Analysis of Complex Mixtures by Chemosensing NMR Using para-Hydrogen-Induced Hyperpolarization

Abstract

Conspectus Nuclear magnetic resonance (NMR) is a powerful technique for chemical analysis. The use of NMR to investigate dilute analytes in complex systems is, however, hampered by its relatively low sensitivity. An additional obstacle is represented by the NMR signal overlap. Because solutes in a complex mixture are usually not isotopically labeled, NMR studies are often limited to 1 H measurements, which, because of the modest dispersion of the 1 H resonances (typically ∼10 ppm), can result in challenging signal crowding. The low NMR sensitivity issue can be alleviated by nuclear spin hyperpolarization (i.e., transiently increasing the differences in nuclear spin populations), which determines large NMR signal enhancements. This has been demonstrated for hyperpolarization methods such as dynamic nuclear polarization, spin-exchange optical pumping and para -hydrogen-induced polarization (PHIP). In particular, PHIP has grown into a fast, efficient, and versatile technique since the recent discovery of non-hydrogenative routes to achieve nuclear spin hyperpolarization. For instance, signal amplification by reversible exchange (SABRE) can generate proton as well as heteronuclear spin hyperpolarization in a few seconds in compounds that are able to transiently bind to an iridium catalyst in the presence of para -hydrogen in solution. The hyperpolarization transfer catalyst acts as a chemosensor in the sense that it is selective for analytes that can coordinate to the metal center, such as nitrogen-containing aromatic heterocycles, sulfur heteroaromatic compounds, nitriles, Schiff bases, diaziridines, carboxylic acids, and amines. We have demonstrated that the signal enhancement achieved by SABRE allows rapid NMR detection and quantification of a mixture of substrates down to low-micromolar concentration. Furthermore, in the transient complex, the spin configuration of p -H 2 can be easily converted to spin hyperpolarization to produce up to 1000-fold enhanced NMR hydride signals. Because the hydrides’ chemical shifts are highly sensitive to the structure of the analyte associating with the iridium complex, they can be employed as hyperpolarized “probes” to signal the presence of specific compounds in the mixture. This indirect detection of the analytes in solution provides important benefits in the case of complex systems, as hydrides resonate in a region of the 1 H spectrum (at ca. −20 ppm) that is generally signal-free. The enhanced sensitivity provided by non-hydrogenative PHIP (nhPHIP), together with the absence of interference from the complex matrix (usually resonating between 0 and 10 ppm), set the detection limit for this NMR chemosensor down to sub-μM concentrations, approximately 3 orders of magnitude lower than for conventional NMR. This nhPHIP approach represents, therefore, a powerful tool for NMR analysis of dilut...

NMR Metabolomics Methods for Investigating Disease

“…Recently, PHIP has been extended to amino acids, which can have far-reaching implications for metabolism studies (Figure ). The discovery of SABRE and SABRE-SHEATH (shield enables alignment transfer to heteronuclei) as non-hydrogenation routes to achieve nuclear spin polarization has enabled high nuclear polarization of both proton and heteronuclear spins in a broad range of metabolites and other compounds and does not require unsaturated target molecules. − An excellent review on the application of these methods that can now selectively detect and quantify analytes down to nanomolar concentrations has been reported recently . Further, using this approach, the analysis of urine metabolites was recently reported, which avoids the need for solid-phase extraction (SPE) and replaces it with a minimal sample manipulation that retains the majority of urinary metabolome.…”

“…11−16 An excellent review on the application of these methods that can now selectively detect and quantify analytes down to nanomolar concentrations has been reported recently. 17 Further, using this approach, the analysis of urine metabolites was recently reported, 18 which avoids the need for solid-phase extraction (SPE) and replaces it with a minimal sample manipulation that retains the majority of urinary metabolome. A persistent challenge, however, is that the hyperpolarization approach does not boost the signal intensity evenly across all molecules in a mixture, which makes the determination of concentrations more difficult.…”

NMR methods for the analysis of mixtures

“…112 The potential of SABRE for mixture analysis has been explored by Tessari and co-workers, who showed that the method can be applied to complex mixtures of molecules with concentrations in the micromolar range, provided that an appropriate co-substrate is also added to the mixture. 113,114 They also showed that the signal intensity changes linearly with concentration in that regime. With a standard-addition method, they were able to quantify several analytes in an artificial mixture.…”

Illuminating the Human Metabolome: Selective Detection of Multiple Metabolites in Unaltered Biofluids via Hyperpolarisation-Enhanced NMR Spectroscopy

“…In another study, Dey et al presented an untargeted NMR-based metabolomic workflow based on dissolution DNP thereby enabling hyperpolarised 13 C metabolomics of plant extracts at natural abundance [8]. Despite of these and several other [9,10] extremely encouraging results, typical liquid-state NMR experiments employing d-DNP-or parahydrogen-based hyperpolarisation will, most likely, have a limited applicability in metabolomics research due the very specific and demanding sample preparation/setup requirements, e.g., harsh hyperpolarisation conditions and/or long polarisation times in combination with the occasional need for expensive additional instrumentation, which render biocompatible and automated high-throughput NMR measurements rather difficult.…”