Abstract
Copper-free click chemistry between cyclooctynes and azide is a mild, fast and selective technology for conjugation of oligonucleotides. However, technology for site-specific introduction of the requisite probes by automated protocols is scarce, while the reported cyclooctynes are large and hydrophobic. In this work, it is demonstrated that the introduction of bicyclo[6.1.0]nonyne (BCN) into synthetic oligonucleotides is feasible by standard solid-phase phosphoramidite chemistry. A range of phosphoramidite building blocks is presented for incoporation of BCN or azide, either on-support or in solution. The usefulness of the approach is demonstrated by the straightforward and high-yielding conjugation of the resulting oligonucleotides, including biotinylation, fluorescent labeling, dimerization and attachment to polymer.
Highlights
Synthetic DNA and RNA oligonucleotides (ONs) are key tools in a broad variety of diagnostic and therapeutic applications, including microarray technology [1], antisense and gene-silencing therapies [2], nanotechnology [3] and materials sciences [4,5]
We reasoned that bicyclo[6.1.0]nonyne (BCN) has particular potential for oligonucleotide conjugation, due to its higher reactivity in comparison to DIBO, its relatively low lipophilicity in comparison all dibenzofused cyclooctynes [36], and its plane-symmetry, which precludes the formation of regioisomers upon cycloaddition
We have successfully demonstrated the suitability of BCN and derivatives thereof for the preparation of oligonucleotide conjugates by (3+2) cycloaddition with azides
Summary
Synthetic DNA and RNA oligonucleotides (ONs) are key tools in a broad variety of diagnostic and therapeutic applications, including microarray technology [1], antisense and gene-silencing therapies [2], nanotechnology [3] and materials sciences [4,5] Such applications require the introduction of a suitable handle in an oligonucleotide to enable selective conjugation to a functionality of interest [6,7,8]. Alternative approaches for the preparation of azide-containing nucleotides—compromised by the incompatibility of azide with phosphoramidite chemistry—involve post-synthetic nucleophilic substitution [27,28,29] or selective diazotransfer reaction [30] or phosphonate-based coupling chemistry [31,32,33,34] a simple and general strategy for the on-support, automated synthesis of oligonucleotides with readily accessible building blocks, and suitable for introduction of any functional group (including cyclooctyne and azide), is still desirable. The ease of operation of copper-free click conjugation is demonstrated for a range of functional groups, by oligonucleotide dimerization, and by the preparation and characterization of an amphiphilic polythiophene-oligonucleotide hybrid polymer
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