Nuclear Magnetic Relaxation Dispersion Measurements Research Articles

Developing High Field MRI Contrast Agents by Tuning the Rotational Dynamics: Bisaqua GdAAZTA‐based Dendrimers

AbstractMonomeric and dimeric AAZTA‐based bifunctional chelators (AAZTA=6‐amino‐6‐methylperhydro‐1,4‐diazepine tetraacetic acid) were attached to different generations (G0, G1 and G2) of ethylenediamine‐cored PAMAM dendrimers (PAMAM=polyamidoamine) to obtain a series of six dendrimeric systems with 4 to 32 chelates at the periphery. These GdIII‐loaded dendrimers have molecular weight ranging from 3.5 to 25 kDa, thus allowing a systematic investigation on the changes in relaxivity (r1) with the variation of the rotational dynamics following the increase in molecular size. Variable‐temperature 17O NMR (on the dimeric building block Gd2L2) and 1H Nuclear Magnetic Relaxation Dispersion measurements at different temperatures indicate that the water exchange lifetime (τM∼90 ns) of the two inner sphere water molecules does not represent a limiting factor to the relaxivity of the systems. The r1 values at 1.5 T (60 MHz) and 298 K increases from 10.2 mM−1 s−1 for the monomer GdL1 to 31.4 mM−1 s−1 for the dendrimer Gd32G2‐32 (+308 %). However, the relaxivity (per Gd) does not show a linear dependence on the molecular mass, but rather the enhancement tends to attenuate markedly for larger systems. This effect has been attributed to the growing decrease in correlation between local rotational motions and global molecular tumbling.

A comparative study of 1H and 19F Overhauser DNP in fluorinated benzenes

Hyperpolarization techniques, such as Overhauser dynamic nuclear polarization (DNP), can provide a dramatic increase in the signal obtained from nuclear magnetic resonance experiments and may therefore enable new applications where sensitivity is a limiting factor. In this contribution, studies of the (1)H and (19)F Overhauser dynamic nuclear polarization enhancements at 345 mT are presented for three different aromatic solvents with the TEMPO radical for a range of radical concentrations. Furthermore, nuclear magnetic relaxation dispersion measurements of the same solutions are analyzed, showing contributions from dipolar and scalar coupling modulated by translational diffusion and different coupling efficiency for different solvents and nuclei. Measurements of the electron paramagnetic resonance linewidth are included to support the analysis of the DNP saturation factor for varying radical concentration. The results of our study give an insight into the characteristics of nitroxide radicals as polarizing agents for (19)F Overhauser DNP of aromatic fluorinated solvents. Furthermore, we compare our results with the findings of the extensive research on Overhauser DNP that was conducted in the past for a large variety of other radicals.

Isoquinoline-Based Lanthanide Complexes: Bright NIR Optical Probes and Efficient MRI Agents

In the objective of developing ligands that simultaneously satisfy the requirements for MRI contrast agents and near-infrared emitting optical probes that are suitable for imaging, three isoquinoline-based polyaminocarboxylate ligands, L1, L2 and L3, have been synthesized and the corresponding Gd(3+), Nd(3+) and Yb(3+) complexes investigated. The specific challenge of the present work was to create NIR emitting agents which (i) have excitation wavelengths compatible with biological applications and (ii) are able to emit a sufficient number of photons to ensure sensitive NIR detection for microscopic imaging. Here we report the first observation of a NIR signal arising from a Ln(3+) complex in aqueous solution in a microscopy setup. The lanthanide complexes have high thermodynamic stability (log K(LnL) =17.7-18.7) and good selectivity for lanthanide ions versus the endogenous cations Zn(2+), Cu(2+), and Ca(2+) thus preventing transmetalation. A variable temperature and pressure (17)O NMR study combined with nuclear magnetic relaxation dispersion measurements yielded the microscopic parameters characterizing water exchange and rotation. Bishydration of the lanthanide cation in the complexes, an important advantage to obtain high relaxivity for the Gd(3+) chelates, has been demonstrated by (17)O chemical shifts for the Gd(3+) complexes and by luminescence lifetime measurements for the Yb(3+) analogues. The water exchange on the three Gd(3+) complexes is considerably faster (k(ex)(298) = (13.9-15.4) × 10(6) s(-1)) than on commercial Gd(3+)-based contrast agents and proceeds via a dissociative mechanism, as evidenced by the large positive activation volumes for GdL1 and GdL2 (+10.3 ± 0.9 and +10.6 ± 0.9 cm(3) mol(-1), respectively). The relaxivity of GdL1 is doubled at 40 MHz and 298 K in fetal bovine serum (r(1) = 16.1 vs 8.5 mM(-1) s(-1) in HEPES buffer), due to hydrophobic interactions between the chelate and serum proteins. The isoquinoline core allows for the optimization of the optical properties of the luminescent lanthanide complexes in comparison to the pyridinic analogues and provides significant shifts of the excitation energies toward lower values which therefore become more adapted for biological applications. L2 and L3 bear two methoxy substituents on the aromatic core in ortho and para positions, respectively, that further modulate their electronic structure. The Nd(3+) and Yb(3+) complexes of the ligand L3, which incorporates the p-dimethoxyisoquinoline moiety, can be excited up to 420 nm. This wavelength is shifted over 100 nm toward lower energy in comparison to the pyridine-based analogue. The luminescence quantum yields of the Nd(3+) (0.013-0.016%) and Yb(3+) chelates (0.028-0.040%) are in the range of the best nonhydrated complexes, despite the presence of two inner sphere water molecules. More importantly, the 980 nm NIR emission band of YbL3 was detected with a good sensitivity in a proof of concept microscopy experiment at a concentration of 10 μM in fetal bovine serum. Our results demonstrate that even bishydrated NIR lanthanide complexes can emit a sufficient number of photons to ensure sensitive detection in practical applications. In particular, these ligands containing an aromatic core with coordinating pyridine nitrogen can be easily modified to tune the optical properties of the NIR luminescent lanthanide complexes while retaining good complex stability and MRI characteristics for the Gd(3+) analogues. They constitute a highly versatile platform for the development of bimodal MR and optical imaging probes based on a simple mixture of Gd(3+) and Yb(3+)/Nd(3+) complexes using an identical chelator. Given the presence of two inner sphere water molecules, important for MRI applications of the corresponding Gd(3+) analogues, this result is particularly exciting and opens wide perspectives not only for NIR imaging based on Ln(3+) ions but also for the design of combined NIR optical and MRI probes.

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.

Copper-Responsive Magnetic Resonance Imaging Contrast Agents

The design, synthesis, and evaluation of the Copper-Gad (CG) family, a new class of copper-activated magnetic resonance imaging (MRI) contrast agents, are presented. These indicators comprise a Gd(3+)-DO3A core coupled to various thioether-rich receptors for copper-induced relaxivity switching. In the absence of copper ions, inner-sphere water binding to the Gd(3+) chelate is restricted, resulting in low longitudinal relaxivity values (r(1) = 1.2-2.2 mM(-1) s(-1) measured at 60 MHz). Addition of Cu(+) to CG2, CG3, CG4, and CG5 and either Cu(+) or Cu(2+) to CG6 triggers marked enhancements in relaxivity (r(1) = 2.3-6.9 mM(-1) s(-1)). CG2 and CG3 exhibit the greatest turn-on responses, going from r(1) = 1.5 mM(-1) s(-1) in the absence of Cu(+) to r(1) = 6.9 mM(-1) s(-1) upon Cu(+) binding (a 360% increase). The CG sensors are highly selective for Cu(+) and/or Cu(2+) over competing metal ions at cellular concentrations, including Zn(2+) at 10-fold higher concentrations. (17)O NMR dysprosium-induced shift and nuclear magnetic relaxation dispersion measurements support a mechanism in which copper-induced changes in the coordination environment of the Gd(3+) core result in increases in q and r(1). T(1)-weighted phantom images establish that the CG sensors are capable of visualizing changes in copper levels by MRI at clinical field strengths.

Relaxivity of liposomal paramagnetic MRI contrast agents

Paramagnetic liposomes, spherical particles formed by a lipid bilayer, are able to accommodate a high payload of Gd-containing lipid and therefore can serve as a highly potent magnetic resonance imaging contrast agent. In this paper the relaxation properties of paramagnetic liposomes were studied as a function of composition, temperature and magnetic field strength. The pegylated liposomes with a diameter of approximately 100 nm were designed for favorable pharmacokinetic properties in vivo. The proton relaxivity, i.e. the T1 relaxation rate per mmol of Gd(III) ions, of liposomes with unsaturated DOPC phospholipids was higher than those with saturated DSPC lipids. Addition of cholesterol was essential to obtain monodisperse liposomes and led to a further, although smaller, increase of the relaxivity. Nuclear magnetic relaxation dispersion measurements showed that the relaxivity was limited by water exchange. These results show that these paramagnetic liposomes are very effective contrast agents, making them excellent candidates for many applications in magnetic resonance imaging.

Surface changes and role of buried water molecules during the sulfane sulfur transfer in rhodanese from Azotobacter vinelandii: a fluorescence quenching and nuclear magnetic relaxation dispersion spectroscopic study.

The Azotobacter vinelandii rhodanese is a sulfurtransferase enzyme that catalyzes the transfer of the outer sulfur atom from thiosulfate to cyanide. Recently, investigations by NMR relaxation on the (15)N-enriched protein reported that interdomain contacts are rigidly maintained upon the sulfane sulfur transfer from the enzyme to the substrate. The modality of the enzymatic mechanism is then confined to a surface interaction, including dynamics of water molecules buried in the tertiary structure. Thus, investigations have been carried out by fluorescence, circular dichroism, and nuclear magnetic relaxation dispersion measurements. The comparison of circular dichroism spectra of the persulfurated enzyme and the sulfur-free form indicated that small changes occur. Fluorescence quenching studies have been performed to evaluate the conformational changes during catalysis using the fluorescent probe 8-anilinonaphthalene-2-sulfonic acid, and acrylamide, iodide, and cesium ions as quenchers. Changes in exchange dynamics of water molecules buried in the structure with bulk water, observed by nuclear magnetic relaxation dispersion, are due to local conformational transitions, likely involving residues around the active site, and are consistent with the global correlation time found by (15)N relaxation. These results, taken together, provide important information for elucidating the conformational features of the mechanism of action of the enzyme either in the role of a selective donor of a sulfur atom to small-sized substrates (i.e., to cyanide, transforming it into thiocyanate) or in the role of sulfur insertase for the formation of the Fe(2)S(2) iron-sulfur cluster in sulfur-deprived ferredoxins.

Investigation of the active site of Escherichia coli Cu,Zn superoxide dismutase reveals the absence of the copper-coordinated water molecule. Is the water molecule really necessary for the enzymatic mechanism?

The active site of the Cu,Zn superoxide dismutase from Escherichia coli in the oxidized Cu(II) state has been studied by nuclear magnetic relaxation dispersion (NMRD), optical and nuclear magnetic resonance spectroscopy. The orientation of some metal ligands is different with respect to all the other Cu,Zn superoxide dismutases. Moreover, NMRD measurements demonstrate the lack of a copper-coordinated water molecule. In spite of these differences the enzymatic activity is still high. Azide also binds copper with normal affinity and induces modifications in the active site comparable to those previously observed in the eukaryotic enzymes. Our results suggest that, in this enzyme, the copper-coordinated water molecule appears not necessary for the enzymatic reaction. A role for the copper-coordinated water molecule is discussed in the light of recent crystallographic studies.

Water molecules in DNA recognition II: a molecular dynamics view of the structure and hydration of the trp operator

The structure and hydration of the DNA duplex d-(AGCGTACTAGTACGCT)2corresponding to the trp operator fragment used in the crystal structure of the half site complex (PDB entry 1TRR) was studied by a 1.4 ns molecular dynamics simulation in water. The simulation, starting from a B-DNA conformation, used a non-bonded cutoff of 1.4 nm with a reaction field correction and resulted in a stable trajectory. The average DNA conformation obtained was closer to the ones found in the crystal structures of the complexes (PDB entries 1TRO and 1TRR) than to the crystal structure of unbound trp operator (Nucleic Acid Database entry BDJ061). The DNA hydration was characterized in terms of hydrogen bond percentages and corresponding residence times. The residence times of water molecules within 0.35 nm of the DNA non-exchangeable protons were calculated for comparison with NMR measurements of intermolecular water-DNA NOEs and nuclear magnetic relaxation dispersion measurements. No significant difference was found between major and minor groove hydration. The DNA donors and acceptors were hydrogen bonded to water molecules for 77(±19)% of the time on average. The average residence time of the hydrogen bonded water molecules was 11(±11) ps with a maximum of 223 ps. When all water molecules within NOE distance (0.35 nm) of non-exchangeable protons were considered, the average residence times increased to an average of 100(±4) ps and a maximum of 608 ps. These results agree with the experimental NMR results of Sunnerhagen et al. which did not show any evidence for water molecules bound with more than 1 ns residence time on the DNA surface. The exchange of hydration water from the DNA occurred in the major groove primarily through direct exchange with the bulk solvent, while access to and from the minor groove frequently proceeded via pathways involving ribose O3′ and O4′ and phosphate O2P oxygen atoms. The most common water diffusion pathways in the minor groove were perpendicular to the groove direction. In general, water molecules visited only a limited number of sites in the DNA grooves before exiting. The hydrogen bonding sites, where hydrogen bonds could be formed with donor and acceptor groups of the DNA, were filled with water molecules with an average B-factor value of 0.58 mn2. No special values were observed at any of the sites, where water molecules were observed both in the trp repressor/operator co-crystals and in the crystal structure of unbound DNA.

Nuclear Magnetic Relaxation Dispersion Studies on Bentonite Clay Suspensions

Nuclear magnetic relaxation dispersion measurements are reported on aqueous suspensions of bentonite clays which demonstrate that the water-proton spin-lattice relaxation rates are dominated by magnetic interactions with paramagnetic centers entrapped in the mineral matrix. The water-proton spin-lattice relaxation rates are linear in the concentration of the clay mineral. The relaxivity may be increased by the addition of paramagnetic metal ions to the suspension and the shape of the relaxation dispersion profile demonstrates that the mineral behaves as a sequestering agent in that there is no free metal detectable. The temperature dependence of the relaxation rate is consistent with the electron-nuclear coupling being modulated by translational motions of the water molecules; however, the shape of the relaxation dispersion profile does not fit the usual models for nuclear-spin relaxation caused by translational diffusion in a solution containing a paramagnetic cosolute.

Electron Spin Resonance and Nuclear Magnetic Relaxation Dispersion Studies of Commercial Clay Minerals and Zeolites

Nuclear magnetic relaxation dispersion measurements on water protons and EPR spectra are reported for a series of clays and zeolite samples. The paramagnetic components of the samples make strong contributions to the proton spin-lattice relaxation rates at low magnetic field strengths, although the shape of the magnetic relaxation dispersion profile is not accurately fitted by standard relaxation models. For the iron-rich clay minerals, the water-proton spin-lattice relaxation rate is approximately a linear function of the log of the magnetic field strength. The zeolite relaxation profiles are different and may be approximately modeled with a function of the form rate = Av-B , where B ranges from 0.16 to 0.43. The EPR spectra of the clays and zeolites demonstrate significant contributions from iron(III) and manganese(II). The manganese(II) spectra are complicated by zero-field interactions that in some cases add a number of additional transitions to the spectra.

Nuclear Magnetic Relaxation Dispersion Measurements of Ethylenediamine-Tetraacetatoiron(III) Complexes at High pH and Ionic Strength

Abstract Nuclear magnetic relaxation dispersion measurements are reported for aqueous solutions of iron(III)-EDTA complexes at high pH and high ionic strength where the dominant species is the μ-oxo dimer. Formation of the dimeric species leaves only the outer coordination sphere contribution to the paramagnetic relaxation rate of the water protons in solution. The relaxation efficiency is further decreased by the decrease in the effective magnetic moment due to antiferromagnetic coupling. This result is in distinction to dimeric species that form with Gd(HEDTA) complexes at high pH in which antiferromagnetic coupling has not been observed to decrease the effective magnetic moment.

Proton nuclear magnetic relaxation in aqueous copper(II) amine chelate solutions

Nuclear magnetic relaxation dispersion measurements are reported for aqueous solutions of copper(II) ion in solutions of ammonia, 1,2-diaminoethane and triethylenetetraamine. The water proton spin relaxation efficiency is increased dramatically over the hexaaquocopper(II) ion rate when square planar complexes are formed. The major factor contributing to this change is the development of a large hyperfine coupling between the coordinated amine protons and the copper(II) center that dominates low field relaxation rates. The relaxation is generally controlled by the rate of ligand exchange which is catalyzed by high ligand concentration. The triethylenetetraamine complex is substitutionally inert on the time scale of the nuclear spin relaxation and the water proton relaxation induced is dominated by the relative translational motion of the water and the metal center, i.e. outer sphere effects.

Electron spin echo envelope modulation studies of lectins: evidence for a conserved manganese(2+)-binding site

Electron spin echo envelope modulation (ESEEM) experiments have been used to investigate the Mn(2+)-binding site in a series of lectins including concanavalin A, pea lectin (Pisum sativum), isolectin A from lentil (Lens culinaris), soybean agglutinin (Glycine max), Erythrina indica lectin, and Lotus tetragonolobus isoelectin A. Together with model studies, the results provide direct evidence for a single nitrogen atom of a conserved residue bonded directly to Mn2+ in all of them. ESEEM measurements of the lectins exchanged with deuterium oxide, together with model studies, provide evidence for the presence of two water molecules coordinated to the Mn2+ in all of the proteins. In contrast to concanavalin A, the absence of solvent exchange at the Mn2+ site in the pea and lentil lectins demonstrated by nuclear magnetic relaxation dispersion measurements [Bhattacharyya, L., Brewer, C.F., Brown, R. D., III, & Koenig, S. H. (1985) Biochemistry 24, 4985-4990] must therefore be due to slow exchange of the water ligands of the bound Mn2+. Binding of saccharides was observed to have little effect on the structural features of the Mn2+ site in the lectins as determined by ESEEM.

Interactions of asparagine-linked carbohydrates with concanavalin A. Nuclear magnetic relaxation dispersion and circular dichroism studies

By using near-UV circular dichroism (CD) and solvent proton nuclear magnetic relaxation dispersion measurements, three different conformational states have been detected in Ca(2+)-Mn(2+)-concanavalin A upon binding a variety of asparagine-linked carbohydrates. Two of these transitions have been described previously, one for the binding of monosaccharides such as methyl alpha-D-mannopyranoside and oligosaccharides with terminal alpha-Glc or alpha-Man residues, and the second for the binding of oligomannose and complex type carbohydrates (Brewer, C. F., and Bhattacharyya, L. (1986) J. Biol. Chem. 261, 7306-7310). The third transition occurs upon binding a bisected biantennary complex type carbohydrate with terminal GlcNAc residues. Temperature-dependent nuclear magnetic relaxation dispersion and CD measurements have identified regions of the protein near the two metal ion binding sites that are associated with the conformation changes, and Tyr-12, which is part of the monosaccharide binding site, as responsible for the CD changes. The results support our previous conclusions that the rotamer conformation of the (alpha 1,6) arm of bisected complex type oligosaccharides binds to concanavalin A with dihedral angle omega = -60 degrees whereas nonbisected complex type oligosaccharides bind with omega = 180 degrees (Bhattacharyya, L., Haraldsson, M., and Brewer, C. F. (1987) J. Biol. Chem. 262, 1294-1299). The present findings also explain the effects of increasing chain length of bisected complex type carbohydrates on their interactions with the lectin.

Characterization of dicopper-dicobalt- and dicobalt-dicobalt alkaline phosphatase complexes at acidic pH

Alkaline phosphatase (AP) has been shown to bind selectively copper in the A site and cobalt in the B site, at pH 6. Magnesium can be present in the C site. On account of the short distance between A and B sites the electronic relaxation rates of copper decrease, although no magnetic coupling interactions can be detected via magnetic susceptibility measurements at room temperature. The water /sup 1/H nuclear magnetic relaxation dispersion measurements show a dramatic decrease in relaxation rates when cobalt is added to Cu/sub 2/E/sub 2/AP (E = empty) as a result of the decrease of the correlation time of protons feeling the copper electron. The /sup 1/H NMR spectra of the Cu/sub 2/Co/sub 2/Mg/sub 2/AP derivative show well-shaped signals of protons of histidines bound to both copper and cobalt. The assignment is performed through a comparison between the spectra of Cu/sub 2/Co/sub 2/Mg/sub 2/AP and Co/sub 2/Co/sub 2/Mg/sub 2/AP; the histidine NH's are assigned by recording the spectra in H/sub 2/O and D/sub 2/O. In the absence of magnesium the latter protons exchange rapidly on the NMR time scale. 44 refs., 8 figs., 1 tab.