NAD[S], an NAD analogue with reduced susceptibility to phosphodiesterase: Chemical synthesis and enzymic properties

Abstract

The chemical synthesis of adenosine(5′) [α‐thio]diphospho(5′)ribofuranosyl‐nicotinamide (NAD[S]) is described. The product occurs as a pair of diastereomers with different configuration at the sulfur‐bearing phosphorus atom. The diastereomers were separated by high‐performance liquid chromatography and their absolute configuration was determined after chemical degradation to the ADP[αS] diastereomers and chromatographic comparison with enzymically synthesized ADP[αS] diastereomers of known absolute configuration. Additional support for this assignment is based on different rates in the phosphodiesterase‐catalyzed hydrolysis. Furthermore the synthesis of [14C]NAD[S] is described. The coenzyme activity of NAD[S] in the reaction with alchol dehydrogenase from baker's yeast and lactate dehydrogenase from pig heart is very similar to that of β‐NAD. Also, NAD and NAD[S] serve equally well as substrates for NAD glycohydrolase from calf spleen. In contrast, no reaction was detected with NAD pyrophosphorylase, and hydrolysis of the separated NAD[S] diastereomers with snake−venom phosphodiesterase showed a 26‐fold and a 33‐fold slower reaction rate than that of NAD. Nucleotide pyrophosphatase was less sensitive to the S substitution, hydrolyzing NAD[S] 14‐times slower than NAD. Poly(ADP‐ribose) polymerase from Ehrlich ascites tumor cell nuclei accepted NAD[S] as a substrate but the reaction was significantly slower and approached saturation at much lower values than with NAD. Alkaline hydrolysis of the products insoluble in trichloroacetic acid yielded AMP[S] as the main derivative. It is concluded that with NAD[S] as a substrate the nuclear acceptors were nearly exclusively mono(ADP‐ribosyl)ated. Copyright © 1984, Wiley Blackwell. All rights reserved