Site-Specific DNA Functionalization through the Tetrazene-Forming Reaction in Ionic Liquids

Abstract

Development of multiple chemical tools for deoxynucleic acid (DNA) labeling has facilitated wide use of their functionalized conjugates, but significant practical and methodological challenges remain to achievement of site-specific chemical modification of the biomacromolecule. As covalent labeling processes are more challenging in aqueous solution, use of nonaqueous, biomolecule-compatible solvents such as an ionic liquid consisting of a salt with organic molecule architecture, could be remarkably helpful in this connection. Herein, we demonstrate site-specific chemical modification of DNAs through a tetrazene-forming amine-azide coupling reaction using an ionic liquid. This ionic liquid-enhanced reaction process has good functional group tolerance and precise chemoselectivity, and enables incorporation into DNA of various useful functionalities such as biotin, cholesterol and fluorophores which could be incorporated into DNA through this method. A site-specifically labeled single stranded nucleotide, or aptamer interacting with a growth factor receptor (Her2) was successfully used in the fluorescence imaging of breast cancer cell lines. The non-traditional medium-promoted labeling strategy described here provides an alternative design paradigm for future development of chemical tools for applications involving DNA functionalization.

Translation of a Phosphine- and Azide-Based Reaction to Chemical Modification of Biomolecules in Ionic Liquid

“…34 A new set of 15 N NMR data with 15 N isotopes on all the four possible nitrogen atoms of amine and azide groups disclosed that one of the peaks at ca. 300 ppm previously assigned to the N=N group of tetrazene was an artifact, 25 26 and introduction of only two 15 N isotopes out of the four possible nitrogens was observed. The formation of the urea group was confirmed through NMR, mass spectrometry, and infrared spectroscopy experiments.…”

“…23 24 The initial set of our data including mass spectrometry and 1 H/ 15 N NMR studies suggested tetrazene as a potential reaction product (though one of the assigned peaks in 15 N NMR turned out to be an artifact in the later study). 25 26 Based on the set of data, we proposed a phosphazide-based coupling process without loss of N 2 gas as a potential reaction pathway (Scheme 2 C). 15 27 This initial hypothesis was supported by the report of similar electrophilic behavior of phosphazide intermediates for an intramolecular reaction between alkylated phosphazide and enolate species.…”

“…25 26 Based on the set of data, we proposed a phosphazide-based coupling process without loss of N 2 gas as a potential reaction pathway (Scheme 2 C). 15 27 This initial hypothesis was supported by the report of similar electrophilic behavior of phosphazide intermediates for an intramolecular reaction between alkylated phosphazide and enolate species. 28 Alkylazide compounds often behave as soft electrophiles to react with nucleophiles such as thiols and phosphines.…”

“…36 This attribute facilitated site-specific modification of external alkylamine groups of DNA without appreciable side reactivity on the arylamine groups of the nucleobase (Scheme 4 D). 27 Additionally, the incorporation of alkylazides with various functional groups into DNA demonstrated the wide substrate tolerance of the reaction. Modification of azide-tagged biomolecules such as azide-tagged hyaluronic acid proved successful as well.…”