Synthesis of Coenzymically Active Soluble and Insoluble Macromolecularized NAD+ Derivatives

Alkylation at N‐1 of the NAD+ adenine ring with 3,4‐epoxybutanoic acid, followed by chemical reduction to the alkali‐stable NADH form and alkaline Dimroth rearrangement, gave the NADH derivative alkylated at the exocyclic adenine amino group. Enzymic reoxidation of the latter derivative gave nicotinamide–6‐(2‐hydroxy‐3‐carboxypropylamino)purine dinucleotide, a functionalized NAD+ analogue carrying an Ω‐carboxyalkyl side‐chain at the exocyclic adenine amino group. Carbodiimide coupling of the latter derivative to high‐molecular‐weight water‐soluble (polyethyleneimine, polylysine) and insoluble (aminohexyl‐Sepharose) polymers gave the corresponding macromolecularized NAD+ analogues. These derivatives have been shown to be enzymically reducible. The polyethyleneimine and polylysine analogues showed a substantial degree of efficiency relative to free NAD+ with rabbit muscle lactate dehydrogenase (60 and 25% respectively) but a lower one with yeast alcohol dehydrogenase and Bacillus subtilis alanine dehydrogenase (2–7%). The polyethyleneimine derivative entrapped in cellulose triacetate fibres together with the lactate dehydrogenase was operationally stable during repetitive use.