Chemical ligation reactions

Figure 17.25 The native chemical ligation reaction can be used to form larger peptides from smaller peptides, if one contains a cysteine residue at its N-terminal and the other one contains a thioester on its C-terminal. Reaction of the peptide derivatives gives a native peptide (amide) bond.

Figure 17.27 The EPL process involves a fusion protein containing an intein tag plus a CBD. The fusion protein is captured on an immobilized chitin resin and after removal of contaminating proteins, it is eluted using thiophenol, which cleaves at the thioester bond between the intein and the desired expressed protein. This releases a phenylth-ioester-activated protein that can be used in the native chemical ligation reaction with another peptide containing an N-terminal cysteine residue. Conjugation results in a native amide (peptide) bond formed between them.

However, if the expressed protein is treated on the affinity support using thiophenol, this also will release the protein and result in a phenylthioester at its C-terminal, which is the reactive intermediate imminendy suitable for native chemical ligation. Treatment of this activated thioester protein with a N-terminal cysteine peptide induces the native chemical ligation reaction and couples the peptide to the expressed protein through an amide bond (Severinov and Muir, 1998) (Figure 17.27). 

Figure 17.28 EPL reactions can be used to couple a fusion protein to a surface containing a thioester derivative. After cells are grown and the fusion protein expressed, a pH and temperature shift causes intein cleavage with release of the expressed protein with an N-terminal cysteine residue. Reaction with the thioester surface results in a native chemical ligation reaction that forms an amide bond linkage with the expressed protein.

Johnson ECB, Kent SBH (2006) Insights into the mechanism and catalysis of the native chemical ligation reaction. J Am Chem Soc 128 6640-6646... 

Wang C, Guo Q-X, Fu Y (2011) Theoretical analysis of the detailed mechanism of native chemical ligation reactions. Chem Asian J 6 1241-1251... 

---