Publications
2022
Nuclear magnetic resonance (NMR) spectroscopy provides detailed information about dynamic processes through line-shape changes, which are traditionally limited to equilibrium conditions. However, a wealth of information is available by studying chemical reactions under off-equilibrium conditionse.g., in states that arise upon mixing reactants that subsequently undergo chemical changesand in monitoring the reactants and products in real time. Herein, we propose and demonstrate a time-resolved kinetic NMR experiment that combines rapid mixing techniques, continuous flow, and single-scan spectroscopic imaging methods, leading in unison to a 2D spectrotemporal NMR correlation that provides high-quality kinetic information of off-equilibrium chemical reactions. These kinetic 2D NMR spectra possess a high-resolution spectral dimension revealing the individual chemical sites, correlated with a time-independent, steady-state spatial axis that delivers information concerning temporal changes along the reaction coordinate. A comprehensive description of the kinetic, spectroscopic, and experimental features associated with these spectrotemporal NMR analyses is presented. Experimental demonstrations are carried out using an enzymatically catalyzed reaction leading to site- and time-resolved kinetic NMR data, that are in excellent agreement with control experiments and literature values.
The benefits of performing locally low-rank (LLR) reconstructions on subsampled diffusion weighted and diffusion kurtosis imaging data employing spatiotemporal encoding (SPEN) methods, is investigated. SPEN allows for self-referenced correction of motion-induced phase errors in case of interleaved diffusion-oriented acquisitions, and allows one to overcome distortions otherwise observed along EPI's phase-encoded dimension. In combination with LLR-based reconstructions of the pooled imaging data and with a joint subsampling of b-weighted and interleaved images, additional improvements in terms of sensitivity as well as shortened acquisition times are demonstrated, without noticeable penalties. Details on how the LLR-regularized, subspace-constrained image reconstructions were adapted to SPEN are given; the improvements introduced by adopting these reconstruction frameworks for the accelerated acquisition of diffusivity and of kurtosis imaging data in both relatively homogeneous regions like the human brain and in more challenging regions like the human prostate, are presented and discussed within the context of similar efforts in the field.
There are currently no methods for the acquisition of ultra-wideline (UW) solid-state NMR spectra under static conditions that enable reliable separation and resolution of overlapping powder patterns arising from magnetically distinct nuclei. This stands in contrast to the variety of techniques available for spin-1/2 or half-integer quadrupolar nuclei with narrow central transition patterns under magic-angle spinning (MAS). Resolution of overlapping signals is routinely achieved in MRI and solution-state NMR by exploiting relaxation differences between nonequivalent sites. Preliminary studies of relaxation assisted separation (RAS) for separating overlapping UWNMR patterns using pseudo-inverse Laplace Transforms have reported two-dimensional spectra featuring relaxation rates correlated to NMR interaction frequencies. However, RAS methods are inherently sensitive to experimental noise, and require that relaxation rates associated with overlapped patterns be significantly different from one another. Herein, principal component analysis (PCA) denoising is implemented to increase the signal-to-noise ratios of the relaxation datasets and RAS routines are stabilized with truncated singular value decomposition (TSVD) and elastic net (EN) regularization to resolve overlapped patterns with a larger tolerance for differences in relaxation rates. We extend these methods for improved pattern resolution by utilizing 3D frequency-R1R2 correlation spectra. Synthetic and experimental datasets, including 35Cl (I = 3/2), 2H (I = 1), and 14N (I = 1) NMR of organic and biological compounds, are explored with both regularized 2D RAS and 3D RAS; comparison of these data reveal improved resolution in the latter case. These methods have great potential for separating overlapping powder patterns under both static and MAS conditions.
17O and 14N are attractive targets for in vivo NMR spectroscopy and imaging, but low gyromagnetic ratios γ and fast spin relaxation complicate observations. This work explores indirect ways of detecting some of these sites with the help of proton-detected double resonance techniques. As standard coherence transfer methods are of limited use for such indirect detection, alternative routes for probing the quadrupolar spectra on 1H were tested. These centered on modulating the broadening effects imparted onto protons adjacent to the low-γ species through J couplings through either continuous wave or spin-echo double-resonance decoupling/recoupling sequences. As in all cases, the changes imparted by these double-resonance strategies were small due to the fast relaxation undergone by the quadrupoles, the sensitivity of these approaches was amplified by transferring their effects onto the abundant water 1H signal. These amplifications were mediated by the spontaneous exchanges that the labile 1Hs bound to 17O or 14N undergo with the water protons. In experiments designed on the basis of double-resonance spin echoes, these enhancements were imparted by looping the transverse encodings together with multiple longitudinal storage periods, leading to decoupling-recoupling with exchange (D-REX) sequences. In experiments designed on the basis of continuous on/off quadrupolar decoupling, these solvent exchanges were incorporated into chemical-exchange saturation transfer schemes, leading to decoupling-recoupling with saturation transfer (D-REST) sequences. Both of these variants harnessed sizable proportions of the easily detectable water signals, in order to characterize the NMR spectra and/or to image with atomic-site specificity the 17O and 14N species.
Overhauser dynamic nuclear polarization (ODNP) NMR of solutions at high fields is usually mediated by scalar couplings that polarize the nuclei of heavier, electron-rich atoms. This leaves 1H-detected NMR outside the realm of such studies. This study presents experiments that deliver 1H-detected NMR experiments on relatively large liquid volumes (60 ∼ 100 μL) and at high fields (14.1 T), while relying on ODNP enhancements. To this end 13C NMR polarizations were first enhanced by relying on a mechanism that utilizes e--13C scalar coupling interactions; the nuclear spin alignment thus achieved was then passed on to neighboring 1H for observation, by a reverse INEPT scheme relying on one-bond JCH-couplings. Such 13C → 1H polarization transfer ported the 13C ODNP gains into the 1H, permitting detection at higher frequencies and with higher potential sensitivities. For a model solution of labeled 13CHCl3 comixed with a nitroxide-based TEMPO derivative as polarizing agent, an ODNP enhancement factor of ca. 5x could thus be imparted to the 1H signal. When applied to bigger organic molecules like 2-13C-phenylacetylene and 13C8-indole, ODNP enhancements in the 1.2-3x range were obtained. Thus, although handicapped by the lower γ of the 13C, enhancements could be imparted on the 1H thermal acquisitions in all cases. We also find that conventional 1H13C nuclear Overhauser enhancements (NOEs) are largely absent in these solutions due to the presence of co-dissolved radicals, adding negligible gains and playing negligible roles on the scalar e-→13C ODNP transfer. Potential rationalizations of these effects as well as extensions of these experiments, are briefly discussed.
NMR spectroscopy is the only method to access the structural dynamics of biomolecules at high (atomistic) resolution in their native solution state. However, this method's low sensitivity has two important consequences: (i) typically experiments have to be performed at high concentrations that increase sensitivity but are not physiological, and (ii) signals have to be accumulated over long periods, complicating the determination of interaction kinetics on the order of seconds and impeding studies of unstable systems. Both limitations are of equal, fundamental relevance: non-native conditions are of limited pharmacological relevance, and the function of proteins, enzymes and nucleic acids often relies on their interaction kinetics. To overcome these limitations, we have developed applications that involve 'hyperpolarized water' to boost signal intensities in NMR of proteins and nucleic acids. The technique includes four stages: (i) preparation of the biomolecule in partially deuterated buffers, (ii) preparation of 'hyperpolarized' water featuring enhanced H-1 NMR signals via cryogenic dynamic nuclear polarization, (iii) sudden melting of the cryogenic pellet and dissolution of the protein or nucleic acid in the hyperpolarized water (enabling spontaneous exchanges of protons between water and target) and (iv) recording signal-amplified NMR spectra targeting either labile H-1 or neighboring N-15/C-13 nuclei in the biomolecule. Water in the ensuing experiments is used as a universal 'hyperpolarization' agent, rendering the approach versatile and applicable to any biomolecule possessing labile hydrogens. Thus, questions can be addressed, ranging from protein and RNA folding problems to resolving structure-function relationships of intrinsically disordered proteins to investigating membrane interactions.
Quadrupolar solid-state NMR carries a wealth of structural information, including insights about chemical environments arising through the determination of local coupling parameters. Current methods can successfully resolve these parameters for individual sites using sample-spinning methods techniques applicable to quadrupolar I ≥ 1 nuclei, provided second-order central transition broadenings do not exceed by much the spinning rate. For large quadrupolar coupling (CQ) values, however, static acquisitions are often preferable, leading to challenges in extracting local structural information. This study explores the use of two-dimensional QUadrupolar Isotope Correlation SpectroscopY (QUICSY) experiments as a means to increase the NMR spectral resolution and enrich the characterization of quadrupolar NMR patterns under static conditions. QUICSY seeks to correlate the solid-state NMR powder line shapes for two quadrupolar isotopes belonging to the same element via a 2D experiment. In general, two isotopes of the same element will have different nuclear quadrupole moments, gyromagnetic ratios, and spin numbers but essentially identical chemical environments. The possibility then arises of obtaining sharp "ridges"in these 2D correlations, even in static samples showing large quadrupolar effects, which lead to second-order line shapes that are several kilohertz wide. Moreover, pairs of quadrupolar isotopes are recurrent in the periodic table and include important elements such as 35,37Cl, 69,71Ga, 79,81Br, and 85,87Rb. The potential of this approach is explored theoretically and experimentally on two rubidium-containing salts: RbClO4 and Rb2SO4. We find that each compound gives rise to distinctive 2D QUICSY line shapes, depending on the quadrupolar and chemical shift anisotropy (CSA) parameters of its sites. These experimental line shapes show good agreement with analytically derived 2D spectra relying on literature values of the quadrupolar and CSA tensors of these compounds. The approach underlined here paves the way toward better characterization of wideline NMR spectra of quadrupolar nuclei possessing different nuclear isotopes.
Out of all the magnetic resonance communities active in the world today, few are as vibrant and influential as that of India. India has done well both in terms of generating the basis of scientists that drive magnetic resonance research throughout the world, as well as in terms of its own magneticresonance productivity and impact. Prof. Anil Kumar has played a central role in both of these endeavors. As tribute to Prof. Kumar's 80th birthday (June 2021) and to pay homage to India's blossoming magnetic resonance landscape, we are co-editing a special Golden Open Access issue of Journal of Magnetic Resonance Open (JMRO), entitled \u201cCelebrating Magnetic Resonance in India - Today\u201d.
The creation of radicals via electrical discharge is a unique approach capable of facilitating dynamic nuclear polarization (DNP) in solids without the need to co-dissolve them with exogenous polarization agents. This method has previously been shown to generate high proton polarization in a variety of organic and inorganic solid compounds. In this work, we extend the scope of applicability for these so-called electrical discharge-induced radicals (EDIRs), by showing their use in DNP of 13C nuclei of glucose. We achieved significant NMR signal enhancement in low temperature solid-state DNP experiments. This enhancement was further improved by the use of a frequency modulated microwave pump instead of conventional DNP with single-frequency microwaves. We demonstrated successful dissolution DNP experiments with such hyperpolarized solid glucose, achieving 13C polarization levels of up to ∼0.6% in the resulting solution (∼0.95% at the exit of the polarizer). This is to be compared with the ∼6% polarization levels achieved with a trityl radical mixed with glucose in a frozen aqueous solution using the same DNP setup and experimental conditions. It is concluded that the use of EDIRs can serve as a suitable polarizing approach for metabolic MRI using glucose, avoiding the need to employ diluted solutions and eliminating the filtration stage of the polarizing agents.
Both in spectroscopy and imaging, t1-noise arising from instabilities such as temperature alterations, field-related frequency drifts, electronic and sample-spinning instabilities, or motions in in vivo experiments, affects many 2D Magnetic Resonance experiments. This work introduces a post-processing method that aims to attenuate t1-noise, by suitably averaging multiple signals/representations that have been reconstructed from the sampled data. The ensuing Compressed Sensing Multiplicative (CoSeM) denoising starts from a fully sampled 2D MR data set, discards random indirect-domain points, and makes up for these missing, masked data, by a compressed sensing reconstruction of the now incompletely sampled 2D data set. This procedure is repeated for multiple renditions of the masked data some of which will have been more strongly affected by t1-noise than others. This leads to a large set of 2D NMR spectra/images compatible with the collected data; CoSeM chooses out of these those renditions that reduce the noise according to a suitable criterion, and then sums up their spectra/images leading to a reduction in t1-noise. The performance of the method was assessed in synthetic data, as well as in numerous different experiments: 2D solid and solution state NMR, 2D localized MRS of live brains, and 2D abdominal MRI. Throughout all these data, CoSeM processing evidenced 23 fold increases in SNR, without introducing biases, false peaks, or spectral/image blurring. CoSeM also retains a quantitative linearity in the information allowing, for instance, reliable T1 inversion-recovery MRI mapping experiments.
The ongoing pandemic of the respiratory disease COVID-19 is caused by the SARS-CoV-2 (SCoV2) virus. SCoV2 is a member of the Betacoronavirus genus. The 30 kb positive sense, single stranded RNA genome of SCoV2 features 5- and 3-genomic ends that are highly conserved among Betacoronaviruses. These genomic ends contain structured cis-acting RNA elements, which are involved in the regulation of viral replication and translation. Structural information about these potential antiviral drug targets supports the development of novel classes of therapeutics against COVID-19. The highly conserved branched stem-loop 5 (SL5) found within the 5-untranslated region (5-UTR) consists of a basal stem and three stem-loops, namely SL5a, SL5b and SL5c. Both, SL5a and SL5b feature a 5-UUUCGU-3 hexaloop that is also found among Alphacoronaviruses. Here, we report the extensive 1H, 13C and 15N resonance assignment of the 37 nucleotides (nts) long sequence spanning SL5b and SL5c (SL5b + c), as basis for further in-depth structural studies by solution NMR spectroscopy.
Homonuclear isotropic mixing modules allow J-coupled spins to exchange magnetization even when separated by chemical shift offsets that exceed their couplings. This is exploited in TOtal Correlation SpectroscopY (TOCSY) experiments and its variants, which facilitate these homonuclear polarization exchanges by applying broadband RF pulses. These then establish an effective Hamiltonian in which chemical shift offsets are erased, while J-coupling terms including flip-flop components remain active. The polarization that these non-secular terms will transfer among systems of chemically inequivalent sites over the course of a mixing period, are widely used modules in 1D and in multidimensional liquid-state NMR. Homonuclear correlation experiments are also common in solids NMR, particularly among X = 13C or 15N nuclei. Solids NMR experiments are often challenged by high-power RF demands which have led to a family of homonuclear solid-state correlation experiments that avoid pulsing on the nuclei of interest, and focus instead on the 1Hs that are bonded to them. These solid experiments usually reintroduce/strengthen 1H-X dipolar couplings; these, in conjunction with assistance from rotational resonance effects, bring back the truncated X-X dipolar interactions and facilitate the generation of cross peaks. The present study explores whether a similar goal can be achieved for solution-state counterparts, based on the reintroduction of truncated flip-flop terms in the J-coupling Hamiltonian via the pulsing on other, heteronuclear species. A proposal to achieve this is derived, and the resulting HOmonucleaR Recoupling by hEteroNuclear DecOUplingS (HORRENDOUS) approach to provide correlations between like nuclei without pulsing on them, is demonstrated.
Chemical exchange saturation transfer (CEST) is widely used for enhancing the solution nuclear magnetic resonance (NMR) signatures of magnetically dilute spin pools, in particular, species at low concentrations undergoing chemical exchanges with an abundant spin pool. CESTs main feature involves encoding and then detecting weak NMR signals of the magnetically dilute spin pools on a magnetically abundant spin pool of much easier detection, for instance, the protons of H2O. Inspired by this method, we propose and exemplify a methodology to enhance the sensitivity of magic-angle spinning (MAS) solid-state NMR spectra. Our proposal uses the abundant 1H reservoir arising in organic solids as the magnetically abundant spin pool and relies on proton spin diffusion in lieu of chemical exchange to mediate polarization transfer between a magnetically dilute spin pool and this magnetically abundant spin reporter. As an initial test of this idea, we target the spectroscopy of naturally abundant 13C and rely on a Fourier-encoded version of the CEST experiment for achieving broadbandness in coordination with both MAS and heteronuclear decoupling, features normally absent in CEST. Arbitrary evolutions of multiple 13C sites can, thus, be imprinted on the entire 1H reservoir, which is subsequently detected. Theoretical predictions suggest that orders-of-magnitude signal enhancements should be achievable in this manner, on the order of the ratio between the 13C and the 1H reservoirs abundances. Experiments carried out under magic-angle spinning conditions evidenced 510× gains in signal amplitudes. Further opportunities and challenges arising in this Fourier-encoded saturation transfer MAS NMR approach are briefly discussed.
Hadamard encoded saturation transfer can significantly improve the efficiency of NOE-based NMR correlations from labile protons in proteins, glycans and RNAs, increasing the sensitivity of cross-peaks by an order of magnitude and shortening experimental times by ≥100-fold. These schemes, however, fail when tackling correlations within a pool of labile protons for instance imino-imino correlations in RNAs or amide-amide correlations in proteins. Here we analyze the origin of the artifacts appearing in these experiments and propose a way to obtain artifact-free correlations both within the labile pool as well as between labile and non-labile 1Hs, while still enjoying the gains arising from Hadamard encoding and solvent repolarizations. The principles required for implementing what we define as the extended Hadamard scheme are derived, and its clean, artifact-free, sensitivity-enhancing performance is demonstrated on RNA fragments derived from the SARS-CoV-2 genome. Sensitivity gains per unit time approaching an order of magnitude are then achieved in both imino-imino and imino-amino/aromatic protons 2D correlations; similar artifact-free sensitivity gains can be observed when carrying out extended Hadamard encodings of 3D NOESY/HSQC-type experiments. The resulting spectra reveal significantly more correlations than their conventionally acquired counterparts, which can support the spectral assignment and secondary structure determination of structured RNA elements.
Dynamic nuclear polarization (DNP) is widely used to enhance solid state nuclear magnetic resonance (NMR) sensitivity. Its efficiency as a generic signal-enhancing approach for liquid state NMR, however, decays rapidly with magnetic field B0, unless mediated by scalar interactions arising only in exceptional cases. This has prevented a more widespread use of DNP in structural and dynamical solution NMR analyses. This study introduces a potential solution to this problem, relying on biradicals with exchange couplings Jex of the order of the electron Larmor frequency ωE. Numerical and analytical calculations show that in such Jex ≈ ±ωE cases a phenomenon akin to that occurring in chemically induced DNP (CIDNP) happens, leading to different relaxation rates for the biradical singlet and triplet states which are hyperfine-coupled to the nuclear α or β states. Microwave irradiation can then generate a transient nuclear polarization build-up with high efficiency, at all magnetic fields that are relevant in contemporary NMR, and for all rotational diffusion correlation times that occur in small- and medium-sized molecules in conventional solvents.