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

The chemical synthesis of adenosine(5') [alpha-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[alpha S] diastereomers and chromatographic comparison with enzymically synthesized ADP[alpha S] 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 alcohol dehydrogenase from baker's yeast and lactate dehydrogenase from pig heart is very similar to that of beta-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 .