A genetically encoded cyclobutene probe for labelling of live cells

Abstract

We have identified an aminoacyl-tRNA synthetase/tRNA pair for the efficient and site-specific incorporation of a cyclobutene-containing amino acid into proteins in response to amber nonsense codon. Fast and fluorescent labeling of purified proteins and intact proteins in live cells was demonstrated using the inverse electron demand Diels-Alder reaction with a tetrazine.

Addition of Isocyanide-Containing Amino Acids to the Genetic Code for Protein Labeling and Activation

“…[25][26][27][28] Despite the broad application of the IEDDA reactions, most dienophiles used in tetrazine-based cycloaddition reactions are strained hydrophobic alkenes or alkynes, such as TCO, cyclooctyne, norbornene, cyclobutene, cyclopropene, or spirohexene. 13,15,16,29,30 In this report, we incorporated noncanonical amino acids (ncAAs) containing small and hydrophilic isocyano (or isonitrile) groups into proteins in both bacterial and mammalian cells using genetic code expansion technology. Site-specific protein labeling was then achieved via a [4 + 1]-cycloaddition reaction with various types of functional tetrazine moieties.…”

“…The development of bioorthogonal reactions and strategies for introducing “handles” into polypeptides has transformed our ability to study and manipulate proteins. − There have been many improvements in the speed and selectivity of bioorthogonal reactions in the past several decades. − Recent advances in bioorthogonal chemistry have focused on tetrazine-based inverse electron demand Diels–Alder (IEDDA) cycloaddition reactions due to their rapid reaction rates, high selectivity, and product stability. ,− These reactions are particularly useful for precision protein labeling, decaging, and cellular protein imaging. ,,− For example, tetrazine-containing amino acids have been genetically incorporated into proteins, allowing for a rapid bioorthogonal conjugation with trans -cyclooctene (TCO)-labeled biomolecules. , Taking advantage of the high reaction rate and good biocompatibility of IEDDA reactions, Chen and others have redirected this reaction for site-specific protein decaging. − Proteins, including kinases and luciferase, have been caged by TCO-labeled lysine, followed by activation upon reaction with tetrazines. , Besides acting as an IEDDA reaction partner, tetrazine can absorb visible light at around 500–525 nm, which makes it an ideal quencher toward a series of fluorophores. Based on this dual functionality of tetrazine, IEDDA reaction was also employed to design a variety of fluorogenic fluorophores. − Despite the broad application of the IEDDA reactions, most dienophiles used in tetrazine-based cycloaddition reactions are strained hydrophobic alkenes or alkynes, such as TCO, cyclooctyne, norbornene, cyclobutene, cyclopropene, or spirohexene. ,,,, In this report, we incorporated noncanonical amino acids (ncAAs) containing small and hydrophilic isocyano (or isonitrile) groups into proteins in both bacterial and mammalian cells using genetic code expansion technology. Site-specific protein labeling was then achieved via a [4 + 1]-cycloaddition reaction with various types of functional tetrazine moieties.…”

Highly Strained Unsaturated Carbocycles

“…Cyclobutene has also been reported to be a versatile scaffold for use in bioorthogonal reactions due to its small size and excellent stability. Liu and co-workers [85] reported the synthesis and incorporation of cyclobutene acetic acid 72 into amino acids 73 and potentially proteins (Scheme 24).…”

Protein Labeling in Live Cells for Immunological Applications

“…In order to use tetrazine ligation to label proteins in living cells, the bioorthogonal alkene or alkyne must be introduced into the cells where it can react with the tetrazine-linked probe. This is achieved by incorporating unnatural amino acids containing the alkene or alkyne into the POI. − ,, An amber stop codon is introduced into the gene encoding for the POI, and cells producing the POI are supplied with the genes for an engineered tRNA and aminoacyl tRNA synthetase pair that recognizes and suppresses the amber stop codon by incorporating the unnatural amino acid. Unnatural amino acids containing a number of different moieties, including norbornene, cyclooctene, bicyclononyne, and cyclobutene, have been incorporated using this approach. − ,, Methods detailed in the literature cited above can be adapted for labeling of most proteins.…”