Synthesis of Chiral [¹⁸O]Phosphorothioate Analogues of Adenine Nucleotides

Syntheses of R_P-ATPγS,γ¹⁸O₂, R_P- and S_P-ADPβS,β¹⁸O, and R_P- and S_P-AMPS,¹⁸O are described. Coupling of 2′,3′-(methoxymethylidene)-AMP, 1, with S_P-ADPαS,α¹⁸O₂ produced P¹-5′-adenosyl P³-2′,3′-(methoxymethylidene)-5′-adenosyl 1-thio[1-tri¹⁸O₂]phosphate, 3. Upon cleavage of the unprotected ribose ring with IO₄⁻, reduction of IO₃⁻ by HPSO₃²⁻, removal of the 2′,3′-methoxymethylidene protecting group at pH 2, and alkaline elimination of the product from the IO₄⁻-cleaved dialdehyde, R_P-ATPγS,γ¹⁸O₂ was produced and isolated by chromatography in 55–58% yield based on S_P-ADPαS,α¹⁸O₂. Coupling of AMPS,¹⁸O₂ with 1 produced (R_P)- and (S_P)-P¹-5′-adenosyl P²-2′,3′-(methoxymethylidene)-5′-adenosyl 1-thio[1-¹⁸O]pyrophosphate, 4a and 4b, which were separated by ion-exchange chromatography through DEAE-Sephadex A-25. Periodate cleavage, deprotection, and alkaline elimination of 4a and 4b analogous to that described above for 3 produced S_P- and R_P-ADPβS,β¹⁸O, respectively. Coupling of AMPS,¹⁸O₂ with AMP produced (R_P)- and (S_P)-P¹, P²-bis(5′-adenosyl) 1-thio[1-¹⁸O]pyrophosphate, 5a and 5b, which were separated by ion-exchange chromatography through DEAE-Sephadex A-25. Nucleotide pyrophosphatase catalyzed hydrolysis of 5a and 5b produced R_P- and S_P-AMPS,¹⁸O, respectively. These chiral nucleoside and nucleotide [¹⁸O]phosphorothioates are useful as substrates for stereochemical studies of phosphotransferases. These compounds and their precursors should also serve as important synthetic precursors of ADP and ATP stereospecifically enriched with heavy isotopes of oxygen at any desired position in the phosphoanhydride system.