Synthesis of a Fluorescent Cytidine TNA Triphosphate Analogue

Hui Mei; John Chaput

doi:10.1007/978-1-4939-9216-4_3

Synthesis of a Fluorescent Cytidine TNA Triphosphate Analogue

Methods Mol Biol. 2019:1973:27-37. doi: 10.1007/978-1-4939-9216-4_3.

Authors

Hui Mei¹, John Chaput^{2

3

4}

Affiliations

¹ Department of Chemistry, Stanford University, Stanford, CA, USA. huimei@stanford.edu.
² Department of Pharmaceutical Sciences, University of California, Irvine, Irvine, CA, USA. jchaput@uci.edu.
³ Department of Chemistry, University of California, Irvine, Irvine, CA, USA. jchaput@uci.edu.
⁴ Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA, USA. jchaput@uci.edu.

PMID: 31016694
DOI: 10.1007/978-1-4939-9216-4_3

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).

Keywords: 1,3-diaza-2-oxo-phenothiazine; Fluorescence; Nucleoside triphosphate; Threose nucleic acid (TNA); Xeno-nucleic acids (XNA).

Publication types

Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

DNA / chemistry*
DNA-Directed DNA Polymerase / metabolism*
Fluorescent Dyes / chemistry*
Nucleic Acids / chemistry*
Tetroses / chemical synthesis*
Tetroses / chemistry

Substances

Fluorescent Dyes
Nucleic Acids
Tetroses
DNA
DNA-Directed DNA Polymerase