Deep eutectic solvents as a stabilising medium for NAD coenzyme: unravelling the mechanism behind coenzyme stabilisation effect

Nicotinamide adenine dinucleotide (NAD) coenzyme is a vital part of numerous enzymatic reactions that are involved in all major biological processes from energy metabolism to cell survival. Accordingly, it is used in a great number of biocatalytic reactions, as analytical biosensors, and in test kits for diagnostic and analytical purposes. This coenzyme is unstable in aqueous solutions, meaning that the minimization of its degradation during storage, assays, and enzyme-catalysed oxidoreductive reactions is of high importance. Herein, we report on the stabilisation of NAD (NAD(+) as oxidised, and NADH as its reduced form) by deep eutectic solvents (DES), an emerging class of solvents that offer numerous remarkable advantages such as high tunability, and capability to stabilise a wide range of commercially important compounds of natural origin. Preliminary DES screening revealed that out of 7 candidates, choline chloride:urea (ChCl:U) shows the best ability to stabilise both NAD forms. Computational analysis (quantum chemical calculations and molecular dynamics simulations) provided a deeper insight into possible mechanisms behind the observed stabilisation, by identifying geometries and the solvation structure of NAD coenzymes in these solvents, and analysing their possible degradation pathways. Finally, prolonged NAD stability (up to 50 days) in ChCl:U is detected and the system is further confirmed as a stable working NAD solution in a model enzymatic assay.

Automated summary

This study reports the stabilisation of nicotinamide adenine dinucleotide (NAD) coenzyme using deep eutectic solvents (DES). Through DES screening and computational analysis, choline chloride:urea (ChCl:U) was identified as the best solvent for stabilising both NAD forms. The NAD in ChCl:U showed prolonged stability in a model enzymatic assay.