The acidity constants of twofold protonated methyl thiophosphate (MeOPS(2-)) and of monoprotonated uridine 5'- O-thiomonophosphate (UMPS(2-)) have been determined in aqueous solution (25 degrees C; I= 0.1 M, NaNO(3)) by potentiometric pH titration. The stability constants of their 1:1 complexes formed with Pb(2+), i.e. Pb(MeOPS) and Pb(UMPS), have also been measured. The results show that replacement of a phosphate oxygen by a sulfur atom increases the acidity by about 1.4 p K units. On the basis of recently established log versus plots ( = simple phosphate or phosphonate ligands where R is a non-coordinating residue), it is shown that the stability of the Pb(thiophosphate) complexes is by log Delta= 2.43+/-0.09 larger than expected for a Pb(2+)-phosphate interaction. The identity of the stability increase (log Delta) observed for Pb(MeOPS) and Pb(UMPS) shows that the nucleobase residue in the Pb(UMPS) complex has no influence on complex formation. To be able to carry out the mentioned comparisons, we have also determined the stability constant of the complex formed between Pb(2+) and methyl phosphate; the corresponding data for Pb(UMP) were already known from our earlier studies. The present results allow an evaluation of other Pb(2+) complexes formed with thiophosphate derivatives and they are applied now to the Pb(2+) complexes of adenosine 5'- O-thiomonophosphate (AMPS(2-)). The stability constants of the Pb(H;AMPS)(+) and Pb(AMPS) complexes were measured and it is shown that, within the error limits, the stability of the Pb(AMPS) complex is determined by the basicity of the thiophosphate group of AMPS(2-); in other words, no hint for macrochelate formation involving N7 was observed. More important, with the aid of micro-stability-constant considerations it is concluded that the structure of the dominating isomer of the Pb(H;AMPS)(+) species is the one where the proton is located at the N1 site of the adenine residue and Pb(2+) is coordinated to the deprotonated thiophosphate group. The insights gained from this study with regard to thiophosphate-altered single-stranded nucleic acids and their affinity towards Pb(2+) are discussed.

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2-Alkoxy-2-thiono-1,3,2-oxathiaphospholanes are readily transformed into phosphorothioate monoesters of the corresponding alcohols in a one-pot process, involving the reaction with 3-hydroxypropionitrile in the presence of DBU, followed by treatment with aqueous ammonia. In this way a series of nucleoside-3¢-O-and 5¢-O-phosphorothioates were prepared, as well as phosphorothioate derivatives of selected polyols.Nucleoside phosphoromonothioates, originally introduced by Eckstein, 1 became an important tool for studying the mechanisms of action of nucleolytic enzymes 1-3 and are widely employed as intermediates in the synthesis of other phosphorothioate derivatives of nucleosides. 4 The procedure employed by Eckstein 1 involved the treatment of appropriately protected nucleoside with thiophosphoryl tris-imidazolide. The procedure was not very efficient and was limited to the synthesis of nucleoside 5¢-O-phosphorothioates. For the synthesis of this class of compounds much a more convenient methodology was introduced by Murray and Atkinson 2 who treated unprotected adenosine with thiophosphoryl chloride in triethyl phosphate as a solvent. This approach was further extended to other nucleosides in both the ribo-5 and deoxyribonucleoside series. 6 Another example of 5¢-O-thiophosphorylation of unprotected nucleosides was described by Chen and Benkovic 7 who treated nucleoside with phosphorous acid in the presence of DCC, followed by sulfurization of resulting nucleoside 5¢-O-H-phosphonate with elemental sulfur. Other procedures, which were described in the literature for thiophosphorylation of nucleosides required selective protection of reactive groups at the sugar and/or nucleobase moiety. Thus, the synthesis of thymidine 3¢-O-phosphorothioate was accomplished by treatment of 5¢-O-protected thymidine with thiophosphoryl chloride in the presence of pyridine followed by work-up with alkali. 8 An efficient thiophosphorylation of 3¢-O-or 5¢-O-protected nucleosides was also performed by Cook, 9 with S-2-carbamoylethyl phosphorothioate in the presence of DCC as condensing agent. In a similar manner S-2-cyanoethyl phosphorothioate was employed for introducing of phosphorothioate functions into nucleoside moiety. 10 In both cases the S-protecting groups were selectively removed by postsynthetic work-up with alkali. More recently, the phosphoramidite methodology was successfully adopted for both 5¢-O-and 3¢-O-thiophosphorylation of nucleosides. 11,12 The procedure involved phosphitylation of appropriately protected nucleoside with reagents such as bis(2-cyanoethoxy)-N,N-diisopropylaminophosphoramidite, 11 bis[2-(4-nitrophenyl)-ethoxy]-N,N-diisopropylaminophosphoramidite, 11a or 2-cyanoethoxy-2-(dimethoxytrityloxyethylsulfonyl)-ethoxy-N,N-diiso-propylaminophosphoramidite 12 in the presence of tetrazole, followed by sulfurization with elemental sulfur. The phosphorothioate O-protecting groups were removed by b-elimination by treatment with appropriate base. In addition to nucleosides, the phosphoramidite methodology was successf...