Ligation native chemical

Amino acid derivatives for peptide ligation site HS. HS. / 

SCHEME 11.1 Native chemical ligation and a concept using amino acid having thiol at the (i position. Acm, acetamidomethyl PG, protecting group VA-044,1,2 - azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride. 

Peptides typically are prepared for this ligation process using a-alkyl thioesters, because they are simple to make at the time of peptide synthesis. However, due to the relatively slow reaction kinetics of alkyl thioesters, most native chemical ligation processes have been catalyzed through the use of thiol compound additives, such as benzyl mercaptan or thiophenol (Dawson et al., 1997). These compounds react with the initial a-alkyl thioester to form another intermediate, an aryl thioester, which is more reactive toward the N-terminal cysteine on the other peptide to be coupled. A study 

Peptide labeled at C-terminal Peptide labeled at N-terminal 

Another advantage to the use of a thiol additive is that the abundance of free thiol groups in the reaction environment will prevent the oxidation of the cysteine thiol at the N-terminal of the other peptide. Without added thiol transesterification catalysts, disulfide formation resulting in dimerization of the Cys-peptide would be a dominant side reaction in aqueous, oxygenated buffer conditions. 

Native chemical ligation has been used successfully to couple two unprotected peptides together during solid phase synthesis, wherein one of the peptides is attached to the resin using a thioester linkage and the other peptide is introduced containing a cysteine at its N-terminal 

So efficient is this method to link peptide sequences together in a native amide bonded state that complete proteins of various sizes have been synthetically made (Hackeng et al., 1999), 

---

Jung JP, Jones JL, Cronier SA et al (2008) Modulating the mechanical properties of self-assembled peptide hydrogels via native chemical ligation. Biomaterials 29 2143-2151... 

Dose C, Seitz O (2008) Single nucleotide specific detection of DNA by native chemical ligation of fluorescence labeled PNA-probes. Bioorg Med Chem 16 65-77... 

Figure 17.14 An expressed protein containing a thioester intein tag that was subsequently modified by native chemical ligation to contain an alkyne group then can be labeled using an azido-fluorescein probe by the click chemistry reaction in the presence of Cu1+.

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.

Native chemical ligation also can be extended to the conjugation of peptides or proteins to other molecules or surfaces. For instance, Reulen et al. (2007) prepared liposomes that contained cysteine-PEG-phospholipid derivatives and then coupled thioester-modified peptides or proteins to form a protein-liposome conjugate. Using this procedure, approximately 100 molecules of a collagen binding protein could be coupled to the cysteine-containing liposomes. 

In addition, Dose and Seitz (2005) employed native chemical ligation to synthesize peptide nucleic acids (PNAs) by linking shorter segments of PNAs to make long contiguous strands, which could not be made through typical oligo synthesis procedures. 

Muir et al. (1998) realized that the intein reaction could be used to facilitate a native chemical ligation with a synthetic N-terminal cysteine-containing peptide or cysteine-containing molecule. With the discovery of a mutant intein that could form an intermediate thioester but not go on to complete the splice and ligation reaction (Xu and Perler, 1996 Chong et al.,... 

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). 

EPL extends the applicability of native chemical ligation to recombinantly produced proteins using the mutant mini intein vector system. Proteins being expressed using this method will... 

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.

The following protocol for EPL, including purification using a CBD fusion tag followed by native chemical ligation, is based on the methods of Muir et al. (1998), Chong et al. (1997, 1998), Evans et al. (1998), Severinov and Muir (1998), and the NEB instruction manual for the IMPACT-TWIN system. The recombinant protein is recovered from the affinity column as the thioester derivative ready for reaction with a N-terminal Cys peptide or another tag containing a Cys residue. 

Beligere, G.S., and Dawson, P.E. (1999) Synthesis of a three zinc finger protein, Zif268, by native chemical ligation. Biopolymers 51(5), 363-369. 

Dawson, P.E., Muir, T.W., Clark-Lewis, I., and Kent, S.B.H. (1994) Synthesis of proteins by native chemical ligation. Science 266, 776-779. 

Dose, C., and Seitz, O. (2005) Convergent synthesis of peptide nucleic acids by native chemical ligation. Org. Lett. 7(20), 4365-4368. 

Low, D.W., Hill, M.G., Carrasco, M.R., Kent, S.B.H., and Botti, P. (2001) Total synthesis of cytochrome b562 by native chemical ligation using a removable auxiliary. Proc. Natl. Acad. Sci. USA 98, 6554-6559. 

Lu, W., Starovasnik, M.A., and Kent, S.B. (1998) Total chemical synthesis of bovine pancreatic trypsin inhibitor by native chemical ligation. FEBS Lett. 429(1), 31-35. 

Pal, G., Santamaria, F., Kossiakoff, A.A., and Lu, W. (2003) The first semi-synthetic serine protease made by native chemical ligation. Protein Expr. Purif. 29(2), 185-192. 

Reulen, S.W.A., Brusselaars, W.W.T., Langereis, S., Mulder, W.J.M., Breurken, M., and Merkx, M. (2007) Protein-liposome conjugates using cysteine-lipids and native chemical ligation. Bioconjugate Chem. 18(2), 590-596. 

---