Phosphorus-31 Fourier transform nuclear magnetic resonance study of mononucleotides and dinucleotides. 1. Chemical shifts

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Abstract
A 31P NMR study of adenine, uracil and thymine mononucleotides, their cyclic analogs and the corresponding dinucleotides is reported. From the pH dependence of phosphate chemical shifts, pKa values of 6.25-6.30 are found for all 5''-mononucleotides secondary phosphate ionization, independently from the nature of the base and the presence of a hydroxyl group at the 2'' position. Substitution of a H atom for a 2''-OH lowers the pKa of 3''-monoribonucleotides from 6.25 down to 5.71-5.85. This indication of a strong influence of the 2''-hydroxyl group on the 3''-phosphate is confirmed by the existence of a 0.4 to 0.5 ppm downfield shift induced by the 2''-OH on the phosphate resonance of 3''-monoribonucleotides, and 3'',5''-cyclic nucleotides and dinucleotides with respect to the deoxyribosyl analogs. Phosphate chemical shifts and titration curves are affected by the ionization and the type of the base. Typically, deviations from the theoretical Henderson-Hasselbalch plots are observed upon base titration. Purine displays a more deshielding influence than pyrimidine on the phosphate groups of most of the mononucleotides (0.10 to 0.25 ppm downfield shift) with a reverse situation for dinucleotides. These effects and the importance of stereochemical arrangement (furanose ring pucker, furanose-phosphate backbone conformation, O.sbd.P.sbd.O bond angle) on the phosphate chemical shifts are discussed.