Total Chemical Synthesis And Modification Of Proteins

Proteins with non-natural amino acid residues (such as post-translationally modified proteins and proteins functionalized with synthetic probes) are key molecular tools in chemical biology. It is important to develop new chemical ligation strategies for chemically synthesize larger and more complex proteins with higher efficiency.The total chemical synthesis of protein enables the researcher to obtain, at-will, any desired modification in the covalent structure of a protein molecule. And more importantly, total chemical synthetic of protein enables the labeling of a protein with the atom-by-atom precision.The breakthrough for the total chemical synthesis of protein came in1994by Kent with the introduction of native chemical ligation (NCL). The novel concept using unprotected C-terminal thioester and N-terminal Cys peptide segments gives a ligation product by a native amide. It goes under mild aqueous conditions without complications from unprotected side functional groups. The cysteine-based ligation is the most effective stragteyg for chemical synthetic protein.Native chemical ligation requires a Cys at the ligation site. It is a severe limitation of the native chemical ligation approach, because Cys is the least common amino acid found in proteins (1.4%).Chemistries have been developed in an attempt to extend native chemical ligation of unprotected peptides to non-Cys sites. Extension of NCL beyond cysteine have been accomplished by post-ligation desulfurization of the thiolated amino acid residue, affording ligation at Ala, Lys, Phe, Val, Leu, Pro, and Glu.However, there are obstacles for the total chemical synthesis of protein by NCL. The peptide thioesters can be synthesized by Boc-based solid-phase synthesis. However, it’s hard to synthesize glycosylated or phosphorylated peptide thioesters. One of the main obstacles of native chemical ligation is the restricted access to peptide thioesters by Fmoc-based solid-phase synthesis protocols. What’s more, are there new modifications of carboxy C-terminal peptides to substitute thioesters for native chemical ligation? More important, what would be the ideal strategy to the synthesis of more large and complex proteins with post-translational modifications or artificial functionality?To solve the problems, we developed an operationally simple method for the synthesis of peptide thioesters using standard Fmoc solid-phase peptide synthesis procedures. The method relies on the use of a pre-made enamide-containing amino acid which, in the final TFA cleavage step, renders the desired thioester functionality through an irreversible intramolecular N-to-S acyl transfer. Besides, we identified a new one peptide O-ester that can used for peptide ligation via in situ O-to-S acyl shift. These peptide O-esters can be readily prepared through mild Fmoc solid-phase synthesis and thus, extending the practical utility of native chemical ligation. More importantly, a large reactivity difference was observed between the two peptide O-esters. This allows for the design of "one-pot" N-to-C sequential condensation of peptide segments through kinetically controlled ligation.