Proximity-Driven Protein Ligation Beyond the Concentration Limit

Classic chemical protein synthesis is constrained by slow kinetics and millimolar concentration requirements, limiting access to large or hydrophobic proteins because of solubility issues. Existing auxiliary strategies generally demand harsh installation/removal conditions and are incompatible with expressed protein ligation (EPL), limiting their utility for complex targets. To overcome this, a photocleavable picolyl (Pic) linker was developed that enables postsynthetic installation of bio-orthogonal inverse-electron-demand Diels-Alder (IEDDA) reagents onto synthetic peptides and expressed proteins under mild conditions. This strategy uses rapid IEDDA kinetics to enhance effective peptide concentration, enabling efficient ligation at low concentrations. The ligation of expressed G-CSF and SARS-CoV-2 RBD fragments at micromolar concentrations demonstrates the practicality of this method, whereas conventional methods fail to achieve efficient ligation. IEDDA ligation, EPL, desulfurization/Pic linker cleavage can be performed in one pot, overcoming low-concentration synthesis bottlenecks and serving as a powerful tool for accessing challenging hydrophobic or large proteins.