Hydrolytic Reactions of the Diastereomeric Phosphoromonothioate Analogs of Uridylyl(3',5')uridine: Kinetics and Mechanisms for Desulfurization, Phosphoester Hydrolysis, and Transesterification to the 2',5'-Isomers

Abstract

Hydrolytic reactions of the R? and Sp diastereomers of the phosphoromonothioate analog of uridylyl-(3',5')uridine (3',5'-UpU), having a nonbridging oxygen replaced with sulfur, have been followed by HPLC over a wide pH range at 363.2 K. Under neutral and acidic conditions three reactions compete: (i) desulfurization to an equilibrium mixture of 3',5'-and 2',5'-UpU, (ii) hydrolysis to uridine 2'-and 3'-monophosphates with release of uridine (either via a 2',3'-cyclic phosphoromonothioate, or a desulfurized cyclic triester), and (iii) isomerization to the 2',5'-dinucleoside phosphoromonothioate. With both diastereomers, desulfurization predominates over hydrolysis and migration at pH 1-8. Migration proceeds by retention of configuration at phosphorus and is most pronounced in very acidic solutions (Hq < 0.2, i.e., [HC1] > 0.5 mol Lr1), representing 20-30% of the total disappearance of the starting material. At pH 3-6, the proportion of this reaction is less than 10%. In the latter pH range, all the reactions are pH-independent. At lower pH, first-order dependence on acidity is observed, but at Ho < 0.2 desulfurization becomes slower than the competing reactions. The R? diastereomer is at pH < 7 up to three times as reactive as the Sp isomer. Under alkaline conditions (pH > 9), only base-catalyzed hydrolysis to uridine 2'-and 3'thiophosphates with release of uridine takes place. At pH < 1, the thioate analogs are more than 1 order of magnitude more stable than UpU, while at higher pH the reactivities are comparable.

Pharmacological evaluation and chemical stability of 2‐benzylthioether‐5′‐O‐(1‐thiotriphosphate)‐adenosine, a new insulin secretagogue acting through P2Y receptors

“…1). The latter reaction was reported to predominate in several cases of acidic hydrolyses of nucleoside phosphorothioates [Oivanen et al, 1995;Ora et al, 1996a]. In our case, however, for 2-benzylthio-ATP-α-S at pH 1.4/37°C for 18 h, neither desulfurization nor deglycosylation products could be detected by 31 P NMR and HPLC.…”

Thio Effects on the Departure of the 3′‐Linked Ribonucleoside from Diribonucleoside 3′,3′‐Phosphorodithioate Diesters and Triribonucleoside 3′,3′,5′‐Phosphoromonothioate Triesters: Implications for Ribozyme Catalysis

“…The preparation of the diasteromeric uridine 2',3'-cyclic phosphoromonothioates, also used as reference materials, has been described earlier. [12] All the reagents employed were of reagent grade. [22] (5, 0.56 g, 1.00 mmol) was coevaporated three times from anhydrous pyridine.…”

“…[15] For this reason, the cleavage of phosphodiester via a di-A C H T U N G T R E N N U N G anionic transition state usually exhibits a rather small thio effect. [7,10,12] Bearing the importance of solvation in mind, one might expect the thio effects of associative phosphoester cleavage reactions via a phosphorane-like transition state to be sensitive to hydrogen-bonding interactions. In other words, stabilization of a phosphorane intermediate or leaving group by intramolecular hydrogen bonding could well be reflected in the magnitude of the thio effect.…”

Ribozyme speed limits

“…The RNA linkage (1) passes through a pentacoordinate species (2) that degrades into fragments that carry either a 2Ј,3Ј-cyclic phosphate terminus (3) or a 5Ј-hydroxyl terminus (4). The four catalytic strategies that can influence the reaction are identified as follows: ␣, in-line nucleophilic attack (blue); ␤, neutralization of negative charge on a nonbridging phosphate oxygen (purple); ␥, deprotonation of the 2Ј-hydroxyl group (red); and ␦,age of phosphoanhydrides, phosphomonoesters, and phosphoramidates (Benkovic and Schray 1973;Guthrie 1977;Westheimer 1987;Thatcher and Kluger 1989;Admiraal and Herschlag 1995;Oivanen et al 1995;Almer and Strömberg 1996). RNA transesterification occurs spontaneously in the absence of any added catalyst with a first-order rate constant that varies with changing pH (Järvinen 1991;Li and Breaker 1999).…”