Copper-free click chemistry in living animals

Abstract

Chemical reactions that enable selective biomolecule labeling in living organisms offer a means to probe biological processes in vivo. Very few reactions possess the requisite bioorthogonality, and, among these, only the Staudinger ligation between azides and triarylphosphines has been employed for direct covalent modification of biomolecules with probes in the mouse, an important model organism for studies of human disease. Here we explore an alternative bioorthogonal reaction, the 1,3-dipolar cycloaddition of azides and cyclooctynes, also known as “Cu-free click chemistry,” for labeling biomolecules in live mice. Mice were administered peracetylated N-azidoacetylmannosamine (Ac4ManNAz) to metabolically label cell-surface sialic acids with azides. After subsequent injection with cyclooctyne reagents, glycoconjugate labeling was observed on isolated splenocytes and in a variety of tissues including the intestines, heart, and liver, with no apparent toxicity. The cyclooctynes tested displayed various labeling efficiencies that likely reflect the combined influence of intrinsic reactivity and bioavailability. These studies establish Cu-free click chemistry as a bioorthogonal reaction that can be executed in the physiologically relevant context of a mouse.

Selective chemical protein modification

“…The site-specific incorporation of cyclooctynes 97 and biscyclononynes 98 into proteins by amberstop codon suppression has also recently been reported. However, limitations remain, such as occasional difficulty in the synthesis and handling of strained and unstable compounds, while crucially a degree of incompatibility towards cysteine has been reported 99,100 . In addition, these reactions remain relatively slow (Fig.…”

Genetisch kodierte kupferfreie Klick‐Chemie

“…[8] Seitdem wurden diese spannungsgetriebenen AzidAlkin-Cycloadditionen (SPAACs) zunehmend zur Markierung von beispielsweise Kohlenhydraten, [9] Nukleotiden [10] und Lipiden [11] verwendet.…”

The Protein Lipidation and Its Analysis

“…Nevertheless, a copper-free version of the CuAAC has been developed by Bertozzi´s group. This approach which involves the use of the strained, and therefore more reactive, cyclooctyne may overcome this limitation [112]. Regarding the use of bioorthogonal probes, the Huisgen reaction presents some advantages since it permits the use of both azido and alkyne tagged analogs while the Staudinger ligation requires the use of azido-tagged analogs and phosphine derivatives.…”