Protein conjugation has been widely used to generate antibody-drug conjugates, nanomedicines, bioimaging materials, etc. Since proteins are vulnerable to extreme reaction conditions, bioorthogonal chemistries allowing protein conjugation under mild conditions are one of critical components for successful protein conjugation. Recently, inverse electron demand Diels-Alder reaction (IEDDA), a bioorthogonal chemistry, attracts much attention for their fast reaction kinetics between tetrazine containing molecules and transcyclooctene (TCO). A phenylalanine analog containing a methyl tetrazine (Met-Tet) has been site-specifically incorporated to a protein for various protein conjugation in vitro and inside cells. However, for certain protein conjugation applications, a faster reaction kinetics is required due to instability of protein or protein conjugation in vivo. Compared with methyl-tetrazine, hydrogen-tetrazine was known to exhibit a faster reaction kinetics. In order to site-specifically incorporate a phenylalanine analog containing a hydrogen-tetrazine (H-Tet) into a protein, we developed E. coli expression hosts co-expressing engineered pair of tRNA and synthetase recognizing H-Tet. A protein containing H-Tet exhibited a faster reaction rate with a TCO containing molecule than a protein containing Met-Tet. Proteins containing H-Tet would be widely used for protein conjugation, in particular, one requiring very fast conjugation.

Expansion of genetically encoded amino acids containing a tetrazine functional group for fast protein conjugation

2019 Spring National Meeting

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