Assignment of absolute configuration at phosphorus of P-chiral diastereomers of deoxyribonucleoside methanephosphonamidates by means of NMR spectroscopy

Abstract

Recently, we have prepared a novel class of DNA analogues containing the [3′-NH–P(CH 3)(O)–O-5′] methanephosphonamidate linkage. Synthesis of such analogues requires preparation of the dinucleoside methanephosphonamidates N×N, where N is a 2′-deoxyribonucleoside moiety and × is the methanephosphonamidate linkage. Dimers T×T and C×T were obtained in a non-stereospecific manner giving rise to a pair of P-chiral diastereomers. Such diastereomers were effectively separated into fast and slow migrating ones by means of chromatographic methods (TLC). As described in our previous work (Nawrot et al. Nucleic Acids Res. 1998, 26, 2650), the stereochemistry of the phosphorus chiral center of T×T fast migrating diastereomer is R P and of T×T slow migrating diastereomer is S P, as established by means of 2D ROESY experiments. Here we describe assignment of the absolute configuration at the phosphorus center of fast and slow migrating diastereomers of C×T dimer. The 2D ROESY sequence with phosphorus decoupling during acquisition used in these measurements allowed observation of the P–Me group as a singlet instead of a 1H– 31P-coupled doublet. The apparent advantage of this approach was a much better signal to noise ratio and improved resolution in the F1 dimension. For the fast migrating C×T diastereomer an R P and for slow migrating C×T diastereomer an S P configuration was assigned. Conformational analysis of both pairs of diastereomers T×T and C×T indicates significant differences in sugar ring puckering, which strongly depend on the nature of the nucleobase at the 5′-terminus of the dimer. The ribose rings of the 3′-amino-2′,3′-dideoxycytidine moiety of both diastereomers of C×T adopt predominantly a C3′- endo (North) conformation, while thymine-substituted ribofuranoses originating either from C×T or T×T dimers prefer a C2′- endo (South) conformation.