Issue 19, 2022

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

Abstract

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.

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

Supplementary files

Article information

Article type
Paper
Submitted
18 Jul 2022
Accepted
09 Sep 2022
First published
12 Sep 2022

Green Chem., 2022,24, 7661-7674

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

M. Radović, L. Hok, M. Panić, M. Cvjetko Bubalo, R. Vianello, M. Vinković and I. Radojčić Redovniković, Green Chem., 2022, 24, 7661 DOI: 10.1039/D2GC02656B

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