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

Mirror‐image proteins (D‐proteins) are useful in biomedical research for purposes such as mirror‐image screening for D‐peptide drug discovery, but the chemical synthesis of many D‐proteins is often low yielding due to the poor solubility or aggregation of their constituent peptide segments. Here, we report a Lys‐C protease‐cleavable solubilizing tag and its use to synthesize difficult‐to‐obtain D‐proteins. Our tag is easily installed onto multiple amino acids such as DLys, DSer, DThr, and/or the N‐terminal amino acid of hydrophobic D‐peptides, is impervious to various reaction conditions, such as peptide synthesis, ligation, desulfurization, and transition metal‐mediated deprotection, and yet can be completely removed by Lys‐C protease under denaturing conditions to give the desired D‐protein. The efficacy and practicality of the new method were exemplified in the synthesis of two challenging D‐proteins: D‐enantiomers of programmed cell death protein 1 IgV domain and SARS‐CoV‐2 envelope protein, in high yield. This work demonstrates that the enzymatic cleavage of solubilizing tags under denaturing conditions is feasible, thus paving the way for the production of more D‐proteins. An enzyme‐cleavable solubilizing tag was described for the chemical synthesis of mirror‐image proteins. Solubilizing tags were easily installed on the side chain groups of DLys/DSer/DThr and the N‐terminal α‐amino group of D‐peptides via an L‐Lys linker. Solubilizing tags were impervious to various commonly used reagents in D‐protein synthesis. After protein assembly, multiple solubilizing tags can be removed under denaturing conditions.