Cross-Polarization Schemes for Improved Heteronuclear Transfers Involving Labile Protons in Biomolecular Solution NMR.

INEPT-based experiments are widely used for 1 H→15 N transfers, but often fail when involving labile protons due to solvent exchanges. J-based cross polarization (CP) strategies offer a more efficient alternative to perform such transfers, particularly when leveraging the Hwater ↔ ${ \leftrightarrow }$ HN exchange process to boost the 1 H→15 N transfer process. This leveraging, however, demands the simultaneous spin-locking of both Hwater and HN protons by a strong 1 H RF field, while fulfilling the γH B1,H =γN B1,N Hartmann-Hahn matching condition. Given the low value of γN /γH , however, these demands are often incompatible-particularly when experiments are executed by the power-limited cryogenic probes used in contemporary high field NMR. The present manuscript discusses CP alternatives that can alleviate this limitation, and evaluates their performance on urea, amino acids, and intrinsically disordered proteins. These alternatives include new CP variants based on frequency-swept and phase-modulated pulses, designed to simultaneously fulfill the aforementioned conflicting conditions. Their performances vis-à-vis current options are theoretically analyzed with Liouville-space simulations, and experimentally tested with double and triple resonance transfer experiments.

The Extended Hadamard Transform: Sensitivity-Enhanced NMR Experiments Among Labile and Non-Labile 1 Hs of SARS-CoV-2-derived RNAs.

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 1 Hs, 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.

3D Heteronuclear Magnetization Transfers for the Establishment of Secondary Structures in SARS-CoV-2-Derived RNAs.

Multidimensional NOESY experiments targeting correlations between exchangeable imino and amino protons provide valuable information about base pairing in nucleic acids. It has been recently shown that the sensitivity of homonuclear correlations involving RNA's labile imino protons can be significantly enhanced, by exploiting the repolarization brought about by solvent exchanges. Homonuclear correlations, however, are of limited spectral resolution, and usually incapable of tackling relatively large homopolymers with repeating structures like RNAs. This study presents a heteronuclear-resolved version of those NOESY experiments, in which magnetization transfers between the aqueous solvent and the nucleic acid protons are controlled by selecting specific chemical shift combinations of a coupled 1H-15N spin pair. This selective control effectively leads to a pseudo-3D version of HSQC-NOESY, but with cross-peaks enhanced by ∼2-5× as compared with conventional 2D NOESY counterparts. The enhanced signal sensitivity as well as access to both 15N-1H and 1H-1H NOESY dimensions can greatly facilitate RNA assignments and secondary structure determinations, as demonstrated here with the analysis of genome fragments derived from the SARS-CoV-2 virus.

Hadamard acquisition of 13 C-13 C 2-D correlation NMR spectra.

We show that a multiselective excitation with Hadamard encoding is a powerful tool for 2-D acquisition of 13 C─13 C homonuclear correlations. This method is not designed to improve the sensitivity, but rather to reduce the experiment time, provided there is sufficient sensitivity. Therefore, it allows fast acquisition of such 2-D spectra in labeled molecules. The technique has been demonstrated using a U─13 C─15 N histidine hydrochloride monohydrate sample allowing each point of the build-up curves of the 13 C─13 C cross-peaks to be recorded within 4 min 35 s, which is very difficult with conventional methods. Using the U─13 C─15 N f-MLF sample, we have demonstrated that the method can be applied to molecules with 14 13 C resonances with a minimum frequency separation of 240 Hz.

Magnetization Transfer to Enhance NOE Cross-Peaks among Labile Protons: Applications to Imino-Imino Sequential Walks in SARS-CoV-2-Derived RNAs.

2D NOESY plays a central role in structural NMR spectroscopy. We have recently discussed methods that rely on solvent-driven exchanges to enhance NOE correlations between exchangeable and non-exchangeable protons in nucleic acids. Such methods, however, fail when trying to establish connectivities within pools of labile protons. This study introduces an alternative that also enhances NOEs between such labile sites, based on encoding a priori selected peaks by selective saturations. The resulting selective magnetization transfer (SMT) experiment proves particularly useful for enhancing the imino-imino cross-peaks in RNAs, which is a first step in the NMR resolution of these structures. The origins of these enhancements are discussed, and their potential is demonstrated on RNA fragments derived from the genome of SARS-CoV-2, recorded with better sensitivity and an order of magnitude faster than conventional 2D counterparts.

Quantitative Isotopomer Rates in Real-Time Metabolism of Cells Determined by NMR Methods.

Tracer-based metabolism is becoming increasingly important for studying metabolic mechanisms in cells. NMR spectroscopy offers several approaches to measure label incorporation in metabolites, including 13 C- and 1 H-detected spectra. The latter are generally more sensitive, but quantification depends on the proton-carbon 1 JCH coupling constant, which varies significantly between different metabolites. It is therefore not possible to have one experiment optimised for all metabolites, and quantification of 1 H-edited spectra such as HSQCs requires precise knowledge of coupling constants. Increasing interest in tracer-based and metabolic flux analysis requires robust analyses with reasonably small acquisition times. Herein, we compare 13 C-filtered and 13 C-edited methods for quantification and show the applicability of the methods for real-time NMR spectroscopy of cancer-cell metabolism, in which label incorporations are subject to constant flux. We find an approach using a double filter to be most suitable and sufficiently robust to reliably obtain 13 C incorporations from difference spectra. This is demonstrated for JJN3 multiple myeloma cells processing glucose over 24 h. The proposed method is equally well suited for calculating the level of label incorporation in labelled cell extracts in the context of metabolic flux analysis.

Triplet NOAH supersequences optimised for small molecule structure characterisation.

A series of NMR supersequences are presented for the time-efficient structure characterisation of small molecules in the solution state. These triplet sequences provide HMBC, HSQC, and one homonuclear correlation experiment of choice according to the NMR by Ordered Acquisition using 1 H detection principle. The experiments are demonstrated to be compatible with non-uniform sampling schemes and may be acquired and processed under full automation.

2BOB - extracting an H2BC and an HSQC-type spectrum from the same data set, and H2OBC - a fast experiment delineating the protonated 13 C backbone.

Two new related methods, 2BOB and H2OBC, for tracking out the backbone of protonated 13 C nuclei are presented. 2BOB extracts an H2BC and an HSQC-type spectrum from one and the same data set, and the combined information of these two spectra tracks out the molecular backbone. The faster method, H2OBC, typically requiring only a few minutes of instrument time, yields a single spectrum with distinct and different phases imposed on the H2BC and one-bond peaks thus obviating the need to separate them in the absence of complicating spectral overlap. Copyright © 2017 John Wiley & Sons, Ltd.

19 F DOSY diffusion-NMR spectroscopy of fluoropolymers.

A new pulse sequence for obtaining 19 F detected DOSY (diffusion ordered spectroscopy) spectra of fluorinated molecules is presented and used to study fluoropolymers based on vinylidene fluoride and chlorotrifluoroethylene. The performance of 19 F DOSY NMR experiments (and in general any type of NMR experiment) on fluoropolymers creates some unique complications that very often prevent detection of important signals. Factors that create these complications include: (1) the presence of many scalar couplings among 1 H, 19 F and 13 C; (2) the large magnitudes of many 19 F homonuclear couplings (especially 2 JFF ); (3) the large 19 F chemical shift range; and (4) the low solubility of these materials (which requires that experiments be performed at high temperatures). A systematic study of the various methods for collecting DOSY NMR data, and the adaptation of these methods to obtain 19 F detected DOSY data, has been performed using a mixture of low molecular weight, fluorinated model compounds. The best pulse sequences and optimal experimental conditions have been determined for obtaining 19 F DOSY spectra. The optimum pulse sequences for acquiring 19 F DOSY NMR data have been determined for various circumstances taking into account the spectral dispersion, number and magnitude of couplings present, and experimental temperature. Pulse sequences and experimental parameters for optimizing these experiments for the study of fluoropolymers have been studied. Copyright © 2016 John Wiley & Sons, Ltd.

NOAH: NMR Supersequences for Small Molecule Analysis and Structure Elucidation.

Nested NMR experiments combining up to five conventional NMR pulse sequences into one supersequence are introduced. The core 2D NMR techniques routinely employed in small molecule NMR spectroscopy, such as HSQC, HMQC, HMBC, COSY, NOESY, TOCSY, and similar, can be recorded in a single measurement. In this way the data collection time may be dramatically reduced and sample throughput increased for basic NMR applications, such as structure elucidation and verification in synthetic, medicinal, and natural product chemistry.

Parallel NMR spectroscopy with simultaneous detection of (1) H and (19) F nuclei.

Recording NMR signals of several nuclear species simultaneously by using parallel receivers provides more information from a single measurement and at the same time increases the measurement sensitivity per unit time. Here we present a comprehensive series of the most frequently used NMR experiments modified for simultaneous direct detection of two of the most sensitive NMR nuclei - (1) H and (19) F. We hope that the presented material will stimulate interest in and further development of this technique.

Exploiting natural abundance 13C-15N coupling as a method for identification of nitrogen heterocycles: practical use of the HCNMBC sequence.

In this paper, we detail the results of (1)H-(15)N correlation data obtained via (13)C-(15)N coupling at natural abundance on a number of classes of azoles including pyrazoles, imidazoles and triazoles. The experiment produces data that is highly complementary to direct (1)H-(15)N HMBC type correlations in that it can provide (15)N chemical shift data for nitrogen that may not show up in the HMBC. This is particularly advantageous in the triazoles where (15)N chemical shift can be diagnostic of regiochemistry. Because of the consistency in JCN values among the azoles, the experiment produces distinctive correlation patterns that can be used for identification of regiochemistry. The experiment can also be used to directly measure (13)C-(15)N coupling constants.

Fast experiments for structure elucidation of small molecules: Hadamard NMR with multiple receivers.

We propose several significant improvements to the PANSY (Parallel NMR SpectroscopY) experiments-PANSY COSY and PANSY-TOCSY. The improved versions of these experiments provide sufficient spectral information for structure elucidation of small organic molecules from just two 2D experiments. The PANSY-TOCSY-Q experiment has been modified to allow for simultaneous acquisition of three different types of NMR spectra-1D C-13 of non-protonated carbon sites, 2D TOCSY and multiplicity edited 2D HETCOR. In addition the J-filtered 2D PANSY-gCOSY experiment records a 2D HH gCOSY spectrum in parallel with a (1) J-filtered HC long-range HETCOR spectrum as well as offers a simplified data processing. In addition to parallel acquisition, further time savings are feasible because of significantly smaller F1 spectral windows as compared to the indirect detection experiments. Use of cryoprobes and multiple receivers can significantly alleviate the sensitivity issues that are usually associated with the so called direct detection experiments. In cases where experiments are sampling limited rather than sensitivity limited further reduction of experiment time is achieved by using Hadamard encoding. In favorable cases the total recording time for the two PANSY experiments can be reduced to just 40 s. The proposed PANSY experiments provide sufficient information to allow the CMCse software package (Bruker) to solve structures of small organic molecules.

Mapping molecular perturbations by a new form of two-dimensional spectroscopy.

We propose a new general form of two-dimensional spectroscopy where the indirect "evolution" dimension is derived using the Radon transform. This idea is applicable to several types of spectroscopy but is illustrated here for the case of NMR spectroscopy. This "projection spectroscopy" displays characteristic correlation peaks that highlight perturbations of chemical shifts caused by temperature, pressure, solvent, molecular binding, chemical exchange, hydrogen bonding, pH variations, conformational changes, or paramagnetic agents. The results are displayed in a convenient format that allows the chemist to see all of the chemical shift perturbations at a glance and assess their rates of change and directions. As a proof of principle, we present two simple, practical examples that display two-dimensional representations of the effects of temperature and solvent on NMR spectra.

NMR with multiple receivers.

Parallel acquisition NMR spectroscopy (PANSY) is used to detect simultaneously signals from up to four nuclear species, such as H-1, H-2, C-13, N-15, F-19 and P-31. The conventional COSY, TOCSY, HSQC, HMQC and HMBC pulse sequences have been adapted for such applications. Routine availability of NMR systems that incorporate multiple receivers has led to development of new types of NMR experiments. One such scheme named PANACEA allows unambiguous structure determination of small organic molecules from a single measurement and includes an internal field/frequency correction routine. It does not require the conventional NMR lock system and can be recorded in pure liquids. Furthermore, long-range spin-spin couplings can be extracted from the PANACEA spectra and used for three-dimensional structure refinement. In bio-molecular NMR, multi-receiver NMR systems are used for simultaneous recording of H-1 and C-13 detected multi-dimensional spectra. For instance, the 2D (HA)CACO and 3D (HA)CA(CO)NNH experiments can be recorded simultaneously in proteins of moderate size (up to 30 kDa). The multi-receiver experiments can also be used in combination with the fast acquisition schemes such as Hadamard spectroscopy, computer optimized aliasing and projection-reconstruction techniques. In general, experiments that utilize multiple receivers provide significantly more information from a single NMR measurement as compared to the conventional single receiver techniques.

A general scheme for generating NMR supersequences combining high- and low-sensitivity experiments.

NOAH supersequences are a way of collecting multiple 2D NMR experiments in a single measurement. So far, this approach has been limited to experiments with comparable sensitivity. Here, we propose a scheme which overcomes this limitation, combining experiments with very different sensitivities such as 1,1-ADEQUATE, 15N HMBC, and 13C HSQC.

Parallel acquisition of multi-dimensional spectra in protein NMR.

We introduce the use of multiple receivers applied in parallel for simultaneously recording multi-dimensional data sets of proteins in a single experiment. The utility of the approach is established through the introduction of the 2D (15)N,(1)H(N)||(13)CO HSQC experiment in which a pair of two-dimensional (15)N,(1)H(N) and (15)N,(13)CO spectra are recorded. The methodology is further extended to higher dimensionality via the 3D (1)H(N)||(13)CO HNCA in which a pair of data sets recording (13)C(α),(15)N,(1)H(N) and (13)C(α),(15)N,(13)CO chemical shifts are acquired. With the anticipated increases in probe sensitivity it is expected that multiple receiver experiments will become an important approach for efficient recording of NMR data.

Concepts in projection-reconstruction.

The Achilles heel of conventional multidimensional NMR spectroscopy is the long duration of the measurements, set by the Nyquist sampling condition and the resolution requirements in the evolution dimensions. Projection-reconstruction solves this problem by radial sampling of the evolution-domain signals, relying on Bracewell's Fourier transform slice/projection theorem to generate a set of projections at different inclinations. Reconstruction is implemented by one of three possible deterministic back-projection schemes (additive, lowest-value, or algebraic), or by a statistical model-fitting program. For simplicity the treatment focuses principally on the three-dimensional case, and then extends the analysis to four dimensions. The concept of hyperdimensional spectroscopy is described for dealing with even higher dimensions.

Uniform water-mediated saturation transfer: A sensitivity-improved alternative to WaterLOGSY.

In the study of small molecule ligands and candidate macromolecular targets, water spins in long-lived association with macromolecules (proteins or nanoparticles) constitute a remarkable source of magnetization that can be exploited to reveal ligand-target binding. In this work we show how the selective saturation of water spins complemented with adiabatic off-resonance spin-locks can remove the NOE contribution of bulk water in the final difference spectrum, leading to uniformly enhanced signals that reveal weak ligand-target interactions.

Increasing sensitivity and versatility in NMR supersequences with new HSQC-based modules.

The sensitivity-enhanced HSQC, as well as HSQC-TOCSY, experiments have been modified for incorporation into NOAH (NMR by Ordered Acquisition using 1H detection) supersequences, adding diversity for 13C and 15N modules. Importantly, these heteronuclear modules have been specifically tailored to preserve the magnetisation required for subsequent acquisition of other heteronuclear or homonuclear modules in a supersequence. In addition, we present protocols for optimally combining HSQC and HSQC-TOCSY elements within the same supersequences, yielding high-quality 2D spectra suitable for structure characterisation but with greatly reduced experiment durations. We further demonstrate that these time savings can translate to increased detection sensitivity per unit time.