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

Graphical Abstract

Kinetic studies with triribonucleoside 3′,3′,5′-phosphoromonothioates and diribonucleoside 3′,3′-phosphorodithioates suggest that the thiophosphorane intermediate obtained by intramolecular attack of a 2′-OH group on the phosphorus atom (see scheme) is markedly stabilized by hydrogen bonding of the other 2′-OH to the anionic thio ligand. This accelerates both the phosphoester cleavage and phosphate migration. The related thio effects are discussed.

Abstract

To provide a solid chemical basis for the mechanistic interpretations of the thio effects observed for large ribozymes, the cleavage of triribonucleoside 3′,3′,5′-phosphoromonothioate triesters and diribonucleoside 3′,3′-phosphorodithioate diesters has been studied. To elucidate the role of the neighboring hydroxy group of the departing 3′-linked nucleoside, hydrolysis of 2′,3′-O-methyleneadenosin-5′-yl bis[5′-O-methyluridin-3′-yl] phosphoromonothioate (1 a) has been compared to the hydrolysis of 2′,3′-O-methyleneadenosin-5′-yl 5′-O-methyluridin-3′-yl 2′,5′-di-O-methyluridin-3′-yl phosphoromonothioate (1 b) and the hydrolysis of bis[uridin-3′-yl] phosphorodithioate (2 a) to the hydrolysis of uridin-3′-yl 2′,5′-di-O-methyluridin-3′-yl phosphorodithioate (2 b). The reactions have been followed by RP HPLC over a wide pH range. The phosphoromonothioate triesters 1 a,b undergo two competing reactions: the starting material is cleaved to a mixture of 3′,3′- and 3′,5′-diesters, and isomerized to 2′,3′,5′- and 2′,2′,5′-triesters. With phosphorodithioate diesters 2 a,b, hydroxide-ion-catalyzed cleavage of the PO3′ bond is the only reaction detected at pH >6, but under more acidic conditions desulfurization starts to compete with the cleavage. The 3′,3′-diesters do not undergo isomerization. The hydroxide-ion-catalyzed cleavage reaction with both 1 a and 2 a is 27 times as fast as that compared with their 2′-O-methylated counterparts 1 b and 2 b. The hydroxide-ion-catalyzed isomerization of the 3′,3′,5′-triester to 2′,3′,5′- and 2′,2′,5′-triesters with 1 a is 11 times as fast as that compared with 1 b. These accelerations have been accounted for by stabilization of the anionic phosphorane intermediate by hydrogen bonding with the 2′-hydroxy function. Thio substitution of the nonbridging oxygens has an almost negligible influence on the cleavage of 3′,3′-diesters 2 a,b, but the hydrolysis of phosphoromonothioate triesters 1 a,b exhibits a sizable thio effect, k_PO/k_PS=19. The effects of metal ions on the rate of the cleavage of diesters and triesters have been studied and discussed in terms of the suggested hydrogen-bond stabilization of the thiophosphorane intermediates derived from 1 a and 2 a.