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, tC^fTP) 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 tC^fTP, 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 (tC^fTP).