A benzylic linker promotes methyltransferase catalyzed norbornene transfer for rapid bioorthogonal tetrazine ligation

Site-specific alkylation of complex biomolecules is critical for late-stage product diversification as well as post-synthetic labeling and manipulation of proteins and nucleic acids. Promiscuous methyltransferases in combination with analogs of S -adenosyl- l -methionine (AdoMet) can functionalize all major classes of biomolecules. We show that benzylic moieties are transferred by Ecm1 with higher catalytic efficiency than the natural AdoMet. A relative specificity of up to 80% is achieved when a norbornene moiety is placed in para -position, enabling for the first time enzymatic norbornene transfer to specific positions in DNA and RNA- even in cell lysate. Subsequent tetrazine ligation of the stable norbornene moiety is fast, efficient, biocompatible and - in combination with an appropriate tetrazine - fluorogenic. Benzylic AdoMet analogs enable highly efficient enzymatic transfer of norbornenes to nucleic acids and subsequent tetrazine ligation.