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 (original) (raw)
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Tetrahedron, 1989
0-aryl-O,S-dialkylphsphorot~oates, such as fully-protected adenylyl(3 '+S 1-S '-thiouridine 11, 0-aryl-0-ethyl-5'-thiouridyi phosphorothioate 24, upon treatment with an excess n-tetrabutylammonium fluoria!e in tetrahydrqturan-pyridine-water (8:l :I vlvlv) underwent a scission of phosphorus-sulfur bond to give the corresponding 0-alRyphosphoromonofluoridates 20 (74%), and 30 (75%). This facile, new preparation of alkylphosphoromon&wridates has been found to be a general reaction which has been exemplified by the conversion of 0-aryl-OS-dialkylphosphorothioates 26, 27,28 and 29 to the corresponding phosphorofluoridates 30 (79%), (31+ 32, together 8S%), 33 (63%). and 34 (90%) The fully-protected adenylyl(3 '+5 1-S 'Aiouridines 12 and 13 were pam'alb &protected to 37 and38, having a phosphorothioate linkage with a bridging sulfur [ribonucleosia'e-3-0-PO,-S-s-ribonucleoside] in order to examine the stability of this internucleotidyl linkage vicinal to a 2 '-hydroql function. The 2 '-O-protected adenylyl(3 '+5*)-S 'thiouridines 37 and 38 were found to be as stable as thymidylyl(3 '-+S J-5 '-thiothymidine 41. Removal of the 2 '-O-protecting group from 37 or 38 however gave only a transiently stable adenylyl(3 '+S 3-S '-thiouridine 39 which promptly decomposed through a phosphorus-sulfur bond cleavage, due to the nucleophilc attack by the vicinal2 'hydroxyl group, both under mildly acidic and neutral conditions, to give the 2 '3 *-cyclic phosphate 40 (-75%), and 5 '-thiouridine derivatives 18 and 19.
Biochemistry, 1999
The hammerhead ribozyme crystal structure identified a specific metal ion binding site referred to as the P9/G10.1 site. Although this metal ion binding site is ∼20 Å away from the cleavage site, its disruption is highly deleterious for catalysis. Additional published results have suggested that the pro-R P oxygen at the cleavage site is coordinated by a metal ion in the reaction's transition state. Herein, we report a study on Cd 2+ rescue of the deleterious phosphorothioate substitution at the cleavage site. Under all conditions, the Cd 2+ concentration dependence can be accounted for by binding of a single rescuing metal ion. The affinity of the rescuing Cd 2+ is sensitive to perturbations at the P9/G10.1 site but not at the cleavage site or other sites in the conserved core. These observations led to a model in which a metal ion bound at the P9/G10.1 site in the ground state acquires an additional interaction with the cleavage site prior to and in the transition state. A titration experiment ruled out the possibility that a second tightbinding metal ion (K d Cd < 10 µM) is involved in the rescue, further supporting the single metal ion model. Additionally, weakening Cd 2+ binding at the P9/G10.1 site did not result in the biphasic binding curve predicted from other models involving two metal ions. The large stereospecific thio-effects at the P9/ G10.1 and the cleavage site suggest that there are interactions with these oxygen atoms in the normal reaction that are compromised by replacement of oxygen with sulfur. The simplest interpretation of the substantial rescue by Cd 2+ is that these atoms interact with a common metal ion in the normal reaction. Furthermore, base deletions and functional group modifications have similar energetic effects on the transition state in the Cd 2+ -rescued phosphorothioate reaction and the wild-type reaction, further supporting the model that a metal ion bridges the P9/G10.1 and the cleavage site in the normal reaction (i.e., with phosphate linkages rather than phosphorothioate linkages). These results suggest that the hammerhead undergoes a substantial conformational rearrangement to attain its catalytic conformation. Such rearrangements appear to be general features of small functional RNAs, presumably reflecting their structural limitations.
The enzymatic hydrolysis of the phosphate ester bond in some thionucleotides
Biochimica et biophysica acta, 1979
We prepared the 5'- and 3'-O-phosphorothioate esters of the antitumor agent O2 : 2'-anhydro-1-beta-D-arabinosylcytosine. We also included in this study esters of 2'-thio-2'-deoxycytidine, namely, 2'-S-dCyd-2' : 3'-P, 2'-S-dCyd-2'-P, and 2'-S-dCyd-3'-P, along with natural nucleotides. These compounds were subjected to the action of Escherichia coli alkaline phosphatase, potato acid phosphatase, and bovine pancreatic ribonuclease A. The data were analyzed by Lineweaver-Burk plots to obtain Km and KI values. Only 2'-S-dCyd-2'-P was a substrate for alkaline phosphatase; the anhydro-araCyt phosphorothioates were good competitive inhibitors, while 2'-S-dCyd-3'-P did not associate with the enzyme. Acid phosphatase hydrolyzed all four monoesters investigated, including the S-phosphorothioate. The cyclic phosphorothioate, 2'-S-dCyd-2' : 3'-P was neither hydrolyzed by, nor associated with, ribonuclease A. ORD spectros...
Angewandte Chemie, 2000
The mechanisms of enzymatic and chemical hydrolysis of RNA have been the subject of intense interest for many years. Early demonstration of a dramatically enhanced hydrolytic reactivity of RNA compared to DNA established a crucial mechanistic role for the 2'-hydroxyl group. Hitherto, it has been accepted that the major role of the 2'-hydroxyl group is nucleophilic catalysis and the 2',3'-cyclic phosphate intermediate is well established. The comparatively recent discovery of RNA catalysis (ribozymes) has stimulated renewed interest in the potential role(s) of the 2'-hydroxyl group in an increasingly diverse range of reactions. Thus, the report by Roussev et al. of the electrophilic catalysis of displacement reactions at a neighboring phosphoryl center by the cis vicinal hydroxyl group, if it is correct, offers a potentially novel mechanism that could be of significance to ribozymes. The importance of this claim prompted the present investigation.
The trans-2-deoxyribosylation of 4-thiouracil ( 4S Ura) and 2-thiouracil ( 2S Ura), as well as 6-azauracil, 6-azathymine and 6-aza-2thiothymine was studied using dG and E. coli purine nucleoside phosphorylase (PNP) for the in situ generation of 2-deoxy-α-Dribofuranose-1-phosphate (dRib-1P) followed by its coupling with the bases catalyzed by either E. coli thymidine (TP) or uridine (UP) phosphorylases. 4S Ura revealed satisfactory substrate activity for UP and, unexpectedly, complete inertness for TP; no formation of 2'-deoxy-2-thiouridine ( 2S Ud) was observed under analogous reaction conditions in the presence of UP and TP. On the contrary, 2S U, 2S Ud, 4S Td and 2S Td are good substrates for both UP and TP; moreover, 2S U, 4S Td and 2'-deoxy-5-azacytidine (Decitabine) are substrates for PNP and the phosphorolysis of the latter is reversible. Condensation of 2S Ura and 5-azacytosine with dRib-1P (Ba salt) catalyzed by the accordant UP and PNP in Tris•HCl buffer gave 2S Ud and 2'-deoxy-5-azacytidine in 27% and 15% yields, respectively. 6-Azauracil and 6-azathymine showed good substrate properties for both TP and UP, whereas only TP recognizes 2-thio-6-azathymine as a substrate. 5-Phenyl and 5-tert-butyl derivatives of 6-azauracil and its 2-thioxo derivative were tested as substrates for UP and TP, and only 5-phenyl-and 5-tert-butyl-6-azauracils displayed very low substrate activity. The role of structural peculiarities and electronic properties in the substrate recognition by E. coli nucleoside phosphorylases is discussed.