The inverse electron demand Diels Alder reaction between tetrazines and strained alkenes is an exceptionally useful tool in functionalizing to biomolecules since it is orthogonal to the chemistry of most living systems and have exceptionally high rate constants. In particular reactions between strained trans-cyclooctenes (sTCO) and tetrazines can achieve second order rate constants of 106 M-1s-1 or greater. This bioorthogonal coupling reaction can be used to quantitatively modify proteins with fluorescent labels, inhibitors, or dimerization agents in live cells with short reaction time. In this work, we explore the use of genetic code expansion to incorporate tetrazine containing amino acids into proteins for the purpose of conjugating sTCO-containing labeling agents to proteins in live cells
In Escherichia coli, the tetrazine containing amino acid, 4-(6-methyl-s-tetrazin-3-yl)phenylalanine (Tet-v2.0) is incorporated into cells through genetic code expansion. An orthogonal amino acyl-tRNA synthetase is generated that can charge tRNACUA using Tet-v2.0 as a substrate allowing for the site-specific incorporation of Tet-v2.0 into proteins. Tet-v2.0 on proteins is shown to react with sTCO with a high rate of reaction of 87,000 M-1s-1 and minimal off-target reactions. To express tetrazine containing proteins in eukaryotic cells, a new set of amino acids based on 3-(6-methyl-s-tetrazin-3-yl)phenylalanine (Tet-v3.0), are developed that can be used as substrate by Methanosarcina. barkeri Pyrrolysine tRNA synthetases (PylRS). The orthogonal Tet-v3.0-amino acyl-tRNA synthetase/tRNA pair was used to incorporate Tet-v3.0 amino acids into proteins in HEK293T cells and shown to react with both fluorescent labels and dimerizing sTCO molecules.
We then explore methods to improve the efficiency of the reaction between tetrazines and sTCO, through minimizing side reactions. These side reactions include the reduction of tetrazines to dihydro-tetrazines and irreversible degradation of tetrazines to a yet to be characterized product. To eliminate off-target reactions the reduction of tetrazines is monitored in cellulo and proteins are oxidized in cellulo through photooxidation. This oxidation reaction has the potential to be an effective method of establishing higher labeling yields. Finally, trapping the tetrazine in the dihydro-state and selectively oxidizing and labeling the tetrazine may be an effective method of minimizing off-target tetrazine reactions in cells by protecting the oxidized amino acid.