Hydrolytic Reactions of Diribonucleoside 3‘,5‘-(3‘-<i>N</i>-Phosphoramidates):  Kinetics and Mechanisms for the P−O and P−N Bond Cleavage of 3‘-Amino-3‘-deoxyuridylyl-3‘,5‘-uridine

Abstract

Hydrolytic reactions of 3'-amino-3'-deoxyuridylyl-3',5'-uridine (2a), an analogue of uridylyl-3',5'-uridine having the 3'-bridging oxygen replaced with nitrogen, have been followed by RP HPLC over a wide pH range. The only reaction taking place under alkaline conditions (pH > 9) is hydroxide ion-catalyzed hydrolysis (first-order in [OH(-)]) to a mixture of 3'-amino-3'-deoxyuridine 3'-phosphoramidate (7) and uridine (4). The reaction proceeds without detectable accumulation of any intermediates. At pH 6-8, a pH-independent formation of 3'-amino-3'-deoxyuridine 2'-phosphate (3) competes with the base-catalyzed cleavage. Both 3 and in particular 7 are, however, rather rapidly dephosphorylated under these conditions to 3'-amino-3'-deoxyuridine (5). In all likelihood, both 3 and 7 are formed by an intramolecular nucleophilic attack of the 2'-hydroxy function on the phosphorus atom, giving a phosphorane-like intermediate or transition state. Under moderately acidic conditions (pH 2-6), the predominant reaction is acid-catalyzed cleavage of the P-N3' bond (first-order in [H(+)]) that yields an equimolar mixture of 5 and uridine 5'-phosphate (6). The reaction is proposed to proceed without intramolecular participation of the neighboring 2'-hydroxyl group. Under more acidic conditions (pH < 2), hydrolysis to 3 and 4 starts to compete with the cleavage of the P-N bond, and this reaction is even the fastest one at pH < 1. Formation of 2'-O,3'-N-cyclic phosphoramidate as an intermediate appears probable, although its appearance cannot be experimentally verified. The rate constants for various partial reactions have been determined. The reaction mechanisms and the effect that replacing the 3'-oxygen with nitrogen has on the behavior of the phosphorane intermediate are discussed.