Scalable Flow Synthesis of Genetically Encodable Tetrazine Amino Acids

The tetrazine–cyclooctene ligation is among the fastest bioorthogonal reactions known, enabling rapid and selective bioconjugation. Site-specific incorporation of tetrazine-bearing noncanonical amino acids (Tet-ncAAs) into proteins via genetic code expansion has expanded the toolkit for applications in chemical biology, therapeutic conjugation, and live-cell imaging. However, existing synthetic routes to Tet-ncAAs rely on small-scale batch reactions that rely on hazardous reagents and are poorly suited for scale-up. Here, we report both hybrid batch-flow and continuous-flow synthetic approaches for Tet-ncAAs that improve process safety, scalability, and reproducibility. The use of aqueous hydrazine in place of anhydrous hydrazine, 3-mercaptopropionic acid as an organic catalyst in place of metal salts, and a compatible solvent system enables the synthesis under safer, milder, and environmentally friendly conditions. Optimization of the temperature for the reaction revealed a two-step process involving rapid hydrazine addition, followed by cyclization at a lower temperature. This modular process supports multigram-scale synthesis of diverse Tet-ncAAs from commercial precursors and integrates a fully continuous oxidation protocol to access the final tetrazine products. The resulting platform offers a safer, scalable, and sustainable route to Tet-ncAAs that supports the growing demand in antibody–drug conjugates, radioimmunoconjugates, antibody-oligonucleotide, and protein–protein conjugates as well as protein labeling and targeted delivery applications.