An Optimized Protocol for the Synthesis of Peptides Containing <i>trans</i> ‐Cyclooctene and Bicyclononyne Dienophiles as Useful Multifunctional Bioorthogonal Probes

Despite the great advances in solid-phase peptide synthesis (SPPS), incorporation of certain functional groups into sequences is restricted by compatibility building blocks with conditions used during SPPS. In particular, introduction highly reactive modern bioorthogonal reactions peptides remains elusive. Here, we present an optimized synthetic protocol enabling installation two strained dienophiles, trans-cyclooctene (TCO) and bicyclononyne (BCN), different sequences. The enable fast modular post-synthetic functionalization peptides, as demonstrate preparation peptide-peptide peptide-drug conjugates. Due to excellent biocompatibility, click-functionalization can be performed directly live cells. We further show that both clickable enables construction smart, multifunctional probes streamline complex chemical biology experiments such visualization pull-down metabolically labeled glycoconjugates. presented strategy will find utility for diverse applications, where high reactivity, efficiency biocompatibility modification step critical.