Hyperpolarized 1H and 13C NMR Spectroscopy in a Single Experiment for Metabolomics.

The application of NMR spectroscopy to complex mixture analysis and, in particular, to metabolomics is limited by the low sensitivity of NMR. We recently showed that dissolution dynamic nuclear polarization (d-DNP) could enhance the sensitivity of 13C NMR for complex metabolite mixtures, leading to the detection of highly sensitive 13C NMR fingerprints of complex samples such as plant extracts or urine. While such experiments provide heteronuclear spectra, which are complementary to conventional NMR, hyperpolarized 1H NMR spectra would also be highly useful, with improved limits of detection and compatibility with the existing metabolomics workflow and databases. In this technical note, we introduce an approach capable of recording both 1H and 13C hyperpolarized spectra of metabolite mixtures in a single experiment and on the same hyperpolarized sample. We investigate the analytical performance of this method in terms of sensitivity and repeatability, and then we show that it can be efficiently applied to a plant extract. Significant sensitivity enhancements in 1H NMR are reported with a repeatability suitable for metabolomics studies without compromising on the performance of hyperpolarized 13C NMR. This approach provides a way to perform both 1H and 13C hyperpolarized NMR metabolomics with unprecedented sensitivity and throughput.

In-line Multidimensional NMR Monitoring of Photochemical Flow Reactions.

This work demonstrates the in-line monitoring of a flow photochemical reaction using 1D and ultrafast 2D NMR methods at high magnetic field. The reaction mixture exiting the flow reactor is flown through the NMR spectrometer and directly analyzed. In the case of simple substrates, suitable information can be obtained through 1D 1 H spectra, but for molecules of higher complexity the use of 2D experiments is key to address signal overlaps and assignment issues. Here we show the usefulness of ultrafast 2D COSY experiments acquired in 70 s or less, for the in-line monitoring of photochemical reactions, and the possibility to obtain reliable quantitative information. This is a powerful framework to, for example, efficiently screen reaction conditions.

Broadband ultrafast 2D NMR spectroscopy for online monitoring in continuous flow.

Flow NMR is a powerful tool to monitor chemical reactions under realistic conditions. Here, we describe ultrafast (UF) 2D NMR schemes that make it possible to acquire broadband homonuclear 2D NMR spectra in 90 seconds or less for a continuously flowing sample. An interleaved acquisition strategy is used to address the spectral width limitation of UF 2D NMR. We show how, for a flowing sample, the use of a transverse axis for spatial encoding makes it possible to achieve the very high scan-to-scan stability required for interleaved acquisition. We also describe an optimised solvent suppression strategy that is effective for interleaved acquisition in continuous flow. These developments open the way to online monitoring with flow 2D NMR at high time resolution, as we illustrate with the monitoring of an organocatalysed condensation reaction.

Theoretical analysis of flow effects in spatially encoded diffusion NMR.

The measurement of translational diffusion coefficients by nuclear magnetic resonance (NMR) spectroscopy is essential in a broad range of fields, including organic, inorganic, polymer, and supramolecular chemistry. It is also a powerful method for mixture analysis. Spatially encoded diffusion NMR (SPEN DNMR)" is a time efficient technique to collect diffusion NMR data, which is particularly relevant for the analysis of samples that evolve in time. In many cases, motion other than diffusion is present in NMR samples. This is, for example, the case of flow NMR experiments, such as in online reaction monitoring and in the presence of sample convection. Such motion is deleterious for the accuracy of DNMR experiments in general and for SPEN DNMR in particular. Limited theoretical understanding of flow effects in SPEN DNMR experiments is an obstacle for their broader experimental implementation. Here, we present a detailed theoretical analysis of flow effects in SPEN DNMR and of their compensation, throughout the relevant pulse sequences. This analysis is validated by comparison with numerical simulation performed with the Fokker-Planck formalism. We then consider, through numerical simulation, the specific cases of constant, laminar, and convection flow and the accuracy of SPEN DNMR experiments in these contexts. This analysis will be useful for the design and implementation of fast diffusion NMR experiments and for their applications.

Single-Scan Ultraselective NMR Experiments with Preserved Sensitivity.

Selective NMR experiments provide rapid access to important structural information, and are essential to tackle the analysis of large molecules and complex mixtures. Single-scan ultraselective experiments are particularly useful, as they can rapidly select signals that overlap with other signals. Here, we describe a novel type of single-scan ultraselective NMR experiments that is robust against the effects of translational molecular diffusion, and thus make it possible to improve significantly the sensitivity of the experiment. This will largely broaden the applicability of this powerful class of experiments.

Hyperpolarized 13 C†NMR Spectroscopy of Urine Samples at Natural Abundance by Quantitative Dissolution Dynamic Nuclear Polarization.

Hyperpolarized nuclear magnetic resonance (NMR) offers an ensemble of methods that remarkably address the sensitivity issues of conventional NMR. Dissolution Dynamic Nuclear Polarization (d-DNP) provides a unique and general way to detect 13 C NMR signals with a sensitivity enhanced by several orders of magnitude. The expanding application scope of d-DNP now encompasses the analysis of complex mixtures at natural 13 C abundance. However, the application of d-DNP in this area has been limited to metabolite extracts. Here, we report the first d-DNP-enhanced 13 C NMR analysis of a biofluid -urine- at natural abundance, offering unprecedented resolution and sensitivity for this challenging type of sample. We also show that accurate quantitative information on multiple targeted metabolites can be retrieved through a standard addition procedure.

Solution-State 2D NMR Spectroscopy of Mixtures HyperpolarizedUsing Optically Polarized Crystals.

The HYPNOESYS method (Hyperpolarized NOE System), which relies on the dissolution of optically polarized crystals, has recently emerged as a promising approach to enhance the sensitivity of NMR spectroscopy in the solution state. However, HYPNOESYS is a single-shot method that is not generally compatible with multidimensional NMR. Here we show that 2D NMR spectra can be obtained from HYPNOESYS-polarized samples, using single-scan acquisition methods. The approach is illustrated with a mixture of terpene molecules and a benchtop NMR spectrometer, paving the way to a sensitive, information-rich and affordable analytical method.

Online Reaction Monitoring with Fast and Flow-Compatible Diffusion NMR Spectroscopy.

Online monitoring by flow NMR spectroscopy is a powerful approach to study chemical reactions and processes, which can provide mechanistic understanding, and drive optimisations. However, some of the most useful methods for mixture analysis and reaction monitoring are not directly applicable in flow conditions. This is the case of classic diffusion-ordered NMR spectroscopy (DOSY) methods, which can be used to separate the spectral information for mixture's components. We describe a fast and flow-compatible diffusion NMR experiment that makes it possible to collect accurate diffusion data for samples flowing at up to 3 mL/min. We use it to monitor the synthesis of a Schiff base with a flow-tube with a time resolution of approximately 2 minutes. The one-shot flow-compatible diffusion NMR described here open many avenues for reaction monitoring applications.

Optimisation of spatially-encoded diffusion-ordered NMR spectroscopy for the analysis of mixtures.

Diffusion-ordered NMR spectroscopy (DOSY NMR) is a widely used method for the analysis of mixtures. It can be used to separate the spectra of a mixture's components and to analyse interactions. The classic implementation of DOSY experiments, based on an incrementation of the diffusion-encoding gradient area, requires several minutes or more to collect a 2D data set. Spatially-encoded (SPEN) DOSY makes it possible to collect a complete data set in less than 1 s, by spatial parallelisation of the effective gradient area. While several short descriptions of SPEN DOSY experiments have been reported, a thorough characterisation of its features and its practical use is missing, and this hinders the use of the method. Here, we present the unusual principles and implementation of the SPEN DOSY experiment, an understanding of which is useful to make optimal use of the method. The encoding and acquisition steps are described, and the parameter relations that govern the setup of SPEN DOSY experiments are discussed. The influence of key parameters, including on sensitivity, is illustrated experimentally on mixtures of small molecules. This study should be useful for the setup of SPEN DOSY experiments, which are particularly useful for systems that evolve in time.

Single-scan measurements of nuclear spin singlet order decay rates.

Measurements of singlet spin order decay rates are time consuming due to the long-lived nature of this form of order and the typical pseudo-2D mode of acquisition. Additionally, this acquisition modality is not ideal for experiments run on hyperpolarized order because of the single-shot nature of hyperpolarization techniques. We present a methodology based on spatial encoding that not only significantly reduces the duration of these experiments but also confers compatibility using spin hyperpolarization techniques. The method condenses in a single shot the variable delay array used to measure decay rates in conventional pseudo-2D relaxation experiments. This results in a substantial time saving factor and, more importantly, makes the experiment compatible with hyperpolarization techniques since only a single hyperpolarized sample is required. Furthermore, the presented method, besides offering savings on time and costs, avoids reproducibility concerns associated with repetition in the hyperpolarization procedure. The method accelerates the measurement and characterization of singlet order decay times, and, when coupled with hyperpolarization techniques, can facilitate the quest for systems with very long decay times.

Hyperpolarized NMR Metabolomics at Natural 13C Abundance.

Metabolomics plays a pivotal role in systems biology, and NMR is a central tool with high precision and exceptional resolution of chemical information. Most NMR metabolomic studies are based on 1H 1D spectroscopy, severely limited by peak overlap. 13C NMR benefits from a larger signal dispersion but is barely used in metabolomics due to ca. 6000-fold lower sensitivity. We introduce a new approach, based on hyperpolarized 13C NMR at natural abundance, that circumvents this limitation. A new untargeted NMR-based metabolomic workflow based on dissolution dynamic nuclear polarization (d-DNP) for the first time enabled hyperpolarized natural abundance 13C metabolomics. Statistical analysis of resulting hyperpolarized 13C data distinguishes two groups of plant (tomato) extracts and highlights biomarkers, in full agreement with previous results on the same biological model. We also optimize parameters of the semiautomated d-DNP system suitable for high-throughput studies.

Online reaction monitoring by single-scan 2D NMR under flow conditions.

Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for reaction monitoring. Several devices have been recently developed for online monitoring, using flow NMR, of batch reactions carried out under realistic experimental conditions in terms of, e.g., agitation and temperature. Here we show that time series of single-scan ultrafast 2D NMR (UF2DNMR) spectra can be collected to monitor solution mixtures that circulate in a flow unit. Fast multidimensional NMR methods have the demonstrated potential to provide kinetic and mechanistic information on reactions. UF2DNMR makes it possible to collect 2D data sets in less than one second, but relies on spatial encoding (SPEN) that is sensitive to sample motion, thus making online monitoring challenging. We characterize the interference between flow and spatial encoding and provide pulse-sequence- and hardware-based solutions. The resulting method is illustrated with the monitoring of a saponification reaction. UF2DNMR with a flow unit is a promising tool for the online monitoring of organic chemical reactions.

Fast quantitative 2D NMR for metabolomics and lipidomics: A tutorial.

Nuclear magnetic resonance (NMR) is a well-known analytical technique for the analysis of complex mixtures. Its quantitative capability makes it ideally suited to metabolomics or lipidomics studies involving large sample collections of complex biological samples. To overcome the ubiquitous limitation of spectral overcrowding when recording 1D NMR spectra on such samples, the acquisition of 2D NMR spectra allows a better separation between overlapped resonances while yielding accurate quantitative data when appropriate analytical protocols are implemented. Moreover, the experiment duration can be considerably reduced by applying fast acquisition methods. Here, we describe the general workflow to acquire fast quantitative 2D NMR spectra in the "omics" context. It is illustrated on three representative and complementary experiments: UF COSY, ZF-TOCSY with nonuniform sampling, and HSQC with nonuniform sampling. After giving some details and recommendations on how to apply this protocol, its implementation in the case of targeted and untargeted metabolomics/lipidomics studies is described.

Single-Scan Diffusion-Ordered NMR Spectroscopy of SABRE-Hyperpolarized Mixtures.

The analysis of complex mixtures of dissolved molecules is a major challenge, especially for systems that gradually evolve, e. g., in the course of a chemical reaction or in the case of chemical instability. 1D NMR is a fast and non-invasive method suitable for detailed molecular analysis, though of low sensitivity. Moreover, the spectral resolution of proton, the most commonly used and most sensitive stable isotope in NMR, is also quite limited. Spatially encoded (SPEN) experiments aim at creating in one acquisition a 2D data set by simultaneously performing different 1D sub-experiments on different slices of the NMR tube, at the price of an extra loss of sensitivity. Choosing translational diffusion coefficients as the additional dimension (the so-called DOSY approach) helps to recover proton spectra of each molecule in a mixture. The sensitivity limitation of SPEN NMR can, on the other hand, be addressed with hyperpolarization methods. Within hyperpolarization methods, signal amplification by reversible exchange (SABRE), based on parahydrogen, is the cheapest and the easiest one to set up, and allows multi-shot experiments. Here we show that the spectra of a mixture's components at millimolar concentration are resolved in few seconds by combining the SABRE, SPEN and DOSY concepts.

Understanding the methyl-TROSY effect over a wide range of magnetic fields.

The use of relaxation interference in the methyl Transverse Relaxation-Optimized SpectroscopY (TROSY) experiment has opened new avenues for the study of large proteins and protein assemblies in nuclear magnetic resonance. So far, the theoretical description of the methyl-TROSY experiment has been limited to the slow-tumbling approximation, which is correct for large proteins on high-field spectrometers. In a recent paper, favorable relaxation interference was observed in the methyl groups of a small protein at a magnetic field as low as 0.33 T, well outside the slow-tumbling regime. Here, we present a model to describe relaxation interference in methyl groups over a broad range of magnetic fields, not limited to the slow-tumbling regime. We predict that the type of multiple-quantum transition that shows favorable relaxation properties change with the magnetic field. Under the condition of fast methyl-group rotation, methyl-TROSY experiments can be recorded over the entire range of magnetic fields from a fraction of 1 T up to 100 T.

Dynamic Nuclear Polarization Opens New Perspectives for NMR Spectroscopy in Analytical Chemistry.

Dynamic nuclear polarization (DNP) can boost sensitivity in nuclear magnetic resonance (NMR) experiments by several orders of magnitude. This Feature illustrates how the coupling of DNP with both liquid- and solid-state NMR spectroscopy has the potential to considerably extend the range of applications of NMR in analytical chemistry.

Acceleration of 3D DOSY NMR by Spatial Encoding of the Chemical Shift.

Diffusion-ordered NMR spectroscopy (DOSY) is a powerful method for the analysis of solution mixtures. With 3D DOSY, the 2D NMR spectra of a mixture's components can be separated according to the translational diffusion coefficient of each component. The acquisition of 3D DOSY data is, however, very time-consuming because of the need to consecutively acquire scans for both the diffusion and the indirect spectral dimensions. We show that spatial encoding of the indirect spectral dimension, of the kind used in ultrafast 2D NMR, can accelerate 3D DOSY experiments by an order of magnitude or more. This is illustrated with homonuclear single-quantum (COSY) and double-quantum (DQS) correlation spectra. Implementations with concatenated and incorporated (iDOSY) diffusion blocks are compared and in both cases, 2D spectra are separated with less than 6 min of experiment time.

Ultrafast Maximum-Quantum NMR Spectroscopy for the Analysis of Aromatic Mixtures.

Maximum-quantum (MaxQ) NMR experiments have been introduced to overcome issues related to peak overlap and high spectral density in the NMR spectra of aromatic mixtures. In MaxQ NMR, spin systems are separated on the basis of the highest-quantum coherence that they can form. MaxQ experiments are however time consuming and methods have been introduced to accelerate them. In this article, we demonstrate the ultrafast, single-scan acquisition of MaxQ NMR spectra using spatial encoding of the multiple-quantum dimension. So far, the spatial encoding methodology has been applied only for the encoding of up to double-quantum coherences, and here we show that it can be extended to higher coherence orders, to yield a massive reduction of the acquisition time of multi-quantum spectra of aromatic mixtures, and also to monitor chemical reactions.

Spatially encoded diffusion-ordered NMR spectroscopy of reaction mixtures in organic solvents.

Diffusion-ordered NMR spectroscopy (DOSY) is a powerful method for the analysis of mixtures. Classic DOSY methods require several minutes of acquisition, and we show here that DOSY experiments can be recorded in less than one second for the challenging case of solution mixtures in low-viscosity solvents. The proposed method relies on a spatial encoding of the diffusion dimension, for which convection-compensation and spectral-selection strategies are introduced. The method is illustrated with the analysis of a reaction mixture, and more accurate estimates of the diffusion coefficients are obtained.

Tailored Microstructured Hyperpolarizing Matrices for Optimal Magnetic Resonance Imaging.

Tailoring the physical features and the porous network architecture of silica-based hyperpolarizing solids containing TEMPO radicals, known as HYPSO (hybrid polarizing solids), enabled unprecedented performance of dissolution dynamic nuclear polarization (d-DNP). High polarization values up to P(1 H)=99 % were reached for samples impregnated with a mixture of H2 O/D2 O and loaded in a 6.7 T polarizer at temperatures around 1.2 K. These HYPSO materials combine the best performance of homogeneous DNP formulations with the advantages of solid polarizing matrices, which provide hyperpolarized solutions free of any-potentially toxic-additives (radicals and glass-forming agents). The hyperpolarized solutions can be expelled from the porous solids, filtered, and rapidly transferred either to a nuclear magnetic resonance (NMR) spectrometer or to a magnetic resonance imaging (MRI) system.