Multinuclear High-Field FlowNMR for Real-Time Monitoring and Analysis of Biocatalytic Reactions in Batch and Flow

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Online high-field nuclear magnetic resonance spectroscopy in continuous or recirculating flow (FlowNMR) offers a powerful yet underutilised approach for monitoring biocatalytic reactions, particularly in challenging aqueous and low-concentration systems. Herein, we demonstrate the application of multinuclear high-resolution FlowNMR at 500 MHz for real-time analysis of both batch and continuous flow biotransformations. Across four case studies run at the typical low concentrations associated with biocatalysis including alcohol oxidation, nitroreductase-mediated reduction, a two-stage enzymatic cascade and a nucleoside phosphorylation, high-resolution spectra were obtained without interference from enzymes or reaction media. This technique enabled direct observation of key reaction intermediates, rapid optimisation of conditions and structure elucidation at sub-millimolar concentrations. Overall, this work demonstrates online high-field NMR as a versatile and information-rich tool for biocatalysis, providing real-time insight and significantly accelerating reaction development, with strong potential for integration into automated flow systems.