Conformational studies of dithymidine boranomonophosphate diastereoisomers

Abstract

The boranophosphate ester nucleotides are a new class of nucleic acid analogues that are isoelectronic and isostructural to normal phosphodiester nucleic acids and that maintain the anionic charge of the nucleic acid backbone. The two P-diastereoisomers of dithymidine boranomonophosphates were separated using reverse phase HPLC; the faster and slower eluting isomers are designated as d(Tp BT)-1 and d(Tp BT)-2, respectively. Conformations of the isomers were studied using circular dichroism (CD) and NMR, and compared to the analogous phosphate diester. d(TpT). This comparison allowed the effects of the borane group and chirality of the boranophosphate linkage on sugar and base conformations to be assessed. The CD spectra of the diastereoisomers are consistent with both having a B-type conformation. Analysis of the 1H- 1H and 1H- 31P coupling constants showed that these conformations are similar to those of the unmodified parent dimer; specifically, the 2′-deoxyribose rings prefer the S (C2′- endo) conformation, and the C4′-C5′ and C5′-O5′ rotamers are primarily in the γ + and β + conformations, respectively. Conformational differences between the diastereoisomers and between the modified and unmodified dimers are manifested by differences in the preferences of the 3′-residues to adopt S sugar pucker and β + conformations. There is reduced preference for the S sugar pucker of the 3′-residue in d(Tp BT)-1 relative to d(Tp BT)-2, which is similar to d(TpT). There is less preference for the β + conformation of the 3′-residue in d(Tp BT)-2 relative to d(Tp BT)-1 and d(TpT). Based on the CD results, the temperature dependences of the thymidine H6 chemical shifts, and the derived sugar ring and backbone conformational parameters, we conclude that the borane group exerts a minimal influence on the sugar conformations and base stacking interactions. Preliminary assignment of the absolute configuration of the pair of S p and R p diastereoisomers to d(Tp BT)-1 and d(Tp BT)-2, respectively, is made on the basis of enzyme selectivity and NOE difference experiments.