Synthesis and Base-Pairing Properties of Oligonucleotides Containing 5′-(R)- and 5′-(S)-C-Aryl-thymidine

Abstract

By applying the Liebeskind-Srogl cross-coupling reaction to pyrimidine nucleosides, a Pd(0)-catalyzed, Cu(I)-mediated process that effectively couples thioesters and boronic acids to form ketones, we previously described an efficient and easy access to 5′-C-acyl nucleosides. By reduction of the 5′-carbonyl group into a secondary alcohol, we report herein the synthesis of new 5′-C-aryl-5′-O-dimethoxytrityl-3′-O-phosphoramidite building blocks ready to be incorporated into oligonucleotides by automated synthesis. We describe the preparation of two sets of (R)- and (S)-diastereoisomers of 5′-C-phenylthymidine (PhT) and 5′-C-pyrenylthymidine (PyT), their conversion to phosphoramidites and incorporation into oligodeoxynucleotides (ODN). We also discuss the challenges faced by the synthesis of PyT-modified ODN, which required to modify the automated cycle to prevent backbone degradation in subsequent steps. The affinity and specificity of several 5′-C-aryl modified-ODN toward their complementary sequence were studied by UV-melting experiments. While thermal melting analysis failed to differentiate between matched and mismatched pairs, fluorescence measurements demonstrated that pyrenyl-modified ODN can effectively distinguish correct from incorrect base pairs. This highlights their potential use in the detection of genetic mutations through structural changes in DNA duplexes.