Ligation thioester

A superspiral consisting of two spirals (coiled coil), known as the leucine zip, is formed in this sequence via dimerisation. The condensation reaction, carried out in the aqueous phase, involves two peptide fragments which contain 15 and 17 amino acid residues respectively. Activation takes place via thioester formation (see Sect. 5.3.1). The ligation to a complete GCN4 matrix gives a new 32 amino acid peptide, which can itself serve as a matrix. The autocatalytic reaction exhibits a parabolic increase in the peptide concentration (caused by product inhibition see Section 6.4). 

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+.

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... 

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... 

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. 

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). 

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. 

Scheme 34 Overview of native chemical ligation (NCL). Two unprotected segments react in a reversible thiol/thioester reaction only the thioester product between the C-terminal thioester and the N-terminal cysteine can react further to form the desired amide bond via nucleophilic attack of the cysteine amine group.

Chemoselective ligation of peptides using the free amino terminal as nucleophile and a thiobenzyl thioester C-terminal amino acid as electrophile provides an efficient approach to the synthesis of proteins with several hundred residues [30]. From this perspective the introduction of non-natural amino acids into proteins becomes a possibility rather than a problem and chemoselective ligation is thus a prospect for the future for the incorporation of new functionality. 

Chemical ligation methods for peptide synthesis using thioester chemistry in solution have been previously documented (see Vol. E 22a, Section 4.1.5). Generalized procedures for solid-phase ligation have been developed that simplify the overall procedure. One method uses a safety-catch acid labile linker at the C-terminus and was used for the synthesis of a 71-amino acid chemokine, vMIP I (Section 5.3.2.1). Another procedure uses a selectively cleavable glycolate ester linkage (Section 5.3.2.2). 

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