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The team is deeply involved in the design of novel tools for total chemical  synthesis of proteins (see our review in Chemical Society Reviews).

Protein chemical synthesis by native peptide ligation of unprotected peptide segments is an interesting complement and potential alternative to the use of living systems for producing proteins. The synthesis of proteins require efficient native peptide ligation methods, which enable the chemoselective formation of a native peptide bond in aqueous solution between unprotected peptide segments.
We have discovered such a method called SEA native peptide ligation. [article 2]

It involves the reaction of a peptide featuring a C-terminal bis(2-sulfanylethyl)amido group, called SEA, with a Cys peptide. This reaction proceeds perhaps through the formation of a transient thioester intermediate, obtained by intramolecular attack of one SEA thiol on the peptide C-terminal carbonyl group as shown in Scheme 1. SEA group belongs to the {N,S}-acyl shift systems, which have been pioneered by the lab. [article 3]

 SEA-ligation

Scheme 1. SEA ligation between a {bis}(2-sulfanylethyl)amido (SEA) peptide  and a cysteinyl or homocysteinyl peptide leads to the chemoselective and regioselective formation of a native peptide bond.

Ligation proceeds efficiently even in the case of difficult junctions such as Pro-Cys junctions. Indeed, our recent article in Organic Letters shows that ligation at proline proceeds efficiently at 65 °C and pH 5.5 while the formation of a deletion side product is minimized when compared to peptide alkylthioesters (Scheme 2). [article 4]

Pro-ligation

Scheme 2. Ligation at proline using SEA peptide segments minimize deletion side product formation.

SEA peptide segments allows many more.
One important property is the possibility to convert a SEA peptide into a peptide thioester,  which are the key segments for protein assembly using another chemoselective native peptide bond reaction called Native Chemical Ligation [Native Chemical Ligation]

The conversion of SEA peptides into peptide thioesters proceeds as shown in Scheme 3. This reaction is an exchange process in which the SEA moiety is replaced by the alkyl thiol such as 3 -mercaptopropionic acid (MPA). The reaction proceeds efficiently at pH 4 and 37 °C in aqueous solution. [article 5]. Note that peptidyl prolyl MPA thioesters such as the one shown in Scheme 2 are readily prepared at 65 °C without racemization.
A recent protocol article from the lab is now published in the Journal of Peptide Science [article 6], and will help you making peptide thioesters using Fmoc-SPPS starting from SEA polystyrene resin, which is commercially available (XProChem).MPA-thioesters

Scheme 3. SEA peptide segments are converted into thioester peptide segments in the presence of an excess of 3-mercaptopropionic acid.

SEA peptides give also access to peptide thioacids by exchanging the bis(2-sulfanylethyl)amino group by triisopropylsilylthiol at neutral pH in water (37 °C)(article 7). Peptide thioacids are valuable building blocks for setting up efficient chemoselective ligations reactions such as the imide ligation which was pionneered by the lab [article 8], and more recently used for designing a novel amide bond forming reaction at asparagine [article 9].

Perhaps one of the most important properties of SEA group is the possibility to switch easily between the open dithiol form called SEAon and the closed cyclic disulfide form called SEAoff as shown in Scheme 4.

SEAon_off

Scheme 4. SEAon/off concept.

SEAon/off concept enables to design novel sequential assembly methods for protein total synthesis. In an Angewandte Chemie paper (article 10), we designed a method to assemble three peptide segments in a one-pot reaction, which enables the synthesis of proteins composed of up to 150-200 amino acid residues.

One-pot
Scheme 5. One-pot three segments assembly process for protein total synthesis in solution.

 

For recent examples of protein total synthesis using the one-pot three segment assembly approach see (click on the image to enlarge it, or on the animation to download a powerpoint ppsx file):

K1K1_animationNN_animation

Recently, we introduced in a Chemical Science 2013 paper (article 11) a novel solid phase method called SEA SPPS for protein total synthesis based also on SEAon/off concept. The method proceeds as shown in Scheme 6.

SEA SPPS

Scheme 6. SEA SPPS.

 


 

References

1. Raibaut, L., Ollivier, N., and Melnyk, O. (2012) Sequential native peptide ligation strategies for total chemical protein synthesis. Chem. Soc. Rev. 41, 7001-7015.
2. Ollivier, N., Dheur, J., Mhidia, R., Blanpain, A., and Melnyk, O. (2010) Bis(2-sulfanylethyl)amino native peptide ligation. Org. Lett. 12, 5238-41.
3. Ollivier, N., Behr, J. B., El-Mahdi, O., Blanpain, A., and Melnyk, O. (2005) Fmoc solid-phase synthesis of peptide thioesters using an intramolecular N,S-acyl shift. Org. Lett. 7, 2647-50.
4. Raibaut, L., Seeberger, P., and Melnyk, O. (2013) Bis(2-sulfanylethyl)amido Peptides Enable Native Chemical Ligation at Proline and Minimize Deletion Side-Product Formation. Org. Lett. 15, 5516–5519.
5. Dheur, J., Ollivier, N., Vallin, A., and Melnyk, O. (2011) Synthesis of peptide alkylthioesters using the intramolecular N,S-acyl shift properties of bis(2-sulfanylethyl)amido peptides. J. Org. Chem. 76, 3194-3202.
This article has been selected as a contribution to the American Chemical Society virtual collection “Peptides”. To read the editorial of William Lubell see the following link: Editorial
Click for accessing to the contect of the Peptide ACS Virtual Collection.
6. Ollivier, N., Raibaut, L., Blanpain, A., Desmet, R., Dheur, J., Mhidia, R., Boll, E., Drobecq, H., Pira, S. L., and Melnyk, O. (2013) Tidbits for the synthesis of bis(2-sulfanylethyl)amido (SEA) polystyrene resin, SEA peptides and peptide thioesters. J. Pep. Sci., 10.1002/psc.2580.
7. Pira, S. L., Boll, E., and Melnyk, O. (2013) Synthesis of peptide thioacids at neutral pH using bis(2-sulfanylethyl)amido peptide precursors. Org. Lett. 15, 5346–5349.
8. Mhidia, R., Beziere, N., Blanpain, A., Pommery, N., and Melnyk, O. (2010) Assembly/disassembly of drug conjugates using imide ligation. Org. Lett. 12, 3982-5.
9. Mhidia, R., Boll, E., Fecourt, F., Ermolenko, M., Ollivier, N., Sasaki, K., Crich, D., Delpech, B., and Melnyk, O. (2013) Exploration of an imide capture/N,N-acyl shift sequence for asparagine native peptide bond formation. Bioorg Med Chem 21, 3479-85.
10. Ollivier, N., Vicogne, J., Vallin, A., Drobecq, H., Desmet, R., El-Mahdi, O., Leclercq, B., Goormachtigh, G., Fafeur, V., and Melnyk, O. (2012) A one-pot three-segment ligation strategy for protein chemical synthesis. Angew. Chem. Int. Ed. 51, 209-213.
11. Raibaut, L., Adihou, H., Desmet, R., Delmas, A. F., Aucagne, V., and Melnyk, O. (2013) Highly efficient solid phase synthesis of large polypeptides by iterative ligations of bis(2-sulfanylethyl)amido (SEA) peptide segments. Chem. Sci. 4, 4061-4066.