Backbone‐Installed Split Intein‐Assisted Ligation for the Chemical Synthesis of Mirror‐Image Proteins

Abstract Membrane‐associated D‐proteins are an important class of synthetic molecules needed for D‐peptide drug discovery, but their chemical synthesis using canonical ligation methods such as native chemical ligation is often hampered by the poor solubility of their constituent peptide segments. Here, we describe a B ackbone‐ I nstalled S plit I ntein‐ A ssisted L igation (BISIAL) method for the synthesis of these proteins, wherein the native L‐forms of the N‐ and C‐intein fragments of the unique consensus‐fast (Cfa) (i.e. L–Cfa N and L–Cfa C ) are separately installed onto the two D‐peptide segments to be ligated via a removable backbone modification. The ligation proceeds smoothly at micromolar (μM) concentrations under strongly chaotropic conditions (8.0 M urea), and the subsequent removal of the backbone modification groups affords the desired D‐proteins without leaving any “ligation scar” on the products. The effectiveness and practicality of the BISIAL method are exemplified by the synthesis of the D‐enantiomers of the extracellular domains of T cell immunoglobulin and ITIM domain (TIGIT) and tropomyosin receptor kinase C (TrkC). The BISIAL method further expands the chemical protein synthesis ligation toolkit and provides practical access to challenging D‐protein targets.