Relaxation Dispersion Profiles Research Articles

Molecular Dynamics, Dielectric Properties, and Textures of Protonated and Selectively Deuterated 4'-Pentyl-4-biphenylcarbonitrile Liquid Crystal.

Using liquid crystals in near-infrared applications suffers from effects related to processes like parasitic absorption and high sensitivity to UV-light exposure. One way of managing these disadvantages is to use deuterated systems. The combined 1H and 2H nuclear magnetic resonance relaxometry method (FFC NMR), dielectric spectroscopy (DS), optical microscopy (POM), and differential scanning calorimetry (DSC) approach was applied to investigate the influence of selective deuteration on the molecular dynamics, thermal properties, self-organization, and electric-field responsiveness to a 4'-pentyl-4-biphenylcarbonitrile (5CB) liquid crystal. The NMR relaxation dispersion (NMRD) profiles were analyzed using theoretical models for the description of dynamics processes in different mesophases. Obtained optical textures of selectively deuterated 5CB showed the occurrence of the domain structure close to the I/N phase transition. The dielectric measurements showed a substantial difference in switching fields between fully protonated/deuterated 5CB and selectively deuterated molecules. The DSC thermograms showed a more complex phase transition sequence for partially deuterated 5CB with respect to fully protonated/deuterated molecules.

Gadopiclenol: A q = 2 Gadolinium-Based MRI Contrast Agent Combining High Stability and Efficacy

Objectives Gadopiclenol is a q = 2 pyclen gadolinium-based contrast agent (GBCA) recently approved by the Food and Drug Administration, European Medicines Agency, and other European countries. The aim of this report is to demonstrate its stability in multiple stressed in vitro conditions and in vivo, in rat kidney, while maintaining its higher relaxivity compared with conventional GBCAs on the market. Materials and Methods Both gadopiclenol and its chemical precursor Pi828-Gd were characterized and compared with q = 1 gadolinium (Gd) complexes. The number of water molecules coordinated to the Gd (the hydration number, q) was determined by luminescence. 17O NMR (Nuclear Magnetic Resonance) measurements gave access to the water residence time τM. These parameters were used for the fitting of the nuclear magnetic relaxation dispersion profiles in water. Proton relaxivities of the complexes were determined in different media at 60 MHz (1.4 T), at different pH and temperature. The kinetic inertness was investigated in human serum, acidic media, under zinc competition in the presence of phosphate, and under ligand competition. The in vivo stability was evaluated in rat kidneys 12 months after repeated injections. Results The presence of 2 inner-sphere water molecules per Gd complex was confirmed for both pyclen derivatives. The high relaxivity of the complexes in water is maintained under physiological conditions, even under stressed conditions (ionic media, extreme pH, and temperature), which guarantees their efficiency in a large range of in vivo situations. Gd release from the q = 2 complexes was investigated in different potentially destabilizing conditions. Either no Gd release or a slower one than with “q = 1” stable macrocyclic GBCA (acidic conditions) was observed. Their kinetic inertness was demonstrated in physiological conditions, and the Gd release was below the lower limit of quantification of 0.1 μM after 12 days at 37°C in human serum. It was also demonstrated that gadopiclenol is stable in vivo in rat kidney 12 months after repeated injections. Conclusions Thanks to its optimized structural design, gadopiclenol is a highly stable and effective macrocyclic q = 2 GBCA.

Existence of Singularities in NMR Relaxation Dispersion Profiles: Implications for Hidden Dynamics.

It is common for NMR relaxation dispersion experiments to suggest an absence of dynamics despite anecdotal indications of conformational dynamics. We explore the potential explanations and approaches to this conundrum. Some inconsistencies have been observed between two relaxation dispersion experiments, Carr-Purcell-Meiboom-Gill (CPMG) and adiabatic relaxation dispersion experiments, in recent dynamic studies of different biomolecules. Theoretical analyses show that such seemingly paradoxical results might come from a complex exchange topology that is concealed by the application of the simple two-site exchange model for interpretation of the relaxation dispersion data. Scenarios are explored and revealed in which the presence of complex millisecond conformational exchange could suppress the amplitude of CPMG relaxation dispersion profiles, even when the exchange rates are within the detectable range of the experiments. With experimental errors, the suppressed relaxation dispersion profiles could lead to the conclusion of "no millisecond conformational exchange". However, such hidden dynamics can potentially be detected by adiabatic relaxation dispersion experiments. Finally, we demonstrate some advantages of adiabatic relaxation dispersion experiments over conventional relaxation dispersion experiments and a simplified computational approach to analyze the adiabatic relaxation dispersion profiles.

Investigation of Ln3+ complexation by a DOTA derivative substituted by an imidazothiadiazole: synthesis, solution structure, luminescence and relaxation properties.

We investigated the coordination properties of original macrocyclic Ln3+ complexes comprising an imidazothiadiazole heterocycle. The thermodynamic stability of the Gd3+ complex was determined by a combination of potentiometric and photophysical measurements. The kinetic inertness was assessed in highly acidic media. The solution structure of the Ln3+ complex was unambiguously determined by a set of photophysical measurements and 1H, 13C, 89Y NMR data in combination with DFT calculations, which proved coordination of the heterocycle to Ln3+. The ability of the imidazothiadiazole moiety to sensitize Tb3+ luminescence was investigated. Finally, the relaxation properties were investigated by recording 1H nuclear magnetic relaxation dispersion (NMRD) profiles and 17O measurements. The water exchange rate is similar to that of GdDOTA as the less negative charge of the ligand is compensated for by the presence of a bulky heterocycle. Relaxivity is constant over a large range of pH values, demonstrating the favorable properties of the complex for imaging purposes.

Water Dynamics in Dextran-Based Hydrogel Micro/Nanoparticles Studied by NMR Diffusometry and Relaxometry.

Water dynamics in mesoporous dextran hydrogel micro/nanoparticles was investigated by means of nuclear magnetic resonance (NMR) techniques. High-resolution 1H NMR spectra and pulsed field gradient (PFG) NMR diffusometry measurements obtained on swollen state dextran micro/nanogel revealed the existence of different fractions of water molecules based on their interaction with the gel matrix. In addition to the translational diffusion of bulk water, two more diffusion processes characterized with self-diffusion coefficients 1 and 2 orders of magnitude smaller than that of bulk water were identified. 1H spin-lattice relaxation dispersion profiles obtained for a broad range of Larmor frequencies using fast field cycling (FFC) and conventional NMR relaxometry techniques allowed us to further clarify the mechanisms of molecular motion. According to the water proton pool fractions and associated self-diffusion coefficients, it is shown that the relaxation contribution associated with reorientation-mediated translational motions (RMTDs) dominates the relaxation dispersion observed at intermediate frequencies. At very low frequencies, the spin-lattice relaxation rate is dominated by the slow solid-gel dynamics probed by the water molecules interacting with the pores' surface hydroxyl groups due to the rapid chemical exchange between surface hydroxyl groups and free water. The correlation time for the thumbling-like motion of the dextran gel was found to be in the submillisecond range. The values of the self-diffusion and coherence lengths associated with motion of water molecules interacting with the solid-gel particles are consistent with the particle size and pore size distributions obtained for the studied dextran gels.

A methyl-TROSY based 13C relaxation dispersion NMR experiment for studies of chemical exchange in proteins.

A methyl Transverse Relaxation Optimized Spectroscopy (methyl-TROSY) based, multiple quantum (MQ) 13C Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR experiment is described. The experiment is derived from the previously developed MQ 13C-1H CPMG scheme (Korzhnev in J Am Chem Soc 126: 3964-73, 2004) supplemented with a CPMG train of refocusing 1H pulses applied with constant frequency and synchronized with the 13C CPMG pulse train. The optimal 1H 'decoupling' scheme that minimizes the amount of fast-relaxing methyl MQ magnetization present during CPMG intervals, makes use of an XY-4 phase cycling of the refocusing composite 1H pulses. For small-to-medium sized proteins, the MQ 13C CPMG experiment has the advantage over its single quantum (SQ) 13C counterpart of significantly reducing intrinsic, exchange-free relaxation rates of methyl coherences. For high molecular weight proteins, the MQ 13C CPMG experiment eliminates complications in the interpretation of MQ 13C-1H CPMG relaxation dispersion profiles arising from contributions to exchange from differences in methyl 1H chemical shifts between ground and excited states. The MQ 13C CPMG experiment is tested on two protein systems: (1) a triple mutant of the Fyn SH3 domain that interconverts slowly on the chemical shift time scale between the major folded state and an excited state folding intermediate; and (2) the 82-kDa enzyme Malate Synthase G (MSG), where chemical exchange at individual Ile δ1 methyl positions occurs on a much faster time-scale.

Plant defensins as a model to study the thermostability of surface hydrophobic clusters: The Pisum sativum defensin 2 (Psd2)

Plant defensins (PDs) display a CSαβ-fold that lacks a canonical hydrophobic core. They display almost all hydrophobic residues on the protein surface. The exposed hydrophobic residues form surface clusters stabilized by the vicinity of hydrophilic residues and the hydration shell. Here, we used Psd2 as a model to study the formation and stabilization of these local foldons named surface hydrophobic clusters (SHC). We characterized the temperature dependence of 15N CPMG relaxation dispersion profiles to describe the complex dynamics of Psd2 and indirectly study the thermodynamics of SHCs in PDs. We show a correlation between residues undergoing conformational exchange and the SHCs. Chemical shift changes between the native ground state and the first thermally accessible excited state enabled us to map the major conformational changes in Psd2 conformational equilibrium. The observation of a cold-driven excited state revealed that SHCs are stabilized by hydrophobic contacts, which are exposed at low temperatures, leading to a favorable decrease in enthalpy compensated by an unfavorable entropy reduction. At higher temperatures, we detected another excited conformer that may play a role in membrane-specific interaction, as previously described for other defensins.

Characterization of the Fe(III)-Tiron System in Solution through an Integrated Approach Combining NMR Relaxometric, Thermodynamic, Kinetic, and Computational Data.

The Fe(III)-Tiron system (Tiron = 4,5-dihydroxy-1,3-benzenedisulfonate) was investigated using a combination of 1H and 17O NMR relaxometric studies at variable field and temperature and theoretical calculations at the DFT and NEVPT2 levels. These studies require a detailed knowledge of the speciation in aqueous solution at different pH values. This was achieved using potentiometric and spectrophotometric titrations, which afforded the thermodynamic equilibrium constants characterizing the Fe(III)-Tiron system. A careful control of the pH of the solution and the metal-to-ligand stoichiometric ratio allowed the relaxometric characterization of [Fe(Tiron)3]9-, [Fe(Tiron)2(H2O)2]5-, and [Fe(Tiron)(H2O)4]- complexes. The 1H nuclear magnetic relaxation dispersion (NMRD) profiles of [Fe(Tiron)3]9- and [Fe(Tiron)2(H2O)2]5- complexes evidence a significant second-sphere contribution to relaxivity. A complementary 17O NMR study provided access to the exchange rates of the coordinated water molecules in [Fe(Tiron)2(H2O)2]5- and [Fe(Tiron)(H2O)4]- complexes. Analyses of the NMRD profiles and NEVPT2 calculations indicate that electronic relaxation is significantly affected by the geometry of the Fe3+ coordination environment. Dissociation kinetic studies indicated that the [Fe(Tiron)3]9- complex is relatively inert due to the slow release of one of the Tiron ligands, while the [Fe(Tiron)2(H2O)2]5- complex is considerably more labile.

Nuclear magnetic resonance relaxometry to monitor chromium (VI) reduction by hydrogen peroxide, ascorbic acid, and aluminum powder.

The reduction of K2 Cr2 O7 solutions by H2 O2 was studied by nuclear magnetic resonance (NMR) relaxometry and UV-vis spectroscopy in HCl/KCl buffer (pH 2), NaCl/glycine/HCl buffer (pH 3), and sodium acetate/acetic acid buffer (pH 4). Because of Cr(III) paramagnetism, 1/T1 and 1/T2 of the solutions increase during the reduction of diamagnetic Cr(VI). This increase is proportional to the produced Cr(III) concentration. Using different initial H2 O2 concentrations, partially reduced Cr(VI) samples were prepared and studied by T1 and T2 relaxometry and by UV-vis spectroscopy. The correlation between the relaxation rates and the concentration of Cr(VI) remaining in the sample, measured by spectroscopy, was excellent. It was possible, thanks to the measurement of T2 , to study the kinetics of the reduction of K2 Cr2 O7 by H2 O2 in the pH 3 and pH 4 buffers. The reduction of Cr(VI) by ascorbic acid was successfully monitored by NMR relaxometry in the pH 2 buffer. The presence of complexing molecules/ions was shown to drastically influence the nuclear magnetic relaxation dispersion profiles of reduced K2 Cr2 O7 solutions: Both relaxation rates are divided by ~5 when citrate or acetate ions are present and by ~3 in the presence of ascorbic acid. Therefore, the comparison of relaxation results obtained in different reaction mixtures must be done carefully. When all the solutions are set to pH 0, which prevents any complexation, the longitudinal and transverse relaxation rates of all samples become comparable. Finally, as a proof of concept for a turbid solution, the kinetics of the reduction of a K2 Cr2 O7 solution by aluminum powder in the pH 2 buffer was successfully monitored.

Assessing the applicability of 19F labeled tryptophan residues to quantify protein dynamics

Nuclear magnetic resonance (NMR) spectroscopy is uniquely suited to study the dynamics of biomolecules in solution. Most NMR studies exploit the spins of proton, carbon and nitrogen isotopes, as these atoms are highly abundant in proteins and nucleic acids. As an alternative and complementary approach, fluorine atoms can be introduced into biomolecules at specific sites of interest. These labels can then be used as sensitive probes for biomolecular structure, dynamics or interactions. Here, we address if the replacement of tryptophan with 5-fluorotryptophan residues has an effect on the overall dynamics of proteins and if the introduced fluorine probe is able to accurately report on global exchange processes. For the four different model proteins (KIX, Dcp1, Dcp2 and DcpS) that we examined, we established that 15N CPMG relaxation dispersion or EXSY profiles are not affected by the 5-fluorotryptophan, indicating that this replacement of a proton with a fluorine has no effect on the protein motions. However, we found that the motions that the 5-fluorotryptophan reports on can be significantly faster than the backbone motions. This implies that care needs to be taken when interpreting fluorine relaxation data in terms of global protein motions. In summary, our results underscore the great potential of fluorine NMR methods, but also highlight potential pitfalls that need to be considered.

Engineered Nonviral Protein Cages Modified for MR Imaging.

Diagnostic medical imaging utilizes magnetic resonance (MR) to provide anatomical, functional, and molecular information in a single scan. Nanoparticles are often labeled with Gd(III) complexes to amplify the MR signal of contrast agents (CAs) with large payloads and high proton relaxation efficiencies (relaxivity, r1). This study examined the MR performance of two structurally unique cages, AaLS-13 and OP, labeled with Gd(III). The cages have characteristics relevant for the development of theranostic platforms, including (i) well-defined structure, symmetry, and size; (ii) the amenability to extensive engineering; (iii) the adjustable loading of therapeutically relevant cargo molecules; (iv) high physical stability; and (v) facile manufacturing by microbial fermentation. The resulting conjugates showed significantly enhanced proton relaxivity (r1 = 11-18 mM-1 s-1 at 1.4 T) compared to the Gd(III) complex alone (r1 = 4 mM-1 s-1). Serum phantom images revealed 107% and 57% contrast enhancements for Gd(III)-labeled AaLS-13 and OP cages, respectively. Moreover, proton nuclear magnetic relaxation dispersion (1H NMRD) profiles showed maximum relaxivity values of 50 mM-1 s-1. Best-fit analyses of the 1H NMRD profiles attributed the high relaxivity of the Gd(III)-labeled cages to the slow molecular tumbling of the conjugates and restricted local motion of the conjugated Gd(III) complex.

NMR Relaxation Dispersion Experiments to Study Phosphopeptide Recognition by SH2 Domains: The Grb2-SH2-Phosphopeptide Encounter Complex.

Protein interactions are at the essence of life. Proteins evolved not to have stable structures, but rather to be specialized in participating in a network of interactions. Every interaction involving proteins comprises the formation of an encounter complex, which may have two outcomes: (i) the dissociation or (ii) the formation of the final specific complex. Here, we present a methodology to characterize the encounter complex of the Grb2-SH2 domain with a phosphopeptide. This method can be generalized to other protein partners. It consists of the measurement of 15N CPMG relaxation dispersion (RD) profiles of the protein in the free state, which describes the residues that are in conformational exchange. We then acquire the dispersion profiles of the proteinat a semisaturated concentration of the ligand. At this condition, the chemical exchange between the free and bound state leads to the observation of dispersion profiles in residues that are not in conformational exchange in the free state. This is due to fuzzy interactions that are typical of the encounter complexes. The transient "touching" of the ligand in the protein partner generates these new relaxation dispersion profiles. For the Grb2-SH2 domain, we observed a wider surface at SH2 for the encounter complex than the phosphopeptide (pY) binding site, which might explain the molecular recognition of remote phosphotyrosine. The Grb2-SH2-pY encounter complex is dominated by electrostatic interactions, which contribute to the fuzziness of the complex, but also have contribution of hydrophobic interactions.

A "Steady-State" Relaxation Dispersion Nuclear Magnetic Resonance Experiment for Studies of Chemical Exchange in Degenerate 1H Transitions of Methyl Groups.

Degenerate spin-systems consisting of magnetically equivalent nuclear spins, such as a 1H3 spin-system in selectively 13CH3-labeled proteins, present considerable challenges for the design of Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR experiments to characterize chemical exchange on the micro-to-millisecond time-scale. Several approaches have been previously proposed for the elimination of deleterious artifacts observed in methyl 1H CPMG relaxation dispersion profiles obtained for (13C)1H3 groups. We describe an alternative, experimentally simple solution and design a "steady-state" methyl 1H CPMG scheme, where 90° or acute-angle (<90°) 1H radiofrequency pulses are applied after each CPMG echo in-phase with methyl 1H magnetization, resulting in the establishment of a "steady-state" for effective rates of magnetization decay. A simple computational procedure for quantitative analysis of the "steady-state" CPMG relaxation dispersion profiles is developed. The "steady-state" CPMG methodology is applied to two protein systems where exchange between major and minor species occurs in different regimes on the chemical shift time-scale.

Preclinical Profile of Gadoquatrane: A Novel Tetrameric, Macrocyclic High Relaxivity Gadolinium-Based Contrast Agent.

ObjectivesThe aim of this report was to characterize the key physicochemical, pharmacokinetic (PK), and magnetic resonance imaging (MRI) properties of gadoquatrane (BAY 1747846), a newly designed tetrameric, macrocyclic, extracellular gadolinium-based contrast agent (GBCA) with high relaxivity and stability.Materials and MethodsThe r1-relaxivities of the tetrameric gadoquatrane at 1.41 and 3.0 T were determined in human plasma and the nuclear magnetic relaxation dispersion profiles in water and plasma. The complex stability was analyzed in human serum over 21 days at pH 7.4 at 37°C and was compared with the linear GBCA gadodiamide and the macrocyclic GBCA (mGBCA) gadobutrol. In addition, zinc transmetallation assay was performed to investigate the kinetic inertness. Protein binding and the blood-to-plasma ratio were determined in vitro using rat and human plasma. The PK profile was evaluated in rats (up to 7 days postinjection). Magnetic resonance imaging properties were investigated using a glioblastoma (GS9L) rat model.ResultsThe new chemical entity gadoquatrane is a macrocyclic tetrameric Gd complex with one inner sphere water molecule per Gd (q = 1). Gadoquatrane showed high solubility in buffer (1.43 mol Gd/L, 10 mM Tris-HCl, pH 7.4), high hydrophilicity (logP −4.32 in 1-butanol/water), and negligible protein binding. The r1-relaxivity of gadoquatrane in human plasma per Gd of 11.8 mM−1·s−1 (corresponding to 47.2 mM−1·s−1 per molecule at 1.41 T at 37°C, pH 7.4) was more than 2-fold (8-fold per molecule) higher compared with established mGBCAs. Nuclear magnetic relaxation dispersion profiles confirmed the more than 2-fold higher r1-relaxivity in human plasma for the clinically relevant magnetic field strengths from 0.47 to 3.0 T. The complex stability of gadoquatrane at physiological conditions was very high. The observed Gd release after 21 days at 37°C in human serum was below the lower limit of quantification. Gadoquatrane showed no Gd3+ release in the presence of zinc in the transmetallation assay. The PK profile (plasma elimination, biodistribution, recovery) was comparable to that of gadobutrol. In MRI, the quantitative evaluation of the tumor-to-brain contrast in the rat glioblastoma model showed significantly improved contrast enhancement using gadoquatrane compared with gadobutrol at the same Gd dose administered (0.1 mmol Gd/kg body weight). In comparison to gadoterate meglumine, similar contrast enhancement was reached with gadoquatrane with 75% less Gd dose. In terms of the molecule dose, this was reduced by 90% when compared with gadoterate meglumine. Because of its tetrameric structure and hence lower number of molecules per volume, all prepared formulations of gadoquatrane were iso-osmolar to blood.ConclusionsThe tetrameric gadoquatrane is a novel, highly effective mGBCA for use in MRI. Gadoquatrane provides favorable physicochemical properties (high relaxivity and stability, negligible protein binding) while showing essentially the same PK profile (fast extracellular distribution, fast elimination via the kidneys in an unchanged form) to established mGBCAs on the market. Overall, gadoquatrane is an excellent candidate for further clinical development.

Rigidified Derivative of the Non-macrocyclic Ligand H4OCTAPA for Stable Lanthanide(III) Complexation.

The stability constants of lanthanide complexes with the potentially octadentate ligand CHXOCTAPA4–, which contains a rigid 1,2-diaminocyclohexane scaffold functionalized with two acetate and two picolinate pendant arms, reveal the formation of stable complexes [log KLaL = 17.82(1) and log KYbL = 19.65(1)]. Luminescence studies on the Eu3+ and Tb3+ analogues evidenced rather high emission quantum yields of 3.4 and 11%, respectively. The emission lifetimes recorded in H2O and D2O solutions indicate the presence of a water molecule coordinated to the metal ion. 1H nuclear magnetic relaxation dispersion profiles and 17O NMR chemical shift and relaxation measurements point to a rather low water exchange rate of the coordinated water molecule (kex298 = 1.58 × 106 s–1) and relatively high relaxivities of 5.6 and 4.5 mM–1 s–1 at 20 MHz and 25 and 37 °C, respectively. Density functional theory calculations and analysis of the paramagnetic shifts induced by Yb3+ indicate that the complexes adopt an unprecedented cis geometry with the two picolinate groups situated on the same side of the coordination sphere. Dissociation kinetics experiments were conducted by investigating the exchange reactions of LuL occurring with Cu2+. The results confirmed the beneficial effect of the rigid cyclohexyl group on the inertness of the Lu3+ complex. Complex dissociation occurs following proton- and metal-assisted pathways. The latter is relatively efficient at neutral pH, thanks to the formation of a heterodinuclear hydroxo complex.

Longitudinal and transverse NMR relaxivities of Ln(III)-DOTA complexes: A comprehensive investigation.

Longitudinal and transverse 1H nuclear magnetic resonance relaxivities of Ln(III)-DOTA complexes (with Ln = Gd, Tb, Dy, Er; DOTA = 1,4,7,10-tetraazacyclododecane-N,N',N″,N‴-tetraacetic acid) and Mn(II) aqueous solutions were measured in a wide range of frequencies, 10kHz to 700MHz. The experimental data were interpreted by means of models derived from the Solomon-Bloembergen-Morgan theory. The data analysis was performed assuming the orbital angular momentum L = 0 for Gd-DOTA and the aqua ion [Mn(H2O)6]2+ and L ≠ 0 for Dy-, Tb-, and Er-DOTA. A refined estimation of the zero-field-splitting barrier Δ and of the modulation correlation time τv was obtained for [Mn(H2O)6]2+ by extending the fitting of nuclear magnetic relaxation dispersion profiles to the low-field regime. The Gd-DOTA fitting parameters resulted in good agreement with the literature, and the fit of transverse relaxivity data confirmed the negligibility of the scalar interaction in the nuclear relaxation mechanism. Larger transverse relaxivities of Dy-DOTA and Tb-DOTA (∼10 mM-1s-1) with respect to Er-DOTA (∼1 mM-1s-1) were observed at 16T. Such higher values are suggested to be due to a shorter residence time τm that is possibly linked to the fluctuations of the hyperfine interaction and the different shape of the magnetic anisotropy. The possible employment of Dy-DOTA, Tb-DOTA, and Er-DOTA as negative magnetic resonance imaging contrast agents for high-field applications was envisaged by collecting spin-echo images at 7T. Particularly in Dy- and Tb-derivatives, the transverse relaxivity at 16T is of the order of the Gd-one at 1.5T.

1H R1\u03c1 relaxation dispersion experiments in aromatic side chains

Aromatic side chains are attractive probes of protein dynamic, since they are often key residues in enzyme active sites and protein binding sites. Dynamic processes on microsecond to millisecond timescales can be studied by relaxation dispersion experiments that attenuate conformational exchange contributions to the transverse relaxation rate by varying the refocusing frequency of applied radio-frequency fields implemented as either CPMG pulse trains or continuous spin-lock periods. Here we present an aromatic 1H R1ρ relaxation dispersion experiment enabling studies of two to three times faster exchange processes than achievable by existing experiments for aromatic side chains. We show that site-specific isotope labeling schemes generating isolated 1H–13C spin pairs with vicinal 2H–12C moieties are necessary to avoid anomalous relaxation dispersion profiles caused by Hartmann–Hahn matching due to the 3JHH couplings and limited chemical shift differences among 1H spins in phenylalanine, tyrosine and the six-ring moiety of tryptophan. This labeling pattern is sufficient in that remote protons do not cause additional complications. We validated the approach by measuring ring-flip kinetics in the small protein GB1. The determined rate constants, kflip, agree well with previous results from 13C R1ρ relaxation dispersion experiments, and yield 1H chemical shift differences between the two sides of the ring in good agreement with values measured under slow-exchange conditions. The aromatic1H R1ρ relaxation dispersion experiment in combination with the site-selective 1H–13C/2H–12C labeling scheme enable measurement of exchange rates up to kex = 2kflip = 80,000 s–1, and serve as a useful complement to previously developed 13C-based methods.

Lanthanide DO3A-Complexes Bearing Peptide Substrates: The Effect of Peptidic Side Chains on Metal Coordination and Relaxivity.

Two DO3A-type ligands conjugated to substrates of urokinase (L3) and caspase-3 (L4) via a propyl-amide linker were synthesized and their lanthanide(III) (Ln3+) complexes studied. A model compound without peptide substrate (L2) and an amine derivative ligand mimicking the state after enzymatic cleavage (L1) were also prepared. Proton Nuclear Magnetic Relaxation Dispersion (NMRD) profiles recorded on the gadolinium(III) (Gd3+) complexes, complemented with the assessment of hydration numbers via luminescence lifetime measurements on the Eu3+ analogues, allowed us to characterize the lanthanide coordination sphere in the chelates. These data suggest that the potential donor groups of the peptide side chains (carboxylate, amine) interfere in metal coordination, leading to non-hydrated LnL3 and LnL4 complexes. Nevertheless, GdL3 and GdL4 retain a relatively high relaxivity due to an important second-sphere contribution generated by the strongly hydrophilic peptide chain. Weak PARACEST effects are detected for the amine-derivative EuL1 and NdL1 chelates. Unfortunately, the GdL3 and GdL4 complexes are not significantly converted by the enzymes. The lack of enzymatic recognition of these complexes can likely be explained by the participation of donor groups from the peptide side chain in metal coordination.

Expanding the Ligand Classes Used for Mn(II) Complexation: Oxa-aza Macrocycles Make the Difference.

We report two macrocyclic ligands based on a 1,7-diaza-12-crown-4 platform functionalized with acetate (tO2DO2A2−) or piperidineacetamide (tO2DO2AMPip) pendant arms and a detailed characterization of the corresponding Mn(II) complexes. The X−ray structure of [Mn(tO2DO2A)(H2O)]·2H2O shows that the metal ion is coordinated by six donor atoms of the macrocyclic ligand and one water molecule, to result in seven-coordination. The Cu(II) analogue presents a distorted octahedral coordination environment. The protonation constants of the ligands and the stability constants of the complexes formed with Mn(II) and other biologically relevant metal ions (Mg(II), Ca(II), Cu(II) and Zn(II)) were determined using potentiometric titrations (I = 0.15 M NaCl, T = 25 °C). The conditional stabilities of Mn(II) complexes at pH 7.4 are comparable to those reported for the cyclen-based tDO2A2− ligand. The dissociation of the Mn(II) chelates were investigated by evaluating the rate constants of metal exchange reactions with Cu(II) under acidic conditions (I = 0.15 M NaCl, T = 25 °C). Dissociation of the [Mn(tO2DO2A)(H2O)] complex occurs through both proton− and metal−assisted pathways, while the [Mn(tO2DO2AMPip)(H2O)] analogue dissociates through spontaneous and proton-assisted mechanisms. The Mn(II) complex of tO2DO2A2− is remarkably inert with respect to its dissociation, while the amide analogue is significantly more labile. The presence of a water molecule coordinated to Mn(II) imparts relatively high relaxivities to the complexes. The parameters determining this key property were investigated using 17O NMR (Nuclear Magnetic Resonance) transverse relaxation rates and 1H nuclear magnetic relaxation dispersion (NMRD) profiles.

Mn2+ Complexes with Pyclen-Based Derivatives as Contrast Agents for Magnetic Resonance Imaging: Synthesis and Relaxometry Characterization.

Magnetic resonance imaging (MRI) has a leading place in medicine as an imaging tool of high resolution for anatomical studies and diagnosis of diseases, in particular for soft tissues that cannot be accessible by other modalities. Many research works are thus focused on improving the images obtained with MRI. This technique has indeed poor sensitivity, which can be compensated by using a contrast agent (CA). Today, the clinically approved CAs on market are solely based on gadolinium complexes that may induce nephrogenic systemic fibrosis for patients with kidney failure, whereas more recent studies on healthy rats also showed Gd retention in the brain. Consequently, researchers try to elaborate other types of safer MRI CAs like manganese-based complexes. In this context, the synthesis of Mn2+ complexes of four 12-membered pyridine-containing macrocyclic ligands based on the pyclen core was accomplished and described herein. Then, the properties of these Mn(II) complexes were studied by two relaxometric methods, 17O NMR spectroscopy and 1H NMR dispersion profiles. The time of residence (τM) and the number of water molecules (q) present in the inner sphere of coordination were determined by these two experiments. The efficacy of the pyclen-based Mn(II) complexes as MRI CAs was evaluated by proton relaxometry at a magnetic field intensity of 1.41 T near those of most medical MRI scanners (1.5 T). Both the 17O NMR and the nuclear magnetic relaxation dispersion profiles indicated that the four hexadentate ligands prepared herein left one vacant coordination site to accommodate one water molecule, rapidly exchanging, in around 6 ns. Furthermore, it has been shown that the presence of an additional amide bond formed when the paramagnetic complex is conjugated to a molecule of interest does not alter the inner sphere of coordination of Mn, which remains monohydrated. These complexes exhibit r1 relaxivities, large enough to be used as clinical MRI CAs (1.7-3.4 mM-1·s-1, at 1.41 T and 37 °C).