Native peptide bonds

Fig. 4. Scheme of NCL. The mechanism allows the straightforward preparation of small proteins with native backbone structures from fully unprotected synthetic peptide building blocks. The initial tran -thioesterification step includes the chemo-selective reaction between one peptide with a C-terminal a-thioester group (peptide 1) and second peptide with an N-terminal cysteine residue (peptide 2). Generated thio-ester-linked intermediate spontaneously rearranges to form a native peptide bond at the site of ligation. 

The development of chemoselective reactions to give a native peptide bond at the site of hgation allows the synthesis of proteins with little or no modification to the covalent structure. A native structure at the ligation site is often desirable in the middle of protein structural domains (amino acid 60-120). The challenge of this approach is to form an amide bond chemoselectively in the presence of free amine side chains (Lys) and carboxylate side chains (Glu/Asp). Ideally, no protecting groups should be used for any of the amino acid side chains as they limit peptide solubility and require additional deprotection steps that can severely reduce the yield and convenience of the synthesis. 

Fig. 13 a The principle of native chemical ligation. Peptide 1, containing a C-terminal thioester, undergoes a nucleophilic attack by the cysteine residue at the N-terminus of peptide 2. The intermediate rearranges spontaneously to form the native peptide bond, b Native chemical ligation without the requirement of a cysteine residue using the l-phenyl-2-sulfanylethyl auxiliary group... 

Chemoselective Ligation to Form Native Peptide Bonds... 

Expressed protein ligation C-terminal thioester 0 HS. LJ 1 peplkte I 0 0 Native peptide bond Rab7, N-Ras, Rheb, K-Ras4B, D-Ral, YPTl, LC3-PE, lipidated GFP... 

Sortase- mediated protein ligation Protein with a C-terminal LPXTG motif Native peptide bond with additional amino acid sequence K-Ras4B, lipidated GFP, GPI modified GFP... 

As a second approach, Tam et al. demonstrated a selective histidine ligation, in which the C-terminal thiolcarboxyl was activated by Ellman s reagent in situ, captured by the imidazole ring of the N-terminal histidine and subsequently transferred to its a-amine to furnish a native peptide bond (Scheme 9) [145]. The high... 

Li et al. reported a chemoselective ligation between a C-terminal ester of 0-salicylaldehyde and an N-terminal Ser or Thr which after forming an A,0-benzylidene acetal intermediate, undergoes an intra-molecular six-membered transition state-mediated O-to-N acyl shift to generate an amide bond (Scheme 13) [65, 66]. The subsequent post-ligation TFA treatment removes salicylaldehyde and furnishes a native peptide bond. 

Sortases are transpeptidases, which use the linear epitope LPXTjG as a recognition site within a target peptide or protein. The cleavage of the amide bond between the threonine and glycine residue (J.) results in an acyl-enzyme intermediate. Subsequently, the thioester intermediate is attacked by a nucleophilic co-substrate comprised of an N-terminal glycine residue in order to form a native peptide bond. Sortase A (SrtA) derived from Staphylococcus aureus is commonly used for site-specific N- and C-terminal... 

FIGURE 133.4 Incorporation of photonic probes into proteins via expressed protein ligation (F = fluorescent probe). This approach involves a very specific chemical reaction between thioester on one polypeptide fragment and the N-terminal cysteine moiety on the other. Initial intermediate cysteine thioester is converted into native peptide bond by intramolecular reaction. The original free SH group of the cysteine is also regenerated. 

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