Paul R. Vasos

Long-lived states to sustain hyperpolarized magnetization

Major breakthroughs have recently been reported that can help overcome two inherent drawbacks of NMR: the lack of sensitivity and the limited memory of longitudinal magnetization. Dynamic nuclear polarization (DNP) couples nuclear spins to the large reservoir of electrons, thus making it possible to detect dilute endogenous substances in magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI). We have designed a method to preserve enhanced (“hyperpolarized”) magnetization by conversion into long-lived states (LLS). It is shown that these enhanced long-lived states can be generated for proton spins, which afford sensitive detection. Even in complex molecules such as peptides, long-lived proton states can be sustained effectively over time intervals on the order of tens of seconds, thus allowing hyperpolarized substrates to reach target areas and affording access to slow metabolic pathways. The natural abundance carbon-13 polarization has been enhanced ex situ by almost four orders of magnitude in the dipeptide Ala-Gly. The sample was transferred by the dissolution process to a high-resolution magnet where the carbon-13 polarization was converted into a long-lived state associated with a pair of protons. In Ala-Gly, the lifetime TLLS associated with the two nonequivalent Hα glycine protons, sustained by suitable radio-frequency irradiation, was found to be seven times longer than their spin-lattice relaxation time constant (TLLS/T1 = 7). At desired intervals, small fractions of the populations of long-lived states were converted into observable magnetization. This opens the way to observing slow chemical reactions and slow transport phenomena such as diffusion by enhanced magnetic resonance.

Proton NMR of (15)N-choline metabolites enhanced by dynamic nuclear polarization.

Chemical shifts of protons can report on metabolic transformations such as the conversion of choline to phosphocholine. To follow such processes in vivo, magnetization can be enhanced by dynamic nuclear polarization (DNP). We have hyperpolarized in this manner nitrogen-15 spins in (15)N-labeled choline up to 3.3% by irradiating the 94 GHz electron spin resonance of admixed TEMPO nitroxide radicals in a magnetic field of 3.35 T during ca. 3 h at 1.2 K. The sample was subsequently transferred to a high-resolution magnet, and the enhanced polarization was converted from (15)N to methyl- and methylene protons, using the small (2,3)J((1)H,(15)N) couplings in choline. The room-temperature lifetime of nitrogen polarization in choline, T(1)((15)N) approximately 200 s, could be considerably increased by partial deuteration of the molecule. This procedure enables studies of choline metabolites in vitro and in vivo using DNP-enhanced proton NMR.

Scavenging Free Radicals To Preserve Enhancement and Extend Relaxation Times in NMR using Dynamic Nuclear Polarization

This enhance-ment arises from thermal mixing, which is brought about bymicrowavesaturationoftheEPRtransitionsofstableradicalsthat are mixed with the sample under investigation beforefreezing. In dissolution DNP, the sample is usually polarizedat low temperatures and moderate magnetic fields (T=1.2 Kand B

Diffusion coefficients of biomolecules using long-lived spin states.

We report the first observation of long-lived states (LLS) having lifetimes T(LLS) that exceed the corresponding spin-lattice relaxation times T(1) by more than a factor 6 in a protein. Slow diffusion coefficients characteristic of large biomolecules can be determined by combining LLS methods with moderate pulsed field gradients (PFGs) available on commercial probeheads, as the extension of spin memory reduces the strain on the duration and/or strength of the PFGs. No isotope labeling of the biomolecule is necessary.

Proton hyperpolarisation preserved in long-lived states

The polarisation of abundant protons, rather than dilute nuclei with low gyromagnetic ratios, can be enhanced in less than 10 min using dissolution DNP and converted into a long-lived state delocalised over an ensemble of three coupled protons. The process is more straightforward than the hyperpolarisation of heteronuclei followed by magnetisation transfer to protons.

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 States in Multiple-Spin Systems

Long-lived spin states are excited in molecules featuring more than two isolated coupled spins, including amino acids. The figure shows the exponential recovery with the longest time-constant in aspartic acid, T1max=5.842±0.004 s, and of the decay of the long-lived state, TLLS=10.9±0.2 s). An improvement in spin memory by a factor 2 compared to longitudinal spin-lattice relaxation time constants is obtained for most systems.

Long-lived coherences for line-narrowing in high-field NMR.

2010 Elsevier B.V. All rights reserved.Contents1. Introduction . . . 832. The tailored Hamiltonian. . . . . . . . . . . . 843. Definition of long-lived coherences . . . 854. Relaxation of long-lived coherences . . . 855. Designing suitable experiments . . . . . . 866. Long-lived coherences in a small molecule . . . . . . . . . . . . . . . . 867. Long-lived coherences in a protein. . . . 878. Simultaneous excitation of several long-lived coherences . . . . 879. Conclusions. . . . 88Acknowledgements . . . . . . . . . . . . . . . . 89Appendix A . . . . . 89References . . . . 90

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.

Single or triple gradients

Pulsed Field Gradients (PFGs) have become ubiquitous tools not only for Magnetic Resonance Imaging (MRI), but also for NMR experiments designed to study translational diffusion, for spatial encoding in ultra-fast spectroscopy, for the selection of desirable coherence transfer pathways, for the suppression of solvent signals, and for the elimination of zero-quantum coherences. Some of these experiments can only be carried out if three orthogonal gradients are available, while others can also be implemented using a single gradient, albeit at some expense of performance. This paper discusses some of the advantages of triple- with respect to single-gradient probes. By way of examples we discuss (i) the measurement of small diffusion coefficients making use of the long spin-lattice relaxation times of nuclei with low gyromagnetic ratios gamma such as nitrogen-15, and (ii) the elimination of zero-quantum coherences in Exchange or Nuclear Overhauser Spectroscopy (EXSY or NOESY) experiments, as well as in methods relying on long-lived (singlet) states to study very slow exchange or diffusion processes.