Synthesis of thermostable artificial nicotinamide cofactors: carba-NAD⁺ and carba-NADP

Carbocyclic nicotinamide cofactors are attractive NAD(P)⁺ analogues that retain native redox activity while offering substantially enhanced chemical and thermal stability. Their broader use, however, has been limited by demanding multistep chemical syntheses involving complex protection strategies and limited exploration of enzymatic alternatives. In this study, we studied both chemical and enzymatic approaches for the synthesis of carba-analogues. For the enzymatic route, we identify and characterize the key enzymes involved in cofactor assembly, evaluating their tolerance toward non-native, carbocyclic substrates and extending the assembly to further generate the phosphorylated analogue, cNADP⁺. In addition, extending the analysis to cofactor thermostability, carba-NAD⁺ displayed a remarkable halftime (t _1/2 > 1386 h at 50 °C), far exceeding that of NAD⁺ (t _1/2 = 76 h) and is accepted by a broad panel of oxidoreductases. Collectively, this work outlines a modular workflow and details the synthesis landscape for accessing thermostable, synthetic nicotinamide cofactor analogues, cNAD⁺ and cNADP⁺, unveiling new opportunities for their application in cofactor engineering and synthetic biocatalysis.