Si-Click – (Copper-free) Click Chemistry
Mounting labels on proteins in intact cells allows observing and tracking them in their natural environment and represents a powerful tool for studying cell biology and developing drugs. Nowadays, a large variety of approaches exists for protein labeling – differing in size of modification, general scope, as well as the ease of usability and exchangeability.
A major drawback of many genetically encoded tags represents their large size which can be overcome by making use of the fascinating potential of specifically labeling single amino acid residues with small molecule dyes. Genetically encodable unnatural amino acids (UAAs) allowing for biocompatible and even bioorthogonal site-specific labeling of proteins with (fluorogenic) dyes are under deveolopment.
The new lysine-based compounds have (bi)cyclooctyne, trans-cyclooctene or propargyl units that can be grafted into proteins – amongst other of E. coli and mammalian cells. Copper(I)-catalyzed (CuAAC) click chemistry between azides and linear alkynes as well as the two most potent representatives of in vivo bioorthogonal chemical reactions, strain-promoted alkyne-azide cycloaddition (SPAAC) and strain-promoted inverse electron-demand Diels-Alder cycloaddition (SPIEDAC), can be used to efficiently and rapidly attach fluorophores conjugated to azide and tetrazine moieties, respectively. Due to the modularity of the technique, any imaginable probe fused to azide or tetrazine can be attached to any protein modified with a corresponding unnatural side chain with the maximum freedom of placement within the protein under investigation.