Synthesis of a Fluorescent Cytidine TNA Triphosphate Analogue.

Abstract

Threose nucleic acid (TNA) is refractory to nuclease digestion and capable of undergoing Darwinian evolution, which together make it a promising system for diagnostic and therapeutic applications that require high biological stability. Expanding the sequence/chemical diversity of TNA would enable the development of functional TNA molecules with enhanced physicochemical properties. Recently, we have reported the synthesis and polymerase activity of a fluorescent cytidine TNA triphosphate analogue (1,3-diaza-2-oxo-phenothiazine, tCfTP) that maintains Watson-Crick base pairing with guanine. It not only improves TNA synthesis by enabling an engineered TNA polymerase to read through sequential G-repeats in a DNA template but it also introduces new physicochemical properties, such as increased hydrophobic character and fluorescence. Here, we describe the synthesis protocol for tCfTP, which includes three silica gel purifications, two precipitations, and one HPLC purification. Starting from protected threofuranosyl sugar, desired TNA nucleoside was obtained in a Vorbrüggen glycosylation reaction. After deprotection, nucleoside was further converted to 3'-monophosphate, activated by 2-methylimidazole, and subsequently treated with pyrophosphate to afford the desired 3'-triphosphate (tCfTP).