Development of convergent hybrid phase ligation for efficient and convenient total synthesis of proteins

Simple and efficient total synthesis of homogeneous and chemically modified protein samples remains a significant challenge. Here, we report development of a convergent hybrid phase native chemical ligation (CHP‐NCL) strategy for facile preparation of proteins. In this strategy, proteins are split into ~100‐residue blocks, and each block is assembled on solid support from synthetically accessible peptide fragments before ligated together into full‐length protein in solution. With the new method, we increase the yield of CENP‐A synthesis by 2.5‐fold compared to the previous hybrid phase ligation approach. We further extend the new strategy to the total chemical synthesis of 212‐residue linker histone H1.2 in unmodified, phosphorylated, and citrullinated forms, each from eight peptide segments with only one single purification. We demonstrate that fully synthetic H1.2 replicates the binding interactions of linker histones to intact mononucleosomes, as a proxy for the essential function of linker histones in the formation and regulation of higher order chromatin structure.