Site-specific introduction of bioorthogonal handles into biomolecules provides powerful tools for studying and manipulating the structures and functions of proteins. Recent advances in bioorthogonal chemistry demonstrate that tetrazine-based bioorthogonal cycloaddition is a particularly useful methodology due to its high reactivity, biological selectivity, and turn-on property for fluorescence imaging. Despite its broad applications in protein labeling and imaging, utilization of tetrazine-based bioorthogonal cycloaddition has been limited to date by the requirement of a hydrophobic strained alkene reactive moiety. Circumventing this structural requirement, we report the site-specific incorporation of noncanonical amino acids (ncAAs) with a small isocyanide (or isonitrile) group into proteins in both bacterial and mammalian cells. We showed that under physiological conditions and in the absence of a catalyst these isocyanide-containing ncAAs could react selectively with tetrazine molecules via [4 + 1]-cycloaddition, thus providing a versatile bioorthogonal handle for site-specific protein labeling and protein decaging. Significantly, these bioorthogonal reactions between isocyanides and tetrazines also provide a unique mechanism for the activation of tetrazine-quenched fluorophores. The addition of these isocyanide-containing ncAAs to the list of 20 commonly used, naturally occurring amino acids expands our repertoire of reagents for bioorthogonal chemistry, therefore enabling new biological applications ranging from protein labeling and imaging studies to the chemical activation of proteins.
Conflict of interest statement
The authors declare no competing financial interest.
Advances in Tetrazine Bioorthogonal Chemistry Driven by the Synthesis of Novel Tetrazines and Dienophiles.Acc Chem Res. 2018 May 15;51(5):1249-1259. doi: 10.1021/acs.accounts.8b00062. Epub 2018 Apr 11. Acc Chem Res. 2018. PMID: 29638113 Free PMC article.
Site-Specific Bioorthogonal Labeling for Fluorescence Imaging of Intracellular Proteins in Living Cells.J Am Chem Soc. 2016 Nov 2;138(43):14423-14433. doi: 10.1021/jacs.6b08733. Epub 2016 Oct 21. J Am Chem Soc. 2016. PMID: 27768298 Free PMC article.
Site-Specific Protein Labeling with Tetrazine Amino Acids.Methods Mol Biol. 2018;1728:201-217. doi: 10.1007/978-1-4939-7574-7_13. Methods Mol Biol. 2018. PMID: 29405000
Photo-induced and Rapid Labeling of Tetrazine-Bearing Proteins via Cyclopropenone-Caged Bicyclononynes.Angew Chem Int Ed Engl. 2019 Oct 28;58(44):15876-15882. doi: 10.1002/anie.201908209. Epub 2019 Sep 24. Angew Chem Int Ed Engl. 2019. PMID: 31476269 Free PMC article. Review.
Non-canonical amino acid labeling in proteomics and biotechnology.J Biol Eng. 2019 May 22;13:43. doi: 10.1186/s13036-019-0166-3. eCollection 2019. J Biol Eng. 2019. PMID: 31139251 Free PMC article. Review.