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⁺. 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.