Synthesis of pyrrolo-modified nucleotides and their incorporation into DNA via enzymatic extension

Abstract

A series of pyrrole-containing derivatives of the 2-deoxy-pyrimidines, thymidine and cytidine have been prepared by Pd-catalysed cross-coupling between N-alkyl-alkynyl functionalized pyrrole (py), 2-(2-thienyl)pyrrole (tp) and 2,5-bis(2-thienyl)pyrrole (tpt) with 5-iodo-2’-deoxyuridine and 5-iodo-2’-deoxycytidine. The length of the alkyl chain linking the nucleoside and pyrrolyl-containing unit was varied from three to five carbon units. The series of nucleosides were characterized by 1H NMR spectroscopy, 13C NMR spectroscopy, ES-MS, UV-vis spectroscopy, cyclic voltammetry and in some cases single-crystal X-ray diffraction. Cyclic voltammetry revealed that all the py-, tp- and tpt-alkynyl derivatives can be electrochemically polymerized to form conductive materials. Conversion of the tp-modified nucleosides into their corresponding nucleotides was performed by phosphorylation at the 5’-hydroxyl site via phosphorus (V) and phosphorus (III) chemistry to yield dTTP-5-tp (dTTP*) and dCTP-5-tp (dCTP*), respectively. The purified nucleotides were fully characterized by 1H NMR, 31P NMR, ES-MS, MPLC and HPLC. The incorporation of dTTP* and dCTP* into DNA via enzymatic extension was explored. Polyacrylamide gel electrophoresis indicated that DNA polymerases, Pfu Pol B exoand Klenow Fragment exo-, can tolerate dTTP* and dCTP* respectively. Gel electrophoresis revealed the successful incorporation of the modified bases into a primer template duplex of up to 37 base pairs. In comparison to standard nucleotides, dTTP* and dCTP* were incorporated at a slower rate. In order to produce functionalized DNA duplexes of microns in length, attempts were made to incorporate the nucleotides into an extending primer sequence via a slippage mechanism. Analysis via agarose gel electrophoresis demonstrated that the polymerases employed in this study could not read the modified DNA as a template as efficient as standard nucleotides but limited extension was observed.