A novel compound, 2'/3'-O-acetyl-ADP-ribose, is provided. The compound is a mixture of the 2' and 3' regioisomers of O-acetyl-ADP ribose, and is formed nonenzymatically from 2'-O-acetyl-ADP-ribose, which is the newly discovered product of the reaction of Sir2 enzymes with acetylated peptides and NAD.sup.+. Analogs of 2'/3'-O-acetyl-ADP-ribose are also provided. Additionally, methods of preparing 2'/3'-O-acetyl-ADP-ribose, methods of determining whether a test compound is an inhibitor of a Sir2 enzyme, methods of detecting Sir2 activity in a composition, methods of deacetylating an acetylated peptide, and methods of inhibiting the deacetylation of an acetylated peptide are provided. Prodrugs of 2'/3'-O-acetyl-ADP-ribose are also provided.