The chemical reactivity of ribonucleotide analog
2‘-deoxy-2‘-thiouridine 3‘-(p-nitrophenyl phosphate)
(1),
in which the 2‘ hydroxyl is replaced with a 2‘-thiol group, has been
characterized. The major reaction pathway for
1, as monitored by 31P NMR spectroscopy, is
transphosphorylation to afford 2‘,3‘-cyclic phosphorothioate
3, followed
by hydrolysis of 3 to produce 2‘-deoxy-2‘-thiouridine
2‘-phosphorothioate (4). Thus, the reaction pathway of
1 is
similar to that of the hydrolysis of ribonucleotides, yet there are
significant differences. The pH−rate profile for
transphosphorylation of 1 was determined by monitoring the
formation of p-nitrophenol or p-nitrophenolate by
UV−visible spectroscopy. Analysis of the profile reveals the attacking
nucleophile to be thiolate, and the pK
a of
the
2‘-thiol was determined to be 8.3 ± 0.1. At pH 7.4, the
thiol-containing ribonucleotide analog 1 is hydrolyzed at
an
observed rate 27-fold slower than its 2‘-hydroxyl counterpart.
These results indicate that the rate of thiolate
attack
on the adjacent phosphodiester bond is 107-fold slower than
that of the corresponding alkoxide. Thiolate
nucleophiles,
therefore, are remarkably reticent toward attack at electrophilic
phosphate centers. In addition to providing new
information about the reactivity of phosphodiester bonds, our studies
highlight the potential of 2‘-thiol-containing
nucleotides for the study of an array of RNA processes, especially
those in which the 2‘-substituent plays a critical
role.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.