Optimizing Solid-Phase Protein Synthesis: A Systematic Study of Solid Supports and Mild Cleavage Techniques

Solid-phase chemical ligation (SPCL) is a powerful method for simplifying protein synthesis, offering streamlined workflows, improved efficiency, and higher yields. However, the broader application of SPCL, particularly for synthesizing larger proteins, is hindered by the complexity of orthogonal protection strategies, suboptimal solid supports, and limited availability of compatible cleavable linkers. In this study, we systematically evaluated solid supports and cleavable linkers to optimize SPCL. By combining the low loading-controlled pore glass (CPG-2000) support with a nickel-cleavable SNAC-tag linker, we developed an optimized system that enabled the successful assembly of five peptide fragments through SPCL. his approach facilitated the efficient synthesis of a 130-amino-acid de novo designed protein with a yield of approximately 25%.