Measurement of Slow Diffusion Coefficients of Molecules with Arbitrary Scalar Couplings via Long‐Lived Spin States

New experiments are described for the determination of very slow diffusion constants by nuclear magnetic resonance (NMR) using long‐lived (singlet) states. These experiments are suitable for molecules or conformations featuring a wide range of J‐couplings.     

Long‐Lived Spin States for Low‐Field Hyperpolarized Gas MRI

Parahydrogen induced polarization was employed to prepare a relatively long‐lived correlated nuclear spin state between methylene and methyl protons in propane gas. Conventionally, such states are converted into a strong NMR signal enhancement by transferring the reaction product to a high magnetic field in an adiabatic longitudinal transport after dissociation engenders net alignment (ALTADENA) experiment. However, the relaxation time T₁ of ～0.6 s of the resulting hyperpolarized propane is too short for potential biomedical applications. The presented alternative approach employs low‐field MRI to preserve the initial correlated state with a much longer decay time T_LLSS=(4.7±0.5) s. While the direct detection at low‐magnetic fields (e.g. 0.0475 T) is challenging, we demonstrate here that spin‐lock induced crossing (SLIC) at this low magnetic field transforms the long‐lived correlated state into an observable nuclear magnetization suitable for MRI with sub‐millimeter and sub‐second spatial and temporal resolution, respectively. Propane is a non‐toxic gas, and therefore, these results potentially enable low‐cost high‐resolution high‐speed MRI of gases for functional imaging of lungs and other applications.     

Long‐Lived States of Magnetically Equivalent Spins Populated by Dissolution‐DNP and Revealed by Enzymatic Reactions

Hyperpolarization by dissolution dynamic nuclear polarization (D ‐DNP) offers a way of enhancing NMR signals by up to five orders of magnitude in metabolites and other small molecules. Nevertheless, the lifetime of hyperpolarization is inexorably limited, as it decays toward thermal equilibrium with the nuclear spin‐lattice relaxation time. This lifetime can be extended by storing the hyperpolarization in the form of long‐lived states (LLS) that are immune to most dominant relaxation mechanisms. Levitt and co‐workers have shown how LLS can be prepared for a pair of inequivalent spins by D ‐DNP. Here, we demonstrate that this approach can also be applied to magnetically equivalent pairs of spins such as the two protons of fumarate, which can have very long LLS lifetimes. As in the case of para‐hydrogen, these hyperpolarized equivalent LLS (HELLS) are not magnetically active. However, a chemical reaction such as the enzymatic conversion of fumarate into malate can break the magnetic equivalence and reveal intense NMR signals.     

Paramagnetic Relaxation of Long‐Lived Coherences in Solution NMR

Long‐lived coherences (LLCs) are known to have lifetimes much longer than transverse magnetization or single quantum coherences (SQCs). The effect of paramagnetic ions on the relaxation of LLCs is not known. This is particularly important, as LLCs have potential applications in various fields like analytical NMR, in vivo NMR and MR imaging methods. We study here the behaviour of LLCs in the presence of paramagnetic relaxation agents. The stepwise increase in the concentration of the metal ion is followed by measuring various relaxation rates. The effect of paramagnetic ions is analysed in terms of the external random field’s contribution to the relaxation of two coupled protons in 2,3,6‐trichlorobenzaldehyde. The LLCs relax faster than ordinary SQCs in the presence of paramagnetic ions of varying character. This is explained on the basis of an increase in the contribution of the external random field to relaxation due to a paramagnetic relaxation mechanism. Comparison is also made with ordinary Zeeman relaxation rates like R₁, R₂, R_1ρ and also with rate of relaxation of long‐lived states R_LLS which are known to be less sensitive to paramagnetically induced relaxation. Also, the extent of correlation of random fields at two proton sites is studied and is found to be strongly correlated with each other. The obtained correlation constant is found to be independent of the nature of added paramagnetic impurities.     

Long‐lived states in an intrinsically disordered protein domain

Long‐lived states (LLS) are relaxation‐favored spin population distributions of J‐coupled magnetic nuclei. LLS were measured, along with classical ¹H and ¹⁵N relaxation rate constants, in amino acids of the N‐terminal Unique domain of the c‐Src kinase, which is disordered in vitro under physiological conditions. The relaxation rates of LLS can probe motions and interactions in biomolecules. LLS of the aliphatic protons of glycines, with lifetimes approximately four times longer than their spin–lattice relaxation times, are reported for the first time in an intrinsically disordered protein domain. LLS relaxation experiments were integrated with 2D spectroscopy methods, further adapting them for studies on proteins. Copyright © 2013 John Wiley & Sons, Ltd.     

Long‐Lived 1H Nuclear Spin Singlet in Dimethyl Maleate Revealed by Addition of Thiols

Nuclear magnetic resonance (NMR) spectroscopy and magnetic resonance imaging (MRI) have become important techniques in many research areas. One major limitation is the relatively low sensitivity of these methods, which recently has been addressed by hyperpolarization. However, once hyperpolarization is imparted on a molecule, the magnetization typically decays within relatively short times. Singlet states are well isolated from the environment, such that they acquire long lifetimes. We describe herein a model reaction for read‐out of a hyperpolarized long‐lived state in dimethyl maleate using thiol conjugate addition. This type of reaction could lend itself to monitoring oxidative stress or hypoxia by sensitive detection of thiols. Similar reactions could be used in biosensors or assays that exploit molecular switching. Singlet lifetimes of about 4.7 min for ¹H spins in [D₄]MeOH are seen in this system.     

A Nuclear Singlet Lifetime of More than One Hour in Room‐Temperature Solution

Nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) are supremely important techniques with numerous applications in almost all branches of science. However, until recently, NMR methodology was limited by the time constant T₁ for the decay of nuclear spin magnetization through contact with the thermal molecular environment. Long‐lived states, which are correlated quantum states of multiple nuclei, have decay time constants that may exceed T₁ by large factors. Here we demonstrate a nuclear long‐lived state comprising two ¹³C nuclei with a lifetime exceeding one hour in room‐temperature solution, which is around 50 times longer than T₁. This behavior is well‐predicted by a combination of quantum theory, molecular dynamics, and quantum chemistry. Such ultra‐long‐lived states are expected to be useful for the transport and application of nuclear hyperpolarization, which leads to NMR and MRI signals enhanced by up to five orders of magnitude.     

Long-lived states to monitor protein unfolding by proton NMR

The relaxation of long-lived states (LLS) corresponds to the slow return to statistical thermal equilibrium between symmetric and antisymmetric proton spin states. This process is remarkably sensitive to the presence of external spins and can be used to obtain information about partial unfolding of proteins. We detected the appearance of a destabilized conformer of ubiquitin when urea is added to the protein in its native state. This conformer shows increased mobility in the C-terminus, which significantly extends the lifetimes of proton LLS magnetisation in Ser-65. These changes could not be detected by conventional measurements of T(1) and T(2) relaxation times of protons, and would hardly be sensed by carbon-13 or nitrogen-15 relaxation measurements. Conformers with similar dynamic and structural features, as revealed by LLS relaxation times, could be observed, in the absence of urea, in two ubiquitin mutants, L67S and L69S.     

Magnetization Lifetimes Prediction and Measurements Using Long-Lived Spin States in Endogenous Molecules

Nuclear magnetization storage in biologically-relevant molecules opens new possibilities for the investigation of metabolic pathways, provided the lifetimes of magnetization are sufficiently long. Dissolution-dynamic nuclear polarization-based spin-order enhancement, sustained by long-lived states can measure the ratios between concentrations of endogenous molecules on a cellular pathway. These ratios can be used as meters of enzyme function. Biological states featuring intracellular amino-acid concentrations that are depleted or replenished in the course of in-cell or in-vivo tests of drugs or radiation treatments can be revealed. Progressing from already-established long-lived states, we investigated related spin order in the case of amino acids and other metabolites featuring networks of coupled spins counting up to eight nuclei. We detail a new integrated theoretical approach between quantum chemistry simulations, chemical shifts, J-couplings information from databanks, and spin dynamics calculations to deduce a priori magnetization lifetimes in biomarkers. The lifetimes of long-lived states for several amino acids were also measured experimentally in order to ascertain the approach. Experimental values were in fair agreement with the computed ones and prior data in the literature.     

Long‐Lived Charge Carriers in Mn‐Doped CdS Quantum Dots for Photoelectrochemical Cytosensing

Photoelectrochemical (PEC) biosensing with semiconductor quantum dots (QDs) has received great attention because it integrates the advantages of both photo‐excitation and electrochemical detection. During the photon‐to‐electricity conversion in PEC processes, electron–hole (charge) separation competes with electron–hole recombination, and the net effect essentially determines the performance of PEC biosensors. Herein, we propose a new approach for slowing down electron–hole recombination to increase charge separation efficiency for PEC biosensor development. Through doping with Mn²⁺, a pair of d bands (⁴T₁ and ⁶A₁) is inserted between the conduction and valence bands of CdS QDs, which alters the electron–hole separation and recombination dynamics, allowing the generation of long‐lived charge carriers with ms‐scale lifetime that decay about 10⁴–10⁵‐fold more slowly than in the case of undoped QDs. Photocurrent tests indicated that Mn²⁺ doping resulted in an approximately 80 % increase in photocurrent generation compared with undoped CdS QDs. For application, the Mn‐doped CdS QDs were coated on the surface of a glassy carbon electrode and functionalized with a cell surface carbohydrate‐specific ligand (3‐aminophenylboronic acid). In this way, a sensitive cytosensor for K562 leukemia cells was constructed. Moreover, the sugar‐specific binding property of 3‐aminophenylboronic acid allowed the electrode to serve as a switch for the capture and release of cells. This has been further explored with a view to developing a reusable PEC cytosensing platform.     

Kinetics of the chromium(III)/l‐glutamic acid complexation reaction: Formation,decay, and UV‐vis spectrum of a long‐lived intermediate

The kinetics of the aqueous reaction of Cr(III) with either l ‐glutamic acid or sodium hydrogen l ‐glutamate at pH 2.46‐5.87 have been followed by means of absorbance readings. The rate of formation of the reaction products showed acceleration‐deceleration periods, caused by the accumulation and posterior decay of an intermediate in nonnegligible concentration. A double‐exponential integrated rate law allowed obtaining two rate constants for each absorbance‐time experimental series, associated with the appearance (k₁) and decay (k₂) of the long‐lived intermediate. An increase of the initial concentrations of either hydrogen l ‐glutamate (apparent kinetic orders < 1) or hydroxide (kinetic orders = 1) ions resulted in an increase of both k₁ and k₂, but addition of an inert electrolyte (KNO₃) resulted in opposite effects on k₁ (decrease) and k₂ (increase). The experimental activation energies were 83 ± 10 (for k₁) and 95 ± 5 (for k₂) kJ mol⁻¹. The electronic spectrum of the low reactivity detected intermediate resembled more closely to that of the blue/green reactant than that of the violet reaction product. The low number of protons set free by the complexating hydrogen l ‐glutamate ligand seems to suggest that some polymerization of the coordinated amino acid (to form a di‐ or tripeptide) might take place. The available experimental data indicate that the coordination of the organic ligand must be preceded by the breakdown of a strong Cr(III)–H₂O chemical bond in the slow steps of the mechanism.     

Preparation of Selenoinsulin as a Long‐Lasting Insulin Analogue

Synthetic insulin analogues with a long lifetime are current drug targets for the therapy of diabetic patients. The replacement of the interchain disulfide with a diselenide bridge, which is more resistant to reduction and internal bond rotation, can enhance the lifetime of insulin in the presence of the insulin‐degrading enzyme (IDE) without impairing the hormonal function. The [C7U^A,C7U^B] variant of bovine pancreatic insulin (BPIns) was successfully prepared by using two selenocysteine peptides (i.e., the C7U analogues of A‐ and B‐chains, respectively). In a buffer solution at pH 10 they spontaneously assembled under thermodynamic control to the correct insulin fold. The selenoinsulin (Se‐Ins) exhibited a bioactivity comparable to that of BPIns. Interestingly, degradation of Se‐Ins with IDE was significantly decelerated (τ _1/2≈8 h vs. ≈1 h for BPIns). The lifetime enhancement could be due to both the intrinsic stability of the diselenide bond and local conformational changes induced by the substitution.