Oleg Melnyk
Protein total synthesis

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Home > Ligation chemistry > Ligation chemistry. Main concepts

Formation of native peptide bonds

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Synthesis of peptides of moderate size (up to 50 amino acids) is feasible due to robust and efficient chemical methods for peptide bond formation using protected amino acids, the development of efficient solid supports which can be swelled in a variety of solvents and of the solid phase peptide synthesis (SPPS) method, and of course to the development of automated synthesizers. The SPPS of peptides involves the stepwise coupling of the amino acids from the C-terminus to the N-terminuson on a solid support. At the end of the peptide elongation, the peptide is cleaved from the solid support and purified.
It can be easily understood that stepwise methods generate at each chemical step some impurities so that the amount of impurities grows as the power of the number of steps. Consequently, the yield of the desired peptide drops with peptide length. If the mean coupling yield for amino acids is X, the final yield for a peptide composed of n amino acids is Xn. Even if X is as high as 0.98, the yield for a very long peptide is generally low. For a 100 amino acids peptide (0.98)100 equals to 13%! The final yield must take into account the difficulty to separate the target peptide from impurities. This can be a limiting step since long peptides prepared by stepwise synthesis are expected to co-elute with many side-products.

 

These considerations demonstrate that proteins cannot be usually synthesized using conventional stepwise solid phase methods and that there is a need for chemical methods allowing the convergent synthesis of large polypeptides by linking together short peptide fragments in a controlled manner. The great challenge is the necessity to form a native peptide bond using unprotected peptide fragments and in aqueous solution (for solubilising peptide fragments).

 

Our goal is to design and develop novel chemical method for chemoselective native peptide bond formation that can be complementary to existing methods.

 

The peptide chemist tool box contains already a variety of chemoselective ligation methods that allow the assembly of natural proteins or synthetic peptide scaffolds [1-3]. Native chemical ligation (NCL) [4, 5], Staudinger ligation [6] and the decarboxylative condensation of N-alkylhydroxylamines and alpha-ketoacids reported by Bode et al. [7] lead to the formation of a native peptide bond at the ligation site. In particular, NCL is based on the reaction of a peptide thioester group with a cysteinyl peptide. Transthioesterification is followed by an intramolecular S,N-acyl shift that results in the formation of a peptide bond at a X-Cys junction. Application of NCL to selenocysteine was also described [8, 9]. Methods based on the use of N-linked thiol-containing cleavable auxiliaries [10] were used to extend the principle of NCL to sites other than Cys residues. Ligation of peptide thioesters with N-terminal homocysteine [11] or homoselenocysteine [12] peptides followed by methylation permitted the formation of X-Met or X-selenoMet bonds.

1. Tam JP, Xu J, Eom KD. Methods and strategies of peptide ligation. Biopolymers 2001; 60: 194-205.

 

2. Lemieux GA, Bertozzi CR. Chemoselective ligation reactions with proteins, oligosaccharides and cells. Trends Biotechnol 1998; 16: 506-513.

 

3. Casi G, Hilvert D. Convergent protein synthesis. Curr Opin Struct Biol 2003; 13: 589-594.

 

4. Dawson PE, Muir TW, Clark-Lewis I, Kent SB. Synthesis of proteins by native chemical ligation. Science 1994; 266: 776-779.

 

5. Macmillan D. Evolving strategies for protein synthesis converge on native chemical ligation. Angew Chem Int Ed Engl 2006; 45: 7668-7672.

 

6. Saxon E, Armstrong JI, Bertozzi CR. A "traceless" Staudinger ligation for the chemoselective synthesis of amide bonds. Org Lett 2000; 2: 2141-2143.

 

7. Bode JW, Fox RM, Baucom KD. Chemoselective amide ligations by decarboxylative condensations of N-alkylhydroxylamines and alpha-ketoacids. Angew Chem Int Ed Engl 2006; 45: 1248-1252.

 

8. Gieselman MD, Xie L, van Der Donk WA. Synthesis of a selenocysteine-containing peptide by native chemical ligation. Org Lett 2001; 3: 1331-1334.

 

9. Quaderer R, Hilvert D. Selenocysteine-mediated backbone cyclization of unprotected peptides followed by alkylation, oxidative elimination or reduction of the selenol. Chem Commun (Camb) 2002: 2620-2621.

 

10. Botti P, Carrasco MR, Kent SBH. Native chemical ligation using removable N-alpha-(1-phenyl-2-mercaptoethyl) auxiliaries. Tetrahedron Lett. 2001; 42: 1831-1833.

 

11. Tam JP, Yu Q. Methionine ligation strategy in the biomimetic synthesis of parathyroid hormones. Biopolymers 1998; 46: 319-327.

 

12. Roelfes G, Hilvert D. Incorporation of selenomethionine into proteins through selenohomocysteine-mediated ligation. Angew Chem Int Ed Engl 2003; 42: 2275-2277.