A cyclic dinucleotide with a four-carbon 5′-C-to-5′-C connection; synthesis by RCM, NMR-examination and incorporation into secondary nucleic acid structures

Abstract

A 5′-C-allylthymidine derivative was prepared from thymidine by the application of a stereoselective allylation procedure and its 5′(S)-configuration was confirmed. From this nucleoside derivative, appropriately protected building blocks were prepared and coupled using standard phosphoramidite chemistry to afford a dinucleotide with two 5′-C-allylgroups. This molecule was used as a substrate for a ring-closing metathesis (RCM) reaction and after deprotection, a 1 : 1 mixture of E- and Z-isomers of a cyclic dinucleotide with an unsaturated 5′-C-to-5′-C connection was obtained. Alternatively, a hydrogenation of the double bond and deprotection afforded a saturated cyclic dinucleotide. An advanced NMR-examination confirmed the constitution of this molecule and indicated a restriction in its overall conformational freedom. After variation of the protecting group strategy, a phosphoramidite building block of the saturated cyclic dinucleotide with the 5′-O-position protected as a pixyl ether and the phosphate protected as a methyl phosphotriester was obtained. This building block was used in the preparation of two 14-mer oligonucleotides with a central artificial bend due to the cyclic dinucleotide moiety. These were found to destabilise duplexes, slightly destabilise bulged duplexes but, to some extent, stabilise a three-way junction in high Mg²⁺-concentrations.