Synthesis of a Stable Hydrophilic trans ‐Cyclooctene Amino Acid Unlocks Fast Bioorthogonal Click Chemistry for the Generation of Antibody‐Drug Conjugates by Genetic Code Expansion

The strain-promoted inverse electron demand Diels-Alder cycloaddition (SPIEDAC), usually employing trans-cyclooctenes (TCOs) and tetrazines, represents one of the fastest bioorthogonal click reactions currently available within the bioorthogonal "toolbox". TCOs can be conveniently introduced into proteins via genetic code expansion (GCE) technology, enabling a selectable site-specific incorporation of such noncanonical amino acids (ncAAs) at predefined positions. Their application under physiological conditions, however, is limited due their inherent instability toward alkene isomerization to the unreactive cis-isomer. This has limited the use of TCO-containing ncAAs for quantitative labeling applications, for example, of therapeutical bioconjugates. In this study, we report on the synthesis and application of a stable hydrophilic TCO-ncAA for efficient production of a highly efficacious antibody-drug conjugate (ADC) via GCE and subsequent SPIEDAC conjugation.