Attachment of 2-Pyridyltetrazine Affinity Bioorthogonal Chemistry (ABC) Tags to Proteins for Site-Selective...

Site-selective labeling of proteins with small molecules can be performed by using different chemical and enzymatic methods. However, many of these methods lead to incomplete conversion or side products resulting in a mixture where the desired product may be inseparable. Herein, we present a protocol for the site selective conjugation of 3-methyl-6-(2-pyridyl)tetrazine to proteins. 3-methyl-6-(2-pyridyl)tetrazine, in addition to serving a bioorthogonal reagent capable of reacting to strained alkenes such as trans-cyclooctene, also functions as an affinity tag due to its ability to bind to nickel immobilized on resin. This Affinity Bioorthogonal Chemistry (ABC) tag enables the separation of the conjugated product from the unreacted starting material and any side products that may form during the conjugation reaction. This protocol describes the synthesis of the required 3-methyl-6-(2-pyridyl)tetrazines for conjugation to proteins, and the subsequent conjugation and purification steps required for obtaining ABC-tag functionalized protein.