title: 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 (Ac(4)ManNAz) 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.
authors.affiliation: Department of Chemistry, University of California, Berkeley, CA 94720-1460, USA.
authors.forename: Pamela V
authors.initials: PV
authors.lastname: Chang
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Prescher
authors.forename: Ellen M
authors.initials: EM
authors.lastname: Sletten
authors.forename: Jeremy M
authors.initials: JM
authors.lastname: Baskin
authors.forename: Isaac A
authors.initials: IA
authors.lastname: Miller
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Agard
authors.forename: Anderson
authors.initials: A
authors.lastname: Lo
authors.forename: Carolyn R
authors.initials: CR
authors.lastname: Bertozzi
mesh_terms: D000818:Animals; D001386:Azides / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003300:Copper / Q000737:chemistry / Q000378:metabolism; D003500:Cyclization; D034242:Cyclooctanes / Q000737:chemistry / Q000378:metabolism; D006001:Glycoconjugates / Q000737:chemistry / Q000378:metabolism*; D006595:Hexosamines / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D019169:Jurkat Cells; D051379:Mice; D008954:Models, Biological; D015335:Molecular Probes / Q000737:chemistry; D011485:Protein Binding; D012709:Serum Albumin / Q000378:metabolism; D013154:Spleen / Q000166:cytology / Q000378:metabolism
chemical_list: D001386:Azides; D034242:Cyclooctanes; D006001:Glycoconjugates; D006595:Hexosamines; D007202:Indicators and Reagents; D015335:Molecular Probes; C405953:N-azidoacetylmannosamine; D012709:Serum Albumin; D003300:Copper