Computation-Guided Discovery of Diazole Monosubstituted Tetrazines as Optimal Bioorthogonal Tools.

Monosubstituted tetrazines are important bioorthogonal reactive tools due to their rapid ligation with -cyclooctene. However, their application is limited by the reactivity-stability paradox in biological environments. In this study, we demonstrated that steric effects are crucial in resolving this paradox through theoretical methods and developed a simple synthetic route to validate our computational findings, leading to the discovery of 1,3-azole-4-yl and 1,2-azole-3-yl monosubstituted tetrazines as superior bioorthogonal tools.

Noncanonical amino acids as doubly bio-orthogonal handles for one-pot preparation of protein multiconjugates.

Genetic encoding of noncanonical amino acid (ncAA) for site-specific protein modification has been widely applied for many biological and therapeutic applications. To efficiently prepare homogeneous protein multiconjugates, we design two encodable noncanonical amino acids (ncAAs), 4-(6-(3-azidopropyl)-s-tetrazin-3-yl) phenylalanine (pTAF) and 3-(6-(3-azidopropyl)-s-tetrazin-3-yl) phenylalanine (mTAF), containing mutually orthogonal and bioorthogonal azide and tetrazine reaction handles. Recombinant proteins and antibody fragments containing the TAFs can easily be functionalized in one-pot reactions with combinations of commercially available fluorophores, radioisotopes, PEGs, and drugs in a plug-and-play manner to afford protein dual conjugates to assess combinations of tumor diagnosis, image-guided surgery, and targeted therapy in mouse models.

Expanding the Structural Diversity of Protein Building Blocks with Noncanonical Amino Acids Biosynthesized from Aromatic Thiols.

Incorporation of structurally novel noncanonical amino acids (ncAAs) into proteins is valuable for both scientific and biomedical applications. To expand the structural diversity of available ncAAs and to reduce the burden of chemically synthesizing them, we have developed a general and simple biosynthetic method for genetically encoding novel ncAAs into recombinant proteins by feeding cells with economical commercially available or synthetically accessible aromatic thiols. We demonstrate that nearly 50 ncAAs with a diverse array of structures can be biosynthesized from these simple small-molecule precursors by hijacking the cysteine biosynthetic enzymes, and the resulting ncAAs can subsequently be incorporated into proteins via an expanded genetic code.