Characterisation of the artificial coenzyme CL4

A number of NAD(+) analogues based on the structures of triazine dyes have previously been shown to function coenzymically in the oxidation of alcohols by horse liver alcohol dehydrogenase (HLADH). The simplest of these nicotinamide-containing synthetic compounds is the artificial coenzyme CL4, which has been studied in detail in the present work. The structures of the oxidised and reduced forms of CL4 have been determined by H-1 NMR; the reduced form appears to be the 1,4-dihydronicotinamide, similarly to NADH. CL4 has been shown to be stable across a range of pH values from 3 to 9, and the oxidised form has been regenerated from the reduced form using phenazine methosulphate. The spectrophotometric and fluorometric properties of CL4 have been investigated: It has been possible to develop a real-time fluorescence assay for following the enzymic reduction. This assay was used to show that the oxidation of butan-1-ol by HLADH using CL4 as coenzyme proceeds faster with increasing pH and temperature.