Synthesis and configurational analysis of a dinucleoside phosphate isotopically chiral at phosphorus. Stereochemical course of Penicillium citrinum nuclease P1 reaction

(Rp)- and (Sp)-5'-O-thymidyl 3'-O-thymidyl 18Ophosphates have been synthesized by reaction of the respective (Sp)- and (Rp)-phosphorothioate precursors with N-bromosuccinimide in dioxane and H218O. Stereochemical analysis of the product derived from the (Rp)-phosphorothioate by digestion with snake venom phosphodiesterase in H217O and examination of the isotopic chirality of the resulting thymidine 5'-16O,17O,18Ophosphate demonstrate that the replacement reaction has proceeded with inversion of configuration at phosphorus. Inspection of the 31P NMR spectrum of the methyl esters prepared from (Sp)-5'-O-thymidyl 3'-O-thymidyl 18Ophosphate confirms that the replacement reaction has proceeded with very little if any racemization. This spectrum also allows the assignment of the absolute configuration of these methyl triesters. (Rp)-5'-O-Thymidyl 3'-O-thymidyl 18Ophosphate has been used to demonstrate that the stereochemical course of the hydrolytic reaction catalyzed by nuclease P1 from Penicillium citrum proceeds with inversion of configuration at phosphorus and therefore probably does not involve the participation of a covalent enzyme intermediate.