Relaxometric and modelling studies of the binding of a lipophilic Gd-AAZTA complex to fatted and defatted human serum albumin

A new lipophilic gadolinium chelate consisting of a long aliphatic chain bound to the AAZTA coordination cage (Gd-AAZTAC17) has been synthesised. It possesses two coordinated water molecules (q=2) in fast exchange with the solvent (tau298(M) = 67 ns), which yields a relaxivity of 10.2 mM(-1) s(-1). At concentrations greater than 0.1 mM, it forms micelles (average diameter 5.5 nm) characterised by a relaxivity of approximately 30 mM(-1) s(-1) at 20 MHz and 298 K. The latter value appears to be "quenched" by magnetic interactions among the Gd(III) ions on the surface of the micelle that cause a decrease in the electronic relaxation time. A relaxivity of 41 mM(-1) s(-1) was recorded for this micellar system when 98 % of the Gd(III) ions were replaced by diamagnetic Y(III). Gd-AAZTAC17 exhibits a better affinity for fatted human serum albumin (HSA) than for defatted HSA, whereas the relaxivities of the supramolecular adducts are reversed. The relaxivity shown by Gd-AAZTAC17/defatted HSA ({r b(1) (20 MHz, 298 K)=84 mM(-1) s(-1)) is by far the highest relaxivity reported so far for non-covalent paramagnetic adducts with slow-moving substrates. As shown by molecular docking calculations, the gadolinium complex enters a hydrophobic pocket present in fatted HSA more extensively than the corresponding adduct with defatted HSA. Interestingly, no marked difference was observed in either the relaxation enhancement or the binding affinity between fatted and defatted HSA when the binding titrations were carried out at a Gd-AAZTAC17 concentration higher than its critical micellar concentration (cmc). This behaviour has been attributed to the formation of an association between the negatively charged micelle of the lipophilic metal complexes and the positive residues on the surface of the protein.     

Serum Albumin Targeted,pH‐Dependent Magnetic Resonance Relaxation Agents

The objective of this work was the synthesis of serum albumin targeted, Gd^III‐based magnetic resonance imaging (MRI) contrast agents exhibiting a strong pH‐dependent relaxivity. Two new complexes ( Gd‐glu and Gd‐bbu ) were synthesized based on the DO3A macrocycle modified with three carboxyalkyl substituents α to the three ring nitrogen atoms, and a biphenylsulfonamide arm. The sulfonamide nitrogen coordinates the Gd in a pH‐dependent fashion, resulting in a decrease in the hydration state, q, as pH is increased and a resultant decrease in relaxivity (r₁). In the absence of human serum albumin (HSA), r₁ increases from 2.0 to 6.0 mM ^?1 s^?1 for Gd‐glu and from 2.4 to 9.0 mM ^?1 s^?1 for Gd‐bbu from pH 5 to 8.5 at 37 °C, 0.47 T, respectively. These complexes (0.2 mM ) are bound (>98.9 %) to HSA (0.69 mM ) over the pH range 5–8.5. Binding to albumin increases the rotational correlation time and results in higher relaxivity. The r₁ increased 120 % (pH 5) and 550 % (pH 8.5) for Gd‐glu and 42 % (pH 5) and 260 % (pH 8.5) for Gd‐bbu . The increases in r₁ at pH 5 were unexpectedly low for a putative slow tumbling q=2 complex. The Gd‐bbu system was investigated further. At pH 5, it binds in a stepwise fashion to HSA with dissociation constants K_d1=0.65, K_d2=18, K_d3=1360 μM . The relaxivity at each binding site was constant. Luminescence lifetime titration experiments with the Eu^III analogue revealed that the inner‐sphere water ligands are displaced when the complex binds to HSA resulting in lower than expected r₁ at pH 5. Variable pH and temperature nuclear magnetic relaxation dispersion (NMRD) studies showed that the increased r₁ of the albumin‐bound q=0 complexes is due to the presence of a nearby water molecule with a long residency time (1–2 ns). The distance between this water molecule and the Gd ion changes with pH resulting in albumin‐bound pH‐dependent relaxivity.     

Hetero-tripodal hydroxypyridonate gadolinium complexes: syntheses, relaxometric properties, water exchange dynamics, and human serum albumin binding

The synthesis and relaxometric properties of hetero-tripodal hydroxypyridonate-terephthalamide gadolinium (Gd(3+)) chelates with differing structural features for probing human serum albumin (HSA) interactions are reported. The Gd(3+) complexes are divided into two series. The first series (3-5) features a benzyl derivative connected to the hydroxypyridonate (HOPO) moiety. The second series of complexes (6-10) has the common feature of a poly(ethylene glycol) (PEG) attached to the terephthalamide (TAM) moiety and is nonbenzylated. The water exchange of the complexes is in the fast exchange regime with rates (k(ex)) in the range 0.45-1.11 x 10(8) s(-1). The complexes have a moderate interaction with HSA with association constants (K(A)'s) in the range 0.7-8.6 x 10(3) M(-1). Protein binding results in an enhancement in proton relaxivity from 7.7-10.4 mM(-1) s(-1) (r(1p)) to 15-29 mM(-1) s(-1) (r(1p)(b)). It is concluded that the interaction of the complexes with HSA (i) is enhanced by the presence of benzyl groups, (ii) is entropically driven, and (iii) results in a lower hydration number (q).     

Synthesis neutral rare earth complexes of diethylenetriamine-N,N'-bis(acetyl-isoniazid)-N,N',N'-triacetic acid as potential contrast enhancement agents for magnetic resonance imaging

A novel ligand, diethylenetriamine-N,N'-bis(acetyl-isoniazid)-N,N',N'-triacetic acid (H(3)L) has been synthesized from diethylene triamine pentaacetic acid (DTPA) and isoniazid. Ligand and its five neutral rare earth (RE=La, Sm, Eu, Gd, Tb) complexes holding promise of magnetic resonance imaging (MRI) were characterized on the basis of elemental analysis, molar conductivity, (1)H-NMR spectrum, FAB-MS, TG-DTA analysis and IR spectrum. The relaxivity (R(1)) of complexes and Gd(DTPA)(2-) used as a control were determined. The relaxivity of LaL, SmL, EuL, GdL, TbL and Gd(DTPA)(2-) were 0.14, 1.66, 3.14, 6.08, 2.79 and 4.34 l.mmol(-1).s(-1), respectively. The spin-lattice relaxivity of GdL was larger than that of Gd(DTPA)(2-). The relaxivity of GdL had also been investigated in human serum albumin (HSA) solution, the relaxivity of GdL was enhanced from 6.08 l.mmol(-1).s(-1) in water solution to 9.09 l.mmol(-1).s(-1) in HSA solution. In addition, thermodynamics stability constant of GdL complex was determined, the thermodynamic stability constant of GdL complex (K(GdL)=10(20.84)) was a few larger than that of Gd(DTPA)(2-) (K(Gd-DTPA)=10(20.73)). The results showed that complex of GdL may be a prospective MRI contrast agent with low osmotic pressure due to non-ion complex, high spin-lattice relaxivity, good stability and binding affinity for the serum protein.     

When are Two Waters Worse Than One? Doubling the Hydration Number of a Gd–DTPA Derivative Decreases Relaxivity

The synthesis of a novel ligand, based on N-methyl-diethylenetriaminetetraacetate and containing a diphenylcyclohexyl serum albumin binding group (L1) is described and the coordination chemistry and biophysical properties of its Gd(III) complex Gd-L1 are reported. The Gd(III) complex of the diethylenetriaminepentaacetate analogue of the ligand described here (L2) is the MRI contrast agent MS-325. The effect of converting an acetate to a methyl group on metal-ligand stability, hydration number, water-exchange rate, relaxivity, and binding to the protein human serum albumin (HSA) is explored. The complex Gd-L1 has two coordinated water molecules in solution, that is, [Gd(L1)(H2O)2]2- as shown by D-band proton ENDOR spectroscopy and implied by 1H and 17O NMR relaxation rate measurements. The Gd-H(water) distance of the coordinated waters was found to be identical to that found for Gd-L2, 3.08 A. Loss of the acetate group destabilizes the Gd(III) complex by 1.7 log units (log K(ML) = 20.34) relative to the complex with L2. The affinity of Gd-L1 for HSA is essentially the same as that of Gd-L2. The water-exchange rate of the two coordinated waters on Gd-L1 (k(ex) = 4.4x10(5) s(-1)) is slowed by an order of magnitude relative to Gd-L2. As a result of this slow water-exchange rate, the observed proton relaxivity of Gd-L1 is much lower in a solution of HSA under physiological conditions (r1(obs) = 22.0 mM(-1) s(-1) for 0.1 mM Gd-L1 in 0.67 mM HSA, HEPES buffer, pH 7.4, 35 degrees C at 20 MHz) than that of Gd-L2 (r1(obs) = 41.5 mM(-1) s(-1)) measured under the same conditions. Despite having two exchangeable water molecules, slow water exchange limits the potential efficacy of Gd-L1 as an MRI contrast agent.     

Synthesis and characterization of lanthanide complexes of DO3A-alkylphosphonates

Eight DO3A-based lanthanide(III) complexes bearing ester protected and unprotected phosphonate groups at variable distances from the macrocyclic moiety have been synthesized and analyzed. The ligands were made by straightforward four-step synthetic procedures and purified with preparative RP-HPLC, after which they were used to prepare gadolinium(III) and europium(III) complexes. Relaxometric experiments were performed on the Gd(III) complexes at 300 MHz, varying the pH of the solutions or the concentration of human serum albumin (HSA). It was found that when the pH of the medium was changed from neutral to pH 4 the longitudinal relaxivity of GdDO3A-ethylphosphonate and GdDO3A-propylphosphonate complexes increased by 50% and 60%, respectively. Diethyl esters of these complexes did not change longitudinal relaxivity in the same pH range but their transverse relaxivity increased upon binding to HSA. 31P NMR experiments on Eu(III) complexes showed a change in the chemical shift of both acid complexes in the same region where the highest relaxivity changes were observed and proved the stability of the complexes in the investigated pH range, while no shift was observed for the diester complexes. Luminescence studies on europium(III) complexes additionally supported observations obtained by NMR methods. The change in the form of the luminescence emission spectra, and the reduction in the q value upon addition of HSA proved the ternary adduct formation between the charge neutral diester complexes and HSA. Similarly, the change in the emission spectra showing a phosphonate bound structure at pH 7 to a species where the phosphonate oxygen is not coordinated at pH 4 in parallel with the increase of q value is supporting the hypothesis that the deprotonation of phosphonates is the main reason for the distinct relaxivity change from slightly acidic to the neutral solution media.     

Synthesis and relaxivity studies of a tetranuclear gadolinium(III) complex of DO3A as a contrast-enhancing agent for MRI

A tetranuclear gadolinium(III) complex, [Gd4(H2O)8], of DO3A appended onto the pentaerythrityl framework was synthesized to improve the water proton relaxivity for MRI application. The longitudinal relaxivity of [Gd4(H2O)8] is 28.13 mM-1 s-1 (24 MHz, 35+/-0.1 degrees C, pH 5.6) which is 5.86 times higher than that of [Gd(DO3A)(H2O)2]. The relaxivity is based on "molecular" relaxivity of the tetramer and the r1p value is "7 per Gd". The high relaxivity of the tetramer is the result of the decrease in the rotational correlation (tauR) and the presence of eight inner-sphere water molecules (q=8). The complex exhibits pH-dependent longitudinal relaxivity, and the high relaxivity both at low and high pH (r1p=28.13 mM-1 s-1 at pH 5.6 and 16.52 mM-1 s-1 at pH 9.5) indicates that it could be used as a pH-responsive MRI contrast agent. The transverse relaxivity of the tetramer is 129.97 mM-1 s-1 (24 MHz, 35+/-0.1 degrees C, pH 5.6), and the r2p/r1p ratio of 4.6 shows that it could be used as a T2-weighted contrast agent.     

Albumin binding, relaxivity, and water exchange kinetics of the diastereoisomers of MS-325, a gadolinium(III)-based magnetic resonance angiography contrast agent

The amphiphilic gadolinium complex MS-325 ((trisodium-{(2-(R)-[(4,4-diphenylcyclohexyl) phosphonooxymethyl] diethylenetriaminepentaacetato) (aquo)gadolinium(III)}) is a contrast agent for magnetic resonance angiography (MRA). MS-325 consists of two slowly interconverting diastereoisomers, A and B (65:35 ratio), which can be isolated at pH > 8.5 (TyeklAr, Z.; Dunham, S. U.; Midelfort, K.; Scott, D. M.; Sajiki, H.; Ong, K.; Lauffer, R. B.; Caravan, P.; McMurry, T. J. Inorg. Chem. 2007, 46, 6621-6631). MS-325 binds to human serum albumin (HSA) in plasma resulting in an extended plasma half-life, retention of the agent within the blood compartment, and an increased relaxation rate of water protons in plasma. Under physiological conditions (37 degrees C, pH 7.4, phosphate buffered saline (PBS), 4.5% HSA, 0.05 mM complex), there is no statistical difference in HSA affinity or relaxivity between the two isomers (A 88.6 +/- 0.6% bound, r1 = 42.0 +/- 1.0 mM(-1) s(-1) at 20 MHz; B 90.2 +/- 0.6% bound, r1 = 38.3 +/- 1.0 mM(-1) s(-1) at 20 MHz; errors represent 1 standard deviation). At lower temperatures, isomer A has a higher relaxivity than isomer B. The water exchange rates in the absence of HSA at 298 K, kA298 = 5.9 +/- 2.8 x 10(6) s(-1), kB298 = 3.2 +/- 1.8 x 10(6) s(-1), and heats of activation, DeltaHA = 56 +/- 8 kJ/mol, DeltaHB = 59 +/- 11 kJ/mol, were determined by variable-temperature 17O NMR at 7.05 T. Proton nuclear magnetic relaxation dispersion (NMRD) profiles were recorded over the frequency range of 0.01-50 MHz at 5, 15, 25, and 35 degrees C in a 4.5% HSA in PBS solution for each isomer (0.1 mM). Differences in the relaxivity in HSA between the two isomers could be attributed to the differing water exchange rates.     

Synthesis, relaxometric and photophysical properties of a new pH-responsive MRI contrast agent: the effect of other ligating groups on dissociation of a p-nitrophenolic pendant arm

Two gadolinium(III) chelates, GdNP-DO3A (1-methlyene-(p-NitroPhenol)-1,4,7,10-tetraazacycloDOdecane-4,7,10-triAcetate) and GdNP-DO3AM (1-methlyene(p-NitroPhenol)-1,4,7,10-tetraazacycloDOdecane-4,7,10-triacetAMide), containing a single nitrophenolic pendant arm plus either three acetate or three amide pendant arms were synthesized and characterized. The properties of the gadolinium, terbium, and dysprosium complexes of these ligands were examined as a function of pH. The extent and mechanism of the changes in water relaxivity with pH of each gadolinium complex was found to differ substantially for the two complexes. The water relaxivity of Gd(NP-DO3A) increases from 4.1 mM(-1) s(-1) at pH 9 to 7.0 mM(-1) s(-1) at pH 5 as a result of acid-catalyzed dissociation of the nitrophenol from the lanthanide. The nitrophenol group in Gd(NP-DO3AM) does not dissociate from the metal center even at pH 5; therefore, the very modest increase in relaxivity in this complex must be ascribed to an increase in prototropic exchange rate of the bound water and/or phenolic protons.     

Synthesis and physicochemical characterization of two gadolinium(III) TTDA-like complexes and their interaction with human serum albumin

Two novel derivatives of TTDA (3,6,10-tri(carboxymethyl)-3,6,10-triazadodecanedioic acid), TTDA-BOM and TTDA-N'-BOM, each having a benzyloxymethyl group, were synthesized. (17)O NMR longitudinal and transverse relaxation rates and chemical shifts of aqueous solutions of their Gd(III) complexes were measured at variable temperature with a magnetic field strength of 9.4 T. The water exchange rate (k(ex)(298)) values for [Gd(TTDA-BOM)(H(2)O)](2-) (117 x 10(6) s(-1)) and [Gd(TTDA-N'-BOM)(H(2)O)](2-) (131 x 10(6) s(-1)) are significantly higher than those of [Gd(DTPA)(H(2)O)](2-) (4.1 x 10(6) s(-1)) and [Gd(BOPTA)(H(2)O)](2-) (3.45 x 10(6) s(-1)). The rotational correlation time (tau) values for [Gd(TTDA-BOM)(H(2)O)](2-) (119 ps) and [Gd(TTDA-N'-BOM)(H(2)O)](2-) (125 ps) are higher than those of [Gd(DTPA)(H(2)O)](2-) (103 ps) and [Gd(TTDA)(H(2)O)](2-) (104 ps). The stepwise stoichiometric binding constants of [Gd(TTDA-BOM)(H(2)O)](2)(-) and [Gd(TTDA-N'-BOM)(H(2)O)](2)(-) bound to HSA are obtained by ultrafiltration studies. Fluorescent probe displacement studies exhibit that [Gd(TTDA-BOM)(H(2)O)](2-) and [Gd(TTDA-N'-BOM)(H(2)O)](2-) can displace dansylsarcosine from HSA with inhibition constants (K(i)) of 1900 and 1600 microM, respectively; however, they are not able to displace warfarin. These results indicate that [Gd(TTDA-BOM)(H(2)O)](2-) and [Gd(TTDA-N'-BOM)(H(2)O)](2-) have a weak binding to site II on HSA. In addition, the mean bound relaxivity (r(1b)) and bound relaxivity (r(1)(b)) values for the [Gd(TTDA-BOM)(H(2)O)](2-)/HSA and [Gd(TTDA-N'-BOM)(H(2)O)](2-)/HSA adducts are obtained by ultrafiltration and relaxivity studies, respectively. The bound relaxivity of these adducts values are significantly higher than those of [Gd(BOPTA)(H(2)O)](2-)/HSA and [Gd(DTPA-BOM(3))(H(2)O)](2-)/HSA. These results also suggest that bound relaxivity is site dependent. In binding sites studies of Gd(III) chelates to HSA, a significant decrease of the relaxation rates (R(1obs)) was observed for the [Eu(TTDA-BOM)(H(2)O)](2-) complex which was added to the [Gd(TTDA-N'-BOM)(H(2)O)](2-)/HSA solution, and this indicated that these Gd(III) complexes share the same HSA binding site. Finally, as measured by the Zn(II) transmetalation process, the kinetic stability of these Gd(III) complexes are significantly higher than that of [Gd(DTPA-BMA)(H(2)O)].     

Rotational dynamics account for pH-dependent relaxivities of PAMAM dendrimeric, Gd-based potential MRI contrast agents

The EPTPA5) chelate, which ensures fast water exchange in GdIII complexes, has been coupled to three different generations (5, 7, and 9) of polyamidoamine (PAMAM) dendrimers through benzylthiourea linkages (H5EPTPA = ethylenepropylenetriamine-N,N,N',N',N'-pentaacetic acid). The proton relaxivities measured at pH 7.4 for the dendrimer complexes G5-(GdEPTPA)111, G7-(GdEPTPA)253 and G9-(GdEPTPA)1157 decrease with increasing temperature, indicating that, for the first time for dendrimers, slow water exchange does not limit relaxivity. At a given field and temperature, the relaxivity increases from G5 to G7, and then slightly decreases for G9 (r1 = 20.5, 28.3 and 27.9 mM(-1) s(-1), respectively, at 37 degrees C, 30 MHz). The relaxivities show a strong and reversible pH dependency for all three dendrimer complexes. This originates from the pH-dependent rotational dynamics of the dendrimer skeleton, which was evidenced by a combined variable-temperature and multiple-field 17O NMR and 1H relaxivity study performed at pH 6.0 and 9.9 on G5-(GdEPTPA)111. The longitudinal 17O and 1H relaxation rates of the dendrimeric complex are strongly pH-dependent, whereas they are not for the [Gd(EPTPA)(H2O)]2- monomer chelate. The longitudinal 17O and 1H relaxation rates have been analysed by the Lipari-Szabo spectral density functions and correlation times have been calculated for the global motion of the entire macromolecule (tau(gO)) and the local motion of the GdIII chelates on the surface (tau(lO)), correlated by means of an order parameter S2. The dendrimer complex G5-(GdEPTPA)111 has a considerably higher tau(gO) under acidic than under basic conditions (tau(298)gO = 4040 ps and 2950 ps, respectively), while local motions are less influenced by pH (tau(298)lO = 150 and 125 ps). The order parameter, characterizing the rigidity of the macromolecule, is also higher at pH 6.0 than at pH 9.9 (S2 = 0.43 vs 0.36, respectively). The pH dependence of the global correlation time can be related to the protonation of the tertiary amine groups in the PAMAM skeleton, which leads to an expanded and more rigid dendrimeric structure at lower pH. The increase of tau(gO) with decreasing pH is responsible for the pH dependent proton relaxivities. The water exchange rate on G5-(GdEPTPA)111(k(298)ex = 150 x 10(6) s(-1)) shows no significant pH dependency and is similar to the one measured for the monomer [Gd(EPTPA)(H2O)]2-. The proton relaxivity of G5-(GdEPTPA)111 is mainly limited by the important flexibility of the dendrimer structure, and to a small extent, by a faster than optimal water exchange rate.     

Synthesis and evaluation of a high relaxivity manganese(II)-based MRI contrast agent

The manganese(II) ion has many favorable properties that lead to its potential use as an MRI contrast agent: high spin number, long electronic relaxation time, labile water exchange. The present work describes the design, synthesis, and evaluation of a novel Mn(II) complex (MnL1) based on EDTA and also contains a moiety that noncovalently binds the complex to serum albumin, the same moiety used in the gadolinium based contrast agent MS-325. Ultrafiltration albumin binding measurements (0.1 mM, pH 7.4, 37 degrees C) indicated that the complex binds well to plasma proteins (rabbit: 96 +/- 2% bound, human: 93 +/- 2% bound), and most likely to serum albumin (rabbit: 89 +/- 2% bound, human 98 +/- 2% bound). Observed relaxivities (+/- 5%) of the complex were measured (20 MHz, 37 degrees C, 0.1 mM, pH 7.4) in HEPES buffer (r(1) = 5.8 mM(-)(1) s(-)(1)), rabbit plasma (r(1) = 51 mM(-)(1) s(-)(1)), human plasma (r(1) = 46 mM(-)(1) s(-)(1)), 4.5% rabbit serum albumin (r(1) = 47 mM(-)(1) s(-)(1)), and 4.5% human serum albumin (r(1) = 48 mM(-)(1) s(-)(1)). The water exchange rate was near optimal for an MRI contrast agent (k(298) = 2.3 +/- 0.9 x 10(8) s(-)(1)). Variable temperature NMRD profiles indicated that the high relaxivity was due to slow tumbling of the albumin-bound complex and fast exchange of the inner sphere water. The concept of a high relaxivity Mn(II)-based contrast agent was validated by imaging at 1.5 T. In a rabbit model of carotid artery injury, MnL1 clearly delineated both arteries and veins while also distinguishing between healthy tissue and regions of vessel damage.     

A Gd3+-based magnetic resonance imaging contrast agent sensitive to beta-galactosidase activity utilizing a receptor-induced magnetization enhancement (RIME) phenomenon

Magnetic resonance imaging (MRI) permits noninvasive three-dimensional imaging of opaque organisms. Gadolinium (Gd(3+)) complexes have become important imaging tools as MRI contrast agents for MRI studies, though most of them are nonspecific and report solely on anatomy. Recently, MRI contrast agents have been reported whose ability to relax water protons is triggered or greatly enhanced by recognition of a particular biomolecule. This new class of MRI contrast agents could open up the possibility of reporting on the physiological state or metabolic activity deep within living specimens. One possible strategy for this purpose is to utilize the increase in the longitudinal water proton r(1) relaxivity that occurs upon slowing the molecular rotation of a small paramagnetic complex, a phenomenon which is known as receptor-induced magnetization enhancement (RIME), by either binding to a macromolecule or polymerization of the agent itself. Here we describe the design and synthesis of a novel beta-galactosidase-activated MRI contrast agent, the Gd(3+) complex [Gd-5], by using the RIME approach. beta-Galactosidase is commonly used as a marker gene to monitor gene expression. This newly synthesized compound exhibited a 57% increase in the r(1) relaxivity in phosphate-buffered saline (PBS) with 4.5% w/v human serum albumin (HSA) in the presence of beta-galactosidase. Detailed investigations revealed that RIME is the dominant factor in this increase of the observed r(1) relaxivity, based on analysis of Gd(3+) complexes [Gd-5] and [Gd-8], which is generated from [Gd-5] by the activity of beta-galactosidase, and spectroscopic analysis of their corresponding Tb(3+) complexes, [Tb-5] and [Tb-8].     

Protein binding to lanthanide(III) complexes can reduce the water exchange rate at the lanthanide

The GdIII-based magnetic resonance imaging contrast agent MS-325 targets the blood protein serum albumin, resulting in an increased efficacy (relaxivity) as a relaxation agent. MS-325 showed different relaxivities when bound to serum albumin from different species, e.g., r1=30.5 mM-1 s-1 (rabbit) vs 46.3 mM-1 s-1 (human) at 35 degrees C and 0.47 T. To investigate the mechanism for this difference, surrogate complexes were prepared where the GdIII ion was replaced by other LnIII ions. Fluorescence lifetime measurements of the EuIII analogue indicated that the hydration number was q=1 and did not change when bound to either human, rat, rabbit, pig, or dog serum albumin. The YbIII analogue, YbL1, was prepared and characterized by 1H NMR. Line-shape analysis of the paramagnetic-shifted 1H NMR resonances in the presence of increasing amounts of human (HSA) or rabbit (RSA) serum albumin allowed estimation of the transverse relaxation rate, R2, of these resonances for the protein-bound YbL1. The rotational correlation time of YbL1 was calculated from R2, and the Yb-H distance and was tauR=8+/-1 ns when bound to HSA and 13+/-2 ns when bound to RSA. The water exchange rate at the DyIII analogue, DyL1, was determined from variable-temperature R2 measurements at 9.4 T when DyL1 was bound to either HSA or RSA. At 37 degrees C, water exchange at DyL1 was (31+/-5)x10(6) s-1 when bound to HSA but (3.8+/-0.2)x10(6) s-1 when bound to RSA. Slower water exchange upon RSA binding explains the differences in relaxivity observed. The approach of using surrogate lanthanides to identify specific molecular parameters influencing relaxivity is applicable to other protein-targeted GdIII contrast agents.     

Highly relaxing gadolinium based MRI contrast agents responsive to Mg2+ sensing

A Gd complex based on a polyphosphonated pyridyl ligand shows a very high stability in aqueous solution (log?K(EuL) = 25.7), a high relaxivity (8.5 mM(-1) s(-1) at 25 °C and 20 MHz) and a marked and selective relaxivity enhancement (37%) in the presence of Mg(2+), opening interesting perspectives for the design of cation responsive contrast agents.     

A self-assembled complex with a titanium(IV) catecholate core as a potential bimodal contrast agent

A ditopic chelating ligand (H(6)4) that bears catechol and diethylenetriamine-N,N,N',N',N'-pentaacetate (DTPA) has been designed and shown to specifically bind lanthanide(III) ions at the DTPA core ([Ln(H(2)4)(H(2)O)](-)) and further self-assemble with titanium(IV), thereby giving rise to the formation of a supramolecular metallostar complex with a lanthanide(III)-to-titanium(IV) ratio of 3:1, [(Ln4)(3)Ti(H(2)O)(3)](5-) (Ln=La, Eu, Gd). The efficacy of the metallostar complex as a potential bimodal optical/magnetic resonance imaging (MRI) agent has been evaluated. Nuclear magnetic relaxation dispersion (NMRD) measurements for the [(Gd4)(3)Ti(H(2)O)(3)](5-) complex have demonstrated an enhanced r(1) relaxivity that corresponds to 36.9 s(-1) mM(-1) per metallostar molecule at 20 MHz and 310 K, which is a result of a decreased tumbling rate. The ability of the complex to bind to human serum albumin (HSA) was also examined by relaxometric measurements. In addition, upon UV irradiation the [(Gd4)(3)Ti(H(2)O)(3)](5-) complex exhibits broad-band green emission in the range 400-750 nm with a maximum at 490 nm. Taking into account the high relaxivity and luminescence properties, the [(Gd4)(3)Ti(H(2)O)(3)](5-) complex is a good lead compound for the development of efficient bimodal contrast agents.     

Lanthanide(III) complexes that contain a self-immolative arm: potential enzyme responsive contrast agents for magnetic resonance imaging

Enzyme-responsive MRI-contrast agents containing a "self-immolative" benzylcarbamate moiety that links the MRI-reporter lanthanide complex to a specific enzyme substrate have been developed. The enzymatic cleavage initiates an electronic cascade reaction that leads to a structural change in the Ln(III) complex, with a concomitant response in its MRI-contrast-enhancing properties. We synthesized and investigated a series of Gd(3+) and Yb(3+) complexes, including those bearing a self-immolative arm and a sugar unit as selective substrates for β-galactosidase; we synthesized complex LnL(1), its NH(2) amine derivatives formed after enzymatic cleavage, LnL(2), and two model compounds, LnL(3) and LnL(4). All of the Gd(3+) complexes synthesized have a single inner-sphere water molecule. The relaxivity change upon enzymatic cleavage is limited (3.68 vs. 3.15 mM(-1) s(-1) for complexes GdL(1) and GdL(2), respectively; 37 °C, 60 MHz), which prevents application of this system as an enzyme-responsive T(1) relaxation agent. Variable-temperature (17)O NMR spectroscopy and (1)H NMRD (nuclear magnetic relaxation dispersion) analysis were used to assess the parameters that determine proton relaxivity for the Gd(3+) complexes, including the water-exchange rate (k(ex)(298), varies in the range 1.5-3.9×10(6) s(-1)). Following the enzymatic reaction, the chelates contain an exocyclic amine that is not protonated at physiological pH, as deduced from pH-potentiometric measurements (log K(H)=5.12(±0.01) and 5.99(±0.01) for GdL(2) and GdL(3), respectively). The Yb(3+) analogues show a PARACEST effect after enzymatic cleavage that can be exploited for the specific detection of enzymatic activity. The proton-exchange rates were determined at various pH values for the amine derivatives by using the dependency of the CEST effect on concentration, saturation time, and saturation power. A concentration-independent analysis of the saturation-power-dependency data was also applied. All these different methods showed that the exchange rate of the amine protons of the Yb(III) complexes decreases with increasing pH value (for YbL(3), k(ex)=1300 s(-1) at pH 8.4 vs. 6000 s(-1) at pH 6.4), thereby resulting in a diminution of the observed CEST effect.     

Designing Novel Contrast Agents for Magnetic Resonance Imaging. Synthesis and Relaxometric Characterization of three Gadolinium(III) Complexes Based on Functionalized Pyridine‐Containing Macrocyclic Ligands

The three novel pyridine‐containing 12‐membered macrocyclic ligands 1 – 3 were synthesized. The coordinating arms are represented by three acetate moieties in 1 and 3 and by one acetate and two phosphonate moieties in 2 . In all three ligands, the acetate arm in the distal position is substituted, with a benzyl group in 1 and 2 and with an arylmethyl moiety in 3 . The relaxivities r_1p (20 MHz, 25°) of Gd^III complexes are: GD?1 , r_1p=8.3 mM ^?1 s^?1; GD?2 , r_1p8.1 mM ^?1 s^?1; Gd?3 , r_1p10.5 mM ^?1 s^?1. ¹H‐NMRD and ¹⁷O‐NMR T₂ data show that Gd?1 and Gd?3 contain two H₂O molecules in the inner sphere, whereas the presence of two phosphonate arms allows the coordination of only one H₂O molecule in Gd?2 . Interestingly, the exchange lifetime of coordinated H₂O in the three complexes is similar in spite of the difference in the coordination number of the Gd^III ion (i.e., 9 in Gd?1 and Gd?3 , and 8 in Gd?2 ). ¹H‐Relaxometric measurements at different pH and in the presence of lactate and oxalate were carried out to get some insight into the formation of ternary complexes from Gd?1 and Gd?3 . Finally, it was found that binding to human‐serum albumin (HSA) of Gd?1 and Gd?2 , though weak, yields limited relaxivity enhancements, likely as a consequence of effects on the hydration sphere caused by donor atoms on the surface of the protein.     

A new metallostar complex based on an aluminum(iii) 8-hydroxyquinoline core as a potential bimodal contrast agent

A ditopic DTPA monoamide derivative containing an 8-hydroxyquinoline moiety was synthesized and the corresponding gadolinium(iii) complex ([Gd(H5)(H(2)O)](-)) was prepared. After adding aluminum(iii), the 8-hydroxyquinoline part self-assembled into a heteropolymetallic triscomplex [(Gd5)(3)Al(H(2)O)(3)](3-). The magnetic and optical properties of this metallostar compound were investigated in order to classify it as a potential in vitro bimodal contrast agent. The proton nuclear magnetic relaxation dispersion measurements indicated that the relaxivity r(1) of [Gd(H5)(H(2)O)](-) and [(Gd5)(3)Al(H(2)O)(3)](3-) at 20 MHz and 310 K equaled 6.17 s(-1) mM(-1) and 10.9 s(-1) mM(-1) per Gd(iii) ion respectively. This corresponds to a relaxivity value of 32.7 s(-1) mM(-1) for the supramolecular complex containing three Gd(iii) ions. The high relaxivity value is prominently caused by an increase of the rotational tumbling time τ(R) by a factor of 2.7 and 5.5 respectively, in comparison with the commercially used MRI contrast agent Gd(iii)-DTPA (Magnevist?). Furthermore, upon UV irradiation, [(Gd5)(3)Al(H(2)O)(3)](3-) exposes green broad-band emission with a maximum at 543 nm. Regarding the high relaxivity and the photophysical properties of the [(Gd5)(3)Al(H(2)O)(3)](3-) metallostar compound, it can be considered as a lead compound for in vitro bimodal applications.     

Synthesis, thermodynamics stability constant and relaxation properties of neutral Gd(III) complex with derivative from diethylene triamine pentaacetic acid and p-hydroxybenzoyl hydrazine

A novel ligand, diethylenetriamine-N,N'-bis(acetyl-p-hydroxybenzoyl hydrazine)-N,N',N'-triacetic acid (H3L) was synthesized and characterized on the basis of elemental analysis, molar conductivity, 1H-NMR spectrum, FAB-MS, TG-DTA analysis and IR spectrum. Its complex of Gd(III) holding promise of magnetic resonance imaging (MRI) was synthesized, and relaxivity (R1) of complex and Gd(DTPA)2- used as a control was determined in water solution, respectively. The relaxivity of GdL (R1 = 6.39 l.mmol(-1).s(-1)) was larger than that of Gd(DTPA)2- (R1 = 4.34 l.mmol(-1).s(-1)). The relaxivity of GdL has also been investigated in human serum albumin (HSA) solution, the relaxivity of GdL was enhanced from 6.39 l.mmol(-1).s(-1) in water solution to 7.69 l.mmol(-1).s(-1) in HSA solution. In addition, thermodynamics stability constant of GdL was determined. The results showed that complex of GdL is a prospective MRI contrast agent, although the thermodynamic stability constant of GdL complex (K(GdL) = 10(19.56)) was a little less than that of Gd(DTPA)2- (K(Gd-DTPA) = 10(20.73)).