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

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 P--O3' 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.

Intramolecular Participation of Amino Groups in the Cleavage and Isomerization of Ribonucleoside 3′‐Phosphodiesters: The Role in Stabilization of the Phosphorane Intermediate

“…Studies with trinucleoside‐3′,3′,5′‐monophosphates suggest that stabilization of the phosphorane intermediate plays an important role, since replacement of 2′‐OMe with 2′‐OH on the leaving 3′‐linked nucleoside enhances the departure of both the 3′‐ and 5′‐linked nucleosides almost equally 13. This structural change also accelerates isomerization of the 3′,3′,5′‐triester to a mixture of 2′,3′,5′‐ and 2′,2′,5′‐triesters, as exemplified with the more slowly reacting phosphoromonothioate analogues 14. These results are consistent with stabilization of the phosphorane intermediate, although stabilization of the leaving group by inductive effect and/or intramolecular hydrogen bonding undoubtedly contributes appreciably.…”

Understanding Catalysis of Phosphate‐Transfer Reactions by the Large Ribozymes

“…The pro-R P oxygen is another viable candidate for the hydrogen-bond acceptor based on the observed rate retardation by sulfur substitution at this position and the results on small molecular models discussed above. [96,118,120] However, due to the fact that no reaction is detected with the pro-R P substrates regardless of the 2'-substituent, this proposed interaction remains to be confirmed (Figure 9 B). The ribo substrate requirements of the spliceosome parallel those of the group II introns: the 2'-OH of the departing nucleoside is not absolutely necessary in either splicing step, but the second step proceeded approximately an order of magnitude faster with the natural ribo substrate relative to 2'-deoxy or 2'-methoxy substrates.…”

“…[119][120] Theoretical studies on simple, unconstrained model compounds also identify the non-bridging phosphoryl oxygen, rather than the 3'-oxygen, as a hydrogen-bond acceptor. [123] Finally, the 3'-OH of adenosine is only 0.7 pK a units more acidic than the 3'-OH of 2'-O-methyladenosine, indicating that hydrogen-bond stabilization of the departing 3'-oxyanion by the neighboring 2'-OH group could account for no more than a fivefold rate acceleration.…”

“…[122] With the trinucleoside phosphorothioate models, the ratio of P-O3' fission is 88 % for the 2'-OH and, somewhat unexpectedly, 95 % for the 2'-OMe analogue. [120] The fact that cleavage of the P-O3' bond is not specifically retarded upon 2'-methoxy substitution suggests that it is the phosphorane intermediate (Figure 8 A) rather than the 3'-leaving group (Figure 8 B) that is stabilized by the 2'-OH group. This interpretation is further borne out by the observation that methylation of the 2'-oxygen also inhibits the O [b] S [b] OH [a] 1 0.68 OMe [a] The 2'-substituent of the departing nucleoside (see Scheme 5).…”

Participation of an additional 4′-hydroxymethyl group in the cleavage and isomerization of ribonucleoside 3′-phosphodiesters

“…[17][18][19] We have previously 20 tried to quantify the effects that amino groups in the vicinity of the scissile phosphodiester bond may have on the transesterification reactions of an internucleosidic 3′,5′-phosphodiester bond. 22 Finally, the 3′,3′-phosphodiester dimer 4a, having the 2′-hydroxy groups free, is cleaved and isomerized faster than its 2′-O-monomethylated analog 4b. At pH 3-5, i.e.…”