A1 Refereed original research article in a scientific journal
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
Authors: Lonnberg T, Ora M, Virtanen S, Lonnberg H
Publisher: WILEY-V C H VERLAG GMBH
Publication year: 2007
Journal:: Chemistry - A European Journal
Journal name in source: CHEMISTRY-A EUROPEAN JOURNAL
Journal acronym: CHEM-EUR J
Volume: 13
Issue: 16
First page : 4614
Last page: 4627
Number of pages: 14
ISSN: 0947-6539
DOI: https://doi.org/10.1002/chem.200601835
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 (1a) has been compared to the hydrolysis of 2',3'-O-methyleneadenosin-5'-yl 5'-0-methyluridin-Y-yl 2',5'-di-O-methyluridin-3'-yl phosphoromonothioate (1b) and the hydrolysis of bis[uridin-3'-yl] phosphorodithioate (2a) to the hydrolysis of uridin-3'-yl 2',5'-di-O-methyluridin-3'-yl phosphorodithioate (2b). ne reactions have been followed by RP HPLC over a wide pH range. The phosphoromonothioate triesters 1a,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 2a,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 la and 2a is 27 times as fast as that compared with their 2'-O-methylated counterparts 1b and 2b. The hydroxide-ion-catalyzed isomerization of the 3',3',5'-triester to 2',3',5'- and 2',2',5'-triesters with la is It times as fast as that compared with 1b. These accelerations have been accounted for by stabilization of the anionic phosphorane intermediate by hydrogen bonding with the T-hydroxy function. Thio substitution of the nonbridging oxygens has an almost negligible influence on the cleavage of 3',3'-diesters 2a,b, but the hydrolysis of phosphoromonothioate triesters 1a,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 1a and 2a.
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 (1a) has been compared to the hydrolysis of 2',3'-O-methyleneadenosin-5'-yl 5'-0-methyluridin-Y-yl 2',5'-di-O-methyluridin-3'-yl phosphoromonothioate (1b) and the hydrolysis of bis[uridin-3'-yl] phosphorodithioate (2a) to the hydrolysis of uridin-3'-yl 2',5'-di-O-methyluridin-3'-yl phosphorodithioate (2b). ne reactions have been followed by RP HPLC over a wide pH range. The phosphoromonothioate triesters 1a,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 2a,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 la and 2a is 27 times as fast as that compared with their 2'-O-methylated counterparts 1b and 2b. The hydroxide-ion-catalyzed isomerization of the 3',3',5'-triester to 2',3',5'- and 2',2',5'-triesters with la is It times as fast as that compared with 1b. These accelerations have been accounted for by stabilization of the anionic phosphorane intermediate by hydrogen bonding with the T-hydroxy function. Thio substitution of the nonbridging oxygens has an almost negligible influence on the cleavage of 3',3'-diesters 2a,b, but the hydrolysis of phosphoromonothioate triesters 1a,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 1a and 2a.
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