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 Backbone‐Installed Split Intein‐Assisted Ligation (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–CfaN and L–CfaC) 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.

An Enzyme‐Cleavable Solubilizing‐Tag Facilitates the Chemical Synthesis of Mirror‐Image Proteins

“…We envision that this method will be applied to the synthesis of more D‐protein targets to facilitate the development of D‐peptide ligands through mirror‐image display techniques. It is expected that the L–Lys linker/Lys‐C protease system would be expanded to temporarily immobilize a peptide for solid‐supported chemical D‐protein ligation, [27] install reactive groups for template‐mediated ligation of D‐peptides, [6d] introduce glycans for D‐protein folding, [28] or incorporate other groups for D‐proteins. Our study also suggests that the use of natural L‐type enzymes in the chemical synthesis and engineering of D‐proteins is promising and deserves more exploration.…”

Chemical Synthesis of Human Proteoforms and Application in Biomedicine

“…To overcome the challenge of ligating large protein segments at low reaction concentrations, we and others have developed templated ligation strategies that bring the reacting partners into physical proximity to increase the effective molarity of the reactants, thereby enabling ligation to occur even at micro- to nanomolar concentrations. Both noncovalent interactions (e.g., peptide nucleic acid interactions, , DNA pairing interactions, electrostatic interactions, and streptavidin-desthiobiotin binding) and covalent templates (e.g., click chemistry and split-intein splicing chemistry) have been examined. Nonetheless, to synthesize human proteoforms exceeding 50 kDa in size, a more practical approach is to achieve the individual synthesis and folding of the protein domains of the target protein first, followed by transpeptidative domain–domain ligation under nondenaturing conditions (Figure a).…”

Three Rounds of Stability-Guided Optimization and Systematical Evaluation of Oncolytic Peptide LTX-315

“…Then, the canonical N- or/and C- terminal modifications, as well as the inverse-peptide strategy were conducted on LTX-315. Recently, D-type peptides have been considered as promising anticancer agents due to their striking hydrolysis-resistant properties and low immunogenicity, compared with their enantiomeric counterparts. − Thus, we also performed the first D-type amino acid substitution for LTX-315 and derived peptides. Especially, the in vivo and in vitro anticancer tests were conducted to evaluate the anticancer activity of synthetic peptides.…”