The chemical stability of S-(2-acylthioethyl) and S-acyloxymethyl protected thymidylyl-3',5'-thymidine phosphoromonothiolates and their deacylation products in aqueous solution.

The hydrolytic stability of the S-(2-acetylthioethyl) (1a,b), S-(2-pivaloylthioethyl) (2a,b), and S-acetyloxymethyl (3a,b) protected Rp and Sp phosphoromonothiolates of 3',5'-TpT has been studied. Rather unexpectedly, an intramolecular hydroxide ion catalyzed acetyl migration from the protecting group to the nucleoside 3'- and 5'-hydroxy functions was found to compete with the intermolecular displacement of the AcSCH2CH2S- or AcOCH2S-ligand from the phosphorus atom of 1a,b and 3a,b, respectively. With the S-pivaloylthioethyl derivative 2a,b no such reaction took place. Additionally, the kinetics of the cleavage of the S-(2-mercaptoethyl) group from 4a,b, the products of enzymatic deacylation of 1a,b and 2a,b, were studied as a function of pH.

Preparation, hydrolysis and intramolecular transesterification of 3'-deoxy-3'-thioinosine 3'-S-dimethylphosphorothiolate.

The hydrolytic reactions of the dimethyl ester of 3'-deoxy-3'-thioinosine 3'-S-phosphorothiolate have been followed over a wide aciditiy range by HPLC. At pH > 3, only hydroxide ion catalyzed isomerization to the 2'-dimethylphosphate takes place, whereas under more acidic conditions hydrolysis to the 2'-monomethylphosphate and 3'-S-monomethylphosphorothiolate competes. The latter is the only product accumulating in very acidic solutions (1 M hydrochloric acid). Mechanisms of the reactions are discussed.

Hydrolytic reactions of the phosphorodithioate analogue of uridylyl(3',5')uridine: kinetics and mechanisms for the cleavage, desulfurization, and isomerization of the internucleosidic linkage

The hydrolytic reactions of the phosphorodithioate analogue of uridylyl(3',5')uridine [3',5'-Up(s)2U] were followed by HPLC over a wide pH range at 363.2 K. Under acidic and neutral conditions, three reactions compete: (i) desulfurization to a mixture of the (Rp)- and (Sp)-diastereomers of the corresponding 3',5'- and 2',5'-phosphoromonothioates [3',5'- and 2',5'-Up(s)U], which are subsequently desulfurized to a mixture of uridylyl(3',5')- and -(2',5')uridine [3',5'- and 2',5'-UpU], (ii) isomerization to 2',5'-Up(s)2U, and (iii) cleavage to uridine, in all likelihood via a 2',3'-cyclic phosphorodithioate (2',3'-cUMPS2). Under alkaline conditions (pH > 8), only a hydroxide ion catalyzed hydrolysis to uridine via 2',3'-cUMPS2 takes place. At pH 3-7, all three reactions are pH-independent, the desulfurization being approximately 1 order of magnitude faster than the cleavage and isomerization. At pH < 3, all the reactions are hydronium ion catalyzed. On going to very acidic solutions, the cleavage gradually takes over the desulfurization and isomerization. Accordingly, the cleavage overwhelmingly predominates at pH < 0. The overall hydrolytic stability of 3',5'-Up(s)2U is comparable to that of (Sp)- and (Rp)-3',5'-Up(s)U (and to that of 3',5'-UpU, except at pH < 2). The rate of the hydroxide ion catalyzed hydrolysis of 3',5'-Up(s)2U is 37% and 53% of that of (Sp)- and (Rp)-3',5'-Up(s)U, respectively. The reactions, however, differ with the respect of the product accumulation. While the phosphoromonothioates produce a mixture of 2'- and 3'-thiophosphates as stable products, 3',5'-Up(s)2U is hydrolyzed to uridine without accumulation of the corresponding dithiophosphates. At pH < 3, where the hydrolysis is hydronium ion catalyzed, the kinetic thio-effect of the second thio substitution is small: under very acidic conditions (Ho -0.69), (Sp)-3',5'-Up(s)U reacts 1.6 times as fast as 3',5'-Up(s)2U, but the reactivity difference decreases on going to less acidic solutions. In summary, the hydrolytic stability of 3',5'-Up(s)2U closely resembles that of the corresponding phosphoromonothioate. While replacing one of the nonbridging phosphate oxygens of 3',5'-UpU with sulfur stabilizes the phosphodiester bond under acidic conditions by more than 1 order of magnitude, the replacement of the remaining nonbridging oxygen has only a minor influence on the overall hydrolytic stability.

Cleavage of the phosphodiester bond of uridylyl-(3',5')-8-carboxymethylaminoadenosine by hydronium, hydroxide and zinc(II) ions: a model study aimed at elucidating the potential of a carboxylate function as an intramolecular catalyst.

Uridylyl-(3',5')-8-carboxymethylaminoadenosine has been synthesised, and its transesterification to uridine 2',3'-cyclic phosphate in the presence and absence of Zn2+ ion has been studied. The results show that a carboxylate function in the vicinity of the phosphodiester bond accelerates the metal ion promoted cleavage but not the metal ion independent reaction. Under acidic conditions, the predominant reaction is the cleavage of the side chain, giving the 8-amino derivative.

Determination of the nucleotide conformation in the productive enzyme-substrate complexes of RNA-depolymerases.

The aim of this work is to determine the conformation of the nucleobase adjacent to the cleavable phosphodiester bond in the productive enzyme-substrate complex of RNA-depolymerizing enzymes. To this end the kinetic parameters of hydrolysis of UpA, 2'-C-Me- and 3'-C-Me-UpA were determined for RNase A, RNase Pb2, nuclease S1 and snake venom phosphodiesterase. In these derivatives the ranges of the allowed orientation of uridine residues are restricted due to the substitution of methyl groups for the ribose hydrogen atoms. The results described demonstrate that the proposed method is of general value for the estimation of the nucleotide glycoside angles in the productive enzyme-substrate complexes.

The influence of N6-protecting groups on the acid-catalyzed depurination of 2'-deoxyadenosine.

Kinetics for the acidic hydrolysis of several N6-substituted 2'-deoxyadenosines were studied in a wide pH-range. The proportions of the partial reactions proceeding via mono- and di-protonated substrates were estimated on the bases of the rate profiles obtained and the acidity constants determined spectrophotometrically for the monocations. The site of the initial protonation was established by the effects that trifluoroacetic acid exerted on the 15N NMR chemical shifts. The exceptional lability of the monocations of N6-acyl protected compounds is suggested to result from the preferred N7 protonation.