Reversible control of tetrazine bioorthogonal reactivity by naphthotube-mediated host-guest recognition

Abstract

•Naphthotubes can recognize phenyltetrazine-modified biomolecules with high affinity •Reversible control of phenyltetrazine reactivity through molecular recognition •Selective tetrazine residue caging enables sequential IEDDA reactions on proteins •Reversible caging of phenyltetrazine activity in living cells and animals Tetrazine-mediated inverse electron demand Diels-Alder (IEDDA) reactions are widely used ultrafast bioorthogonal reactions, and methods to control such reactions would be extremely useful. Here, we identified a high-affinity host-guest pair between synthetic macrocyclic naphthotubes and phenyltetrazine and developed a molecular-recognition strategy for controlling tetrazine reactions by host caging. Naphthotubes can recognize phenyltetrazine on various biomolecules with low-micromolar and sub-micromolar binding affinities and thus cage their IEDDA reactivity efficiently in a reversible manner. For tetrazine residues on proteins encoded by non-canonical amino acid mutagenesis, their positions could be computationally designed such that exposed residues could be reversibly caged, whereas semiburied residues that were resistant to caging retained their reactivity, thus allowing preparation of site-specific protein dual conjugates and dual protein labeling on living cells with IEDDA reactions. The reactivity of tetrazine can be further regulated in living animals. Our strategy is thus expected to expand the applications of tetrazine chemistry. Tetrazine-mediated inverse electron demand Diels-Alder (IEDDA) reactions are widely used ultrafast bioorthogonal reactions, and methods to control such reactions would be extremely useful. Here, we identified a high-affinity host-guest pair between synthetic macrocyclic naphthotubes and phenyltetrazine and developed a molecular-recognition strategy for controlling tetrazine reactions by host caging. Naphthotubes can recognize phenyltetrazine on various biomolecules with low-micromolar and sub-micromolar binding affinities and thus cage their IEDDA reactivity efficiently in a reversible manner. For tetrazine residues on proteins encoded by non-canonical amino acid mutagenesis, their positions could be computationally designed such that exposed residues could be reversibly caged, whereas semiburied residues that were resistant to caging retained their reactivity, thus allowing preparation of site-specific protein dual conjugates and dual protein labeling on living cells with IEDDA reactions. The reactivity of tetrazine can be further regulated in living animals. Our strategy is thus expected to expand the applications of tetrazine chemistry. Bioorthogonal chemistry: Bridging chemistry, biology, and medicineHartung et al.ChemJune 5, 2023In BriefBioorthogonal chemistry has become a flourishing toolbox of approaches for performing selective reactions in complex biological systems without perturbing natural processes. Through the implementation of this chemistry, chemical biologists have answered previously unanswerable questions, made new and exciting discoveries, and even begun to have an impact on important therapies, including implementing a bioorthogonal reaction at the center of a new cancer-treatment clinical trial. We highlight some key advances and anticipate next steps within this latest Nobel Prize-winning field. Full-Text PDF