To thoroughly understand the biological processes in organisms, scientists developed different strategies to track the fast dynamics of proteins in？vivo.Although the green fluorescent (GFP) and its related variants tagging strategy had been widely used to monitor protein expression and localization in living cells, their large size can cause significant perturbations to a protein’s structure.

Antibody conjugates can also be generated and applied for a wide range of bimolecular imaging. However, the large size and physical properties of these reagents hinders their access to antigens within cells. To resolve this problem, it’s crucial to develop small-molecule mediated orthogonal probes with high efficiency as well as good specificity.

In recent years, an alternative strategy of bioorthogonal chemical reporter mediated protein labeling has emerged. This method combines the simplicity of genetically encoded tags with the specificity of antibody labeling and the versatility of small-molecule probes. This approach allows the introduction of bioorthogonal chemical reporters into proteins through genetic, metabolic, or a growing repertoire of bioorthogonal reactions. While powerful, the bioorthogonal chemical reporter strategy has been limited to only a handful of reporter groups such as ketones, terminal alkynes, organic azides as well as lager reactive bioothogonal groups. Identifying new chemical reporters remains an important, yet challenging goal.

Professor WANG Jiangyunand his colleagues at the Institute of Biophysics, Chinese Academy of Sciences have been making effort to identify genetically encode substrates that are suitable for photoclick chemistry. They envisioned that non-natural amino acids with strained alkenes attached may show higher rate of cycloaddition without undergoing the Michael addition side reactions with biological nucleophiles typically associated with electron-deficient alkenes. They genetically encoded cyclopropene because of its small size and inherently high ring strain. They synthesized a stable cyclopropene amino acid Nε-(1-methylcycloprop- 2-enecarboxamido) lysine (CpK,1). By evolving pyrrolysyl/tRNA synthetase from Methanosarcina barkeri, they incorporated CpK site-specifically into proteins in response to a TAG amber and characterized its reactivity in the photoinduced cycloaddition reaction with two tetrazoles, its site-specific incorporation into proteins both inE. coli and in mammalian cells, and its use in directing bioorthogonal labeling of proteins both in vitro and in vivo.

Compared to other genetically encoded, bioorthogonal labeling reactions reported recently, the main advantage of the cyclopropene-directed photoclick chemistry lies in its potential for spatiotemporally controlled protein labeling in mammalian cells, which requires the development of highly reactive laser-activatable tetrazole reagents using either a single-photon (for example 405 nm) or two-photon laser source. Because of its small size, a cyclopropene moiety can also be readily incorporated into small molecule substrates and inhibitors for the study of proteomes and lipids. The combination of CpK and other chemical reporters may be widely used for biomolecular tracking analysis.