Abstract
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 conditions—e.g., in states that arise upon mixing reactants that subsequently undergo chemical changes—and 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.
Highlights
Nuclear magnetic resonance (NMR) spectroscopy provides detailed information about dynamic processes through line-shape changes, which are traditionally limited to equilibrium conditions
To understand how the combined use of these two modalities allows for the real-time observation of off-equilibrium reactions, consider a situation in which a chemical reaction is initiated by the rapid mixing of reagents
Under conditions of stable, continuous, and plug-like flow, each point located at a defined distance away from the point of mixing will contain a unique set of chemical species that correspond to a particular time point along the reaction coordinate (Fig. 1B)
Summary
Nuclear magnetic resonance (NMR) spectroscopy provides detailed information about dynamic processes through line-shape changes, which are traditionally limited to equilibrium conditions. 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 highquality 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. Stopped-flow NMR has been used for studying chemical reactions in polymer and in biomolecular chemistry (e.g., protein and nucleic acid folding)[8]; stopped-flow NMR has been combined with nuclear hyperpolarization methods, which among other benefits allows for the facile detection of unreceptive nuclei[9] Despite their advantages, stopped-flow NMR methods are limited by the mandatory relaxation-delay and dead-time periods needed for spin polarization, mixing, and sample stabilization, as well as the necessity to acquire data from multiple sample batches: all these demands complicate the method, and decrease its capability to analyze the reaction coordinate. The principles underlying these time- and siteresolved kinetic NMR experiments are discussed in the subsequent sections, including experimental demonstrations that monitored the depletion and formation of 1H methyl resonances evolving from an enzymatically-catalyzed hydrolysis reaction with millisecond time resolution
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