCl. Mass Spectrometry. Mass spectra were obtained on a Q-tof 2 mass spectrometer (Micromass, Manchester, UK). Samples were dissolved in H2O and injected at a rate of 5 µL/min. The cone voltage was 170 V (T ) 80 °C); 850 spectra were accumulated. The reported mass corresponds to the most abundant isotopic peak. Bis(phthaloyl)diethylenetriamine (1). This compound was synthesized according to a known procedure
Lebdusˇkova´ et al.
(14). 1H NMR (DMSO-d6), δ 2.76 and 3.59 (t, 3JHH ) 6.3 Hz, 2 × 4H, CH2), 7.80 (m, 8H, arom). 13C NMR (DMSOd6), δ 37.14 and 46.10 (NCH2CH2N), 122.70, 131.59 and 134.07 (arom), 167.81 (CO). Ethyl p-Nitrobenzylphosphinate (3). An excess of pyridine (3.45 g, 43.6 mmol) was slowly dropped to a suspension of p-nitrobenzylphosphinic acid (2) (13) (5.00 g, 24.9 mmol) and ethylchloroformate (3.47 g, 32.0 mmol) in CHCl3 (200 mL) during a period of 2 h. The mixture obtained was stirred for 42 h at room temperature and then washed with water (3 × 150 mL). The organic layer was dried over Na2SO4 after which the solvents were evaporated off. The excess of chloroformate was removed by codistillation with toluene (2 × 50 mL) and dry ethanol (2 × 50 mL) to yield 5.25 g (92%) of 3 as a light yellow oil. 31P NMR (CDCl3), δ 33.39 (doublet of multiplets, 1JPH ) 551 Hz). 13C NMR (CDCl3), δ 147.08 (5JCP ) 4.4 Hz, C-NO2), 138.28 (2JCP ) 8.2 Hz, Carom-CH2), 130.80 (3JCP ) 6.0 Hz, arom), 123.83 (4JCP ) 2.7 Hz, arom), 63.10 (2JCP ) 7.1 Hz, OCH2), 36.91 (1JCP ) 86.3 Hz, CH2P), 16.21 (3JCP ) 6.0 Hz). 1H NMR (CDCl3) δ 8.2 (AA′BB′ system, 4H, arom), 7.20 (1JPH ) 554 Hz, 1H, PH), 4.15 (m, 2H, OCH2), 3.42 (1JHP ) 19 Hz, 2H, CH2P), 1.33 (m, 3H, CH3). Ethyl N,N′′-Bis(phthaloyl)diethylenetriamine-N′methylene(p-nitrobenzyl)phosphinate (4). Freshly prepared ethyl p-nitrobenzylphosphinate (3) (5.25 g, 22.9 mmol) was dissolved in a mixture of 100 mL of toluene and 30 mL of dry ethanol. Bis(phthaloyl)diethylenetriamine (1) (5.00 g, 13.8 mmol) was added, and the mixture was heated to reflux under a Dean-Stark trap. Paraformaldehyde (0.41 g, 13.8 mmol) was added portionwise during 1.5 h. After the addition was completed, the reaction mixture was refluxed for another 1.5 h. The solvent in the trap was removed, and a second portion of paraformaldehyde (0.41 g) was added. The mixture was then heated for 18 h at 100 °C. The excess of ethyl p-nitrobenzylphosphinate and the byproduct ethyl hydroxomethyl(p-nitrobenzyl)phosphinate were not removed, and the crude product was used in the next step without purification. 13C NMR (CDCl3), δ 16.98 (CH3), 35.23 (1JCP ) 86 Hz, O2NC6H4CH2P), 35.80 (NCH2CH2NPht), 53.05 (NCH2P, 1JCP ) 87); 53.63 (NCH2CH2NPht), 61.87 (POCH2), 123.70, 131.19 and 134.53 (all Pht), 123.75, 132.53, 139.89, 147.27 (arom), 168.66 (CO). Diethylenetriamine-N′-methylene(p-nitrobenzyl)phosphinic-N,N,N′′,N′′-tetraacetic Acid (7). Hydrazine hydrate (2.00 g, 62.4 mmol) was added to a solution of crude 4 in dry ethanol (100 mL), and the mixture was refluxed for 6 h. After cooling, the precipitated phthalhydrazide was filtered off. The solvent was evaporated and water was removed by codistillation with dry ethanol. The crude product 5 was used in the next reaction step without further purification. All material obtained was dissolved in DMF (70 mL). BrCH2CO2Et (20.04 g, 0.12 mol) and K2CO3 (15.12 g, 0.11 mol) were added to the solution obtained. After being stirred at room temperature for 24 h, the solids were filtered off and the mixture was diluted with a saturated solution of NaHCO3 (70 mL). The product was extracted into toluene (150 mL). The organic layer was washed with a saturated solution of NaHCO3 (3 × 100 mL) and with brine (100 mL). The organic phase was dried (Na2SO4), filtered and evaporated until dryness to give crude 6. All material obtained above was dissolved in 200 mL of diluted HCl (1:1). After being refluxed for 21 h, the mixture was filtered and evaporated to dryness. The residue was dissolved in water and poured onto a column of cation exchanger (Dowex 50, 200 mL, H+-form). Nonbasic impurities were removed by elution with water
A New Potential Macromolecular MRI Contrast Agent
(∼1 L). The crude product was eluted using a diluted ammonia (1:3) solution. The orange fraction obtained was evaporated to dryness, dissolved in water, and poured onto a column of an anion exchanger (Dowex 1, 250 mL, OH--form). After being washed with water, some yellowcolored impurities were removed by elution with 10% acetic acid. The product was collected by elution with aqueous HCl (1:3). The fractions containing the product were evaporated to dryness. Trituration with 300 mL of acetone afforded a light yellow solid, which was filtered off, washed with acetone, and dried under vacuum. The product was recrystallized by dissolution in water with a minimal amount of ammonia and acidification with HCl to pH ∼ 0. Overall yield (based on compound (1)): 5.61 g (70%). Elemental analysis showed that the compound contains a small amount of NaCl (calcd for 7‚2H2O, C20H33N4O14P, M ) 584.47) C 41.55 (41.10); H 5.63 (5.69); N 9.86 (9.59); Cl 1.01 (0.00). 31P NMR (D2O, pH ) 0.5), δ 36.85. 13C NMR (D2O, pH ) 0.5), δ 37.08 (d, 1JCP ) 86 Hz, O2NC6H4CH2P), 51.12 (PCH2NCH2), 51.65 (d, 1JCP ) 80 Hz, NCH2P), 52.27 (PCH2NCH2CH2N), 55.37 (NCH2CO), 124.05, 130.91 (d, 3JCP ) 8.0 Hz), 140.95 (d, 2JCP ) 8.2) and 146.66, (arom), 169.75 (CO). 1H NMR (D2O, pH ) 0.5), δ 3.03 (d, 2JHP ) 6.6 Hz, O2NC6H4CH2P), 3.18 and 3.67 (m, CH2 groups of skeleton and bearing phosphorus moiety), 4.01 (s, NCH2CO); 7.38 and 8.08 (arom). Diethylenetriamine-N′-methylene(p-aminobenzyl)phosphinic-N,N,N′′,N′′-tetraacetic Acid (8). Compound 7 (0.5 g, 0.86 mmol) was dissolved in water (10 mL) with addition of a small amount of a solid LiOH. Hydrogenation catalyst (0.05 g of 5% Pd/C) was added, and the mixture was stirred under a H2 atmosphere for 3 days. The reaction mixture was then filtered and purified by chromatography on a column of a strong anion exchanger (Dowex 1, AcO--form, 25 mL). After washing with water, the product was eluted with HCl (1:3). The fractions containing the product were evaporated to dryness. The product was triturated using dry ethanol, collected by filtration, and dried under vacuum. Yield: 0.68 g (92%) Elem. anal. (calcd for (8)‚12H2O‚HCl, C20H56ClN4O22P, M ) 771.16) C 30.98 (31.15); H 5.26 (7.32); N 7.27 (7.27); Cl 2.08 (4.60). 31P NMR (D2O, pH ) 0.5), δ 37.72. 13C NMR (D2O, pH ) 0.5): 36.80 (d, 1JCP ) 87, H2NC6H4CH2P); 51.23 (PCH2NCH2); 51.65 (d, 1JCP ) 90, NCH2P); 52.20 (PCH2NCH2CH2N); 55.50 (NCH2CO); 123.50, 128.73, 131.57 and 133.76 (arom); 169.96 (CO). 1H NMR (D O, pH ) 0.5), 3.05 (d, 2J 2 HP ) 6.9 Hz, H2NC6H4CH2P), 3.18 and 3.39 (m, CH2 groups of skeleton and bearing phosphorus moiety), 4.02 (s, NCH2CO); 7.34 (arom). Diethylenetriamine-N′-methylene(p-isothiocyanatobenzyl)phosphinic-N,N,N′′,N′′-tetraacetic Acid (9). Compound 8 (0.5 g, 0.65 mmol) was dissolved in water (5 mL), and the pH was adjusted to 2 by several drops of concentrated aqueous HCl. A solution of thiophosgene (80 mg in 3 mL CCl4, 0.68 mmol, 1.05 equiv) was added in one portion, and then the mixture was stirred vigorously at room temperature for 48 h. The water phase was separated off and evaporated to dryness. Yield: 0.51 g. 31P NMR (D O, pH ) 0.5), δ 36.16. 13C NMR (D O, pH ) 2 2 0.5), δ 36.85 (d, 1JCP ) 86 Hz, SCNC6H4CH2P), 49.38 (PCH2NCH2), 52.51 (PCH2NCH2CH2N), 52.55 (d, 1JCP ) 102 Hz, NCH2P), 57.29 (NCH2CO), 126.00, 131.14, 134.24 and 134.99 (arom), 170.52 (CO); SCN was not observed, probably due to its very slow relaxation. O-Cyanoethylinulin (11) (15). Inulin (10) (dp 25, 5 g, 31 mmol of sugar units) was dissolved together with 0.26 g of NaOH (6.5 mmol) in water (8.9 mL, 0.49 mmol) by stirring at 45 °C. Then, acrylonitrile (3.28 g, 62 mmol)
Bioconjugate Chem., Vol. 15, No. 4, 2004 883
was added, and the mixture was stirred for 1 h at 45 °C. The mixture was diluted with water (20 mL) and neutralized by addition of 30% HCl (1 mL), followed by small amounts of NaHCO3 (until CO2 bubbles were observable). The reaction mixture was evaporated to dryness, and the residue was dissolved in 50% ethanol (100 mL). Salts were removed by membrane filtration (20 bar, UTC 60 membrane, Torey Industies Inc., Tokyo, Japan). The product was freeze-dried, and integration of the 1H NMR spectrum showed the average degree of substitution to be 1.13. Yield: 5.70 g. 1H NMR (D2O), δ 2.6-2.9 (19% of overall intensity, CH2CN); 3.5-4.4 (81% of overall intensity, remaining protons). O-Aminopropylinulin (12) (16). O-Cyanoethylinulin (11) (1.00 g, 4.5 mmol) was dissolved in a mixture of liquid ammonia (100 mL) and dry ethanol (50 mL). Metallic lithium (0.53 g, 77 mmol) was added portionwise during 30 min at -50 °C. A deep blue color was visible for several seconds after each addition of the metal. The mixture was refluxed for 1 h. Then the ammonia was evaporated off over 3 h using a cold water bath, and the resulting mixture was diluted with water (50 mL). A stream of CO2 was bubbled through the suspension to neutralize LiOH. The mixture was filtered and evaporated to dryness. The product was extracted with water (150 mL). Salts were removed by membrane filtration (UTC 60 membrane), and the filtrate was freeze-dried. The degree of substitution was determined by integration of the 1H NMR spectrum to be 0.99. Yield: 0.88 g. 1H NMR (D2O, pH ) 10.5), δ 1.5-2.0 (15% of overall intensity, CH2CH2NH2), 2.6-2.9 and 2.9-3.1 (15% of overall intensity, CH2CH2NH2); 3.4-4.3 (70% of overall intensity, remaining protons). 1H NMR (D2O, pH ) 0), δ 1.5-2.0 1.65-1.85 (17% of overall intensity, CH2CH2NH2); 2.8-3.0 (16% overall intensity, CH2CH2NH2), 3.34.2 (67% of overall intensity, remaining protons). ESIMS: m/z: 5676 (the mean value cossponds to dp ∼ 26 with ds ∼ 0.99). For MS data see Supporting Information (Figure S1). Conjugate of Ligand 9 and O-Aminopropylinulin, L. O-Aminopropylinulin (12) (0.203 g, 0.93 mmol amino groups) was dissolved in water (7 mL), and the pH was adjusted to 9-10 with diluted KOH. Compound 9 (freshly prepared from 1.0 g of 8, 1.30 mmol, 1.4 equiv) was added, and the reaction mixture was stirred at room temperature for 24 h. The reaction mixture was purified by gel chromatography (Sephadex G-25, 110 × 2 cm) using distilled water for elution. Four fractions of 100 mL were collected. The first two of them contained only inorganic salts and low molecular weight compounds and were discarded. The last two fractions containing the macromolecular material were freeze-dried. Yield: 0.91 g. 31P NMR (D2O), δ 34.18. 1H NMR (D2O), δ 2.0-2.2 (CH2CH2NH2), 2.8-3.0 (CH2CH2NH2), 3.1-3.3 (NCH2CH2N), 3.3-3.6 (NCH2COOH), 3.7-4.2 (inulin H), 7.43 and 7.54 (arom). Comparison of the integrals of the aromatic protons and those of the sugar protons indicated that the degree of substitution was 0.86. Gd(III) Complex of L, GdxL. In following text, for clarity, the abbreviation GdxL will be used for the final conjugate complex without specification of the number of metal ions per inulin molecule, the number of coordinated water molecules, and the charge. Ethylenediaminetetraacetic acid (H4EDTA) (0.29 g, 1 mmol) was suspended in water (3 mL). Concentrated aqueous ammonia was added dropwise until all H4EDTA was dissolved. Then, a solution of 0.22 g of GdCl3‚6H2O (0.59 mmol) in water (3 mL) was added, and the reaction mixture was stirred at room temperature. After 30 min,
884 Bioconjugate Chem., Vol. 15, No. 4, 2004
all Gd(III) was complexed (as checked with Arsenazo III). A solution of L (0.10 g in 2 mL of water) was added, and the reaction mixture was stirred at room temperature for 36 h. The pH was not adjusted, and it had a value of 5.0. The solution was then dialyzed (cellulose membrane, Sigma, retains mol. wt > 12 000). The water used in the dialysis was checked for the presence of Gd(III) by measuring its 17O NMR shift. When the chemical shift of the water signal remained constant, the dialysis was terminated. The final solution was once again checked for the presence of free Gd(III) with Arsenazo III, and no free Gd(III) was detected. The solution of the GdxL complex was freeze-dried. Yield: 0.11 g of white hygroscopic solid. ESI-MS: m/z: 23110 (the mean value corresponds to dp ∼ 27 with ds ∼ 0.86 (number of Gd(III) complexes per sugar unit)). For the MS data see Supporting Information (Figure S1). Variable Temperature 17O NMR Study of the GdxL. A sample of the Gd(III) complex of L was prepared by dissolution of 0.0486 g of GdxL in 0.3621 g of deionized water. Then, 64 mg of 17O enriched water (10% H217O) was added, and the pH was determined to be 5.79. The exact concentration of Gd(III) was determined to be 82 mM from the bulk magnetic susceptibility shift, which was determined using the procedure described previously (17). All NMR spectra were acquired without frequency lock. To correct the 17O NMR shift for a contribution of the BMS, the difference between chemical shifts of proton signals of tert-butyl alcohol in the paramagnetic sample and in pure water was applied according to the procedure described previously (18). Longitudinal (1/T1) and transversal (1/T2) relaxation rates were obtained by the inversion recovery method (19) and the Carr-PurcellMeiboom-Gil pulse sequence (20), respectively. Measurement of 1/T1 1H NMRD Profiles. The sample was prepared by dissolution of the freeze-dried complex GdxL in the appropriate amount of deionized water. The pH value was 7.11 and was not further adjusted. The exact concentration of Gd(III) was determined using the BMS method (17) and from proton relaxivity measurements at 20 MHz and 37 °C after complete hydrolysis in 25% HNO3. The 1/T1 NMRD profiles were measured at 5, 25, and 37 °C at magnetic field strengths between 4.7‚10-4 T and 0.35 T using a Stelar SpinMaster FFC-2000 relaxometer. Measurements at 0.47, 1.42, and 7.05 T were performed on a Bruker Minispec PC-20 and Mq-60 and on a Bruker AMX-300 spectrometer and were included in the profiles. The experimental data were fitted simultaneously with 17O NMR data employing a least-squares fitting procedure using the Micromath Scientist program version 2.0 (Salt Lake City, UT) as described previously (21, 22). Transmetalation. The stability of Gd(III) complex was determined using a transmetalation experiment with ZnCl2 (23). This measurement was done using a buffered solution (phosphate buffer, total concentration 67 mM, pH 7) containing 2.5 mM of GdxL and 2.5 mM of ZnCl2. The transmetalation was followed via the 1H longitudinal relaxation rates of the water using the Bruker Minispec 20 MHz at 37 °C. RESULTS AND DISCUSSION
Synthesis of the Conjugate of the Chelate and Its Gd(III) Complex. The conjugate of inulin and the carboxylic-phosphorus acid derivative of diethylenetriamine (8) was synthesized following the route outlined in Scheme 1. Inulin was chosen as a carrier for the
Lebdusˇkova´ et al.
chelating ligand, because its high flexibility allows the preparation of highly functionalized derivatives suitable for conjugation. In the present case the inulin was functionalized with O-aminopropyl groups by cyanoethylation of inulin with acrylonitrile and base, followed by reduction of the cyano groups with metallic Li in liquid ammonia/methanol at low temperature (15, 16). A degree of substitution (ds, defined as the average number of substituents per monosaccharide unit; the theoretical maximum ds is three corresponding to three free hydroxy groups per sugar unit) of 1.0 was obtained. Previous studies have indicated that the cyanoethyl groups in O-cyanoethyl inulin are distributed uniformly along the inulin chain and that within each fructose unit, the 4-position is the most reactive toward cyanoethylation (15). The Gd(III) chelating ligand (8) was prepared starting from p-nitrobenzylphosphinic acid (2). Its ethyl ester (3) was obtained by esterification using chloroformate and base, employing conditions published for phenylphosphinate (24). The phosphinic ester was attached to phthaloyl-protected diethylenetriamine by a Mannich-type reaction (11) to give intermediate 4. Removal of the phthaloyl protective groups by reaction with hydrazine hydrate followed by alkylation with ethyl bromoacetate and hydrolysis of the ester groups gave nitro derivative 7. The p-amino derivative 8 was obtained by hydrogenation with Pd/C as the catalyst. The latter compound was linked to O-aminopropylinulin (12) by means of a reaction with isothiocyanate to give macromolecular ligand L. The ds obtained was 0.86. Attempts to prepare the Gd(III) complex by mixing aqueous solutions of the ligand and GdCl3 followed by adjusting the pH failed. A precipitate was formed, which did not dissolve even after lowering the pH (4-5) and increasing the temperature to 50 °C. Therefore, the Gd(III) complex was prepared by a transmetalation reaction with NH4[Gd(EDTA)]. Removal of the excess of this complex, free H4EDTA, and salts by means of dialysis using a tubing with a cutoff of 12 000 Da afforded pure Gd(III) complex of L, GdxL. An ESI-mass spectrum showed that the average molecular weight is 23 110 Da, which means that the average dp is 27 and that on the average each molecule carries 24 Gd3+ ions. Evaluation of the Relaxivity and the Parameters Governing It. To assess the efficacy of the novel macromolecular contrast agent, GdxL, we measured the longitudinal relaxation rate enhancements of water protons as a function of the Larmor frequency induced by this complex. In Figure 1, the resulting NMRD profile at 300 K is compared with that of the previously reported Gd(III) complex of the corresponding unconjugated chelate, [Gd(DT4APPh)(H2O)]2- (11) and [Gd(DTPA)(H2O)]2- (6). The shape of the NMRD curve of the GdxL complex, particularly the maximum around 40 MHz, is characteristic for high-molecular-weight complexes and reflects the effect of the relatively low tumbling time of the complex (1, 3). Clinically, MRI exams are performed at a magnetic field between 0.5 and 2 T (corresponding with 1H Larmor frequencies of 20-85 MHz). The relaxivity of the novel contrast agent at these field strengths (20 s-1 mM-1 at 20 mHz and 37 °C) is considerably higher than that of the currently available contrast agents (
