Protein synthesis with the ketoacid-hydroxylamine (KAHA) ligation
The ability to chemically synthesize biologically active proteins in a controlled fashion is one of the greatest achievements of synthetic chemistry in the last 20 years. As part of these efforts, our group reported the α-ketoacid–hydroxylamine (KAHA) ligation as a chemoselective coupling of large, unprotected peptide segments. The KAHA ligation employs C-terminal peptide α-ketoacids (KAs) and N-terminal peptide hydroxylamines (HAs), which react chemoselectively to form amides or esters. This reaction proceeds in aqueous media without reagents or catalysts.
By developing new building blocks and chemical reactions, we can now easily prepare large peptide segments (40+ residues) bearing the necessary reaction partners – C-terminal peptide α-ketoacids and N-terminal peptide hydroxylamines – by standard Fmoc-SPPS. Along with innovative, orthogonal protecting group strategies, we can easily iterate segment ligations for the synthesis of protein targets up to about 200 residues. Recent examples of proteins prepared with the KAHA ligation include Pup, CspA, UFM1, SUMO2, SUMO3, S100A4, Nitrophorin 4 and betatrophin(lipasin).
Our ability to prepare synthetic proteins with any kind of unnatural amino acids, post-translational modification, fluorescent tag, or synthetic handle forms the basis of our increasing efforts in chemical biology and the synthesis of synthetic proteins of therapeutic interest.
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