In contrast to nature, synthetically produced peptides are typically built in the “reverse” direction from the C-terminus towards the N-terminus. This strategy has led to the development of the Merrifield-Synthesis and later the Fmoc/tBu process. Despite ongoing research efforts, today’s solid-phase peptide synthesis still strongly relies on these reagents and protocols which were developed in the 1950-1980s.

But there is more out there! In today’s blog we highlight the ynamide coupling reagent N-methylynetoluenesulfonamide, briefly MYTsA or Zhao-Reagent, as ideal candidate for carboxy group activation in peptide synthesis. It facilitates peptide bond formation in a one-pot, two-step manner using in situ generated α-acyloxyenamide active esters of amino acids as stable intermediates. When the ynamide is substituted with an electron-withdrawing group, e.g., Tosyl in the case of MYTsA, racemization/epimerization during peptide bond formation is not observed, making the reagent superior for couplings compared to carbodiimides.  

Application of MYTsA as replacement for carbodiimides in classical C→N peptide synthesis: A Fmoc-protected amino acid is activated with MYTsA as acyloxyenamide and then used to elongate the peptide chain.

In the case of “classical” C→N peptide synthesis, the alpha-amino group of the next amino acid being added to the growing peptide chain needs to be protected (e.g., with Fmoc) to prevent unwanted side reactions (cyclization and polymerization of the building blocks). The consequence is a rather unfavorable atom economy and undesired chemical waste.

In this context, MYTsA adds another benefit: it also may be used for peptide synthesis in N→C direction, just like in natural protein biosynthesis. This approach is not compatible with carbodiimides due to racemization and is characterized by a far better atom economy and thus by the generation of less waste, because the amino groups of the amino acid building blocks do not require protection and deprotection, thus also no Fmoc and no base (usually piperidine) are needed.

(A) “Traditional“ Fmoc-based peptide synthesis in C→N direction compared to (B) MYTsA-mediated peptide synthesis in N→C direction.

As starting material for solid phase synthesis in N→C direction, 2-CTC resins may be directly loaded with the first amino acid. Also, acid labile carbamate linkers may be used.

Besides, MYTsA enables peptide synthesis in aqueous solvents, where the coupling reaction of activated esters proceeds up to more than ten times faster than in DMF, due to a lowered energy barrier for the aminolysis of the active ester. For such syntheses, hydrophilic resins like, e.g., Tentagel® are available.

In addition, MYTsA is also suitable for the synthesis of esters, thioesters, macrolactones, and macrolactams, and it may be used for the crosslinking of peptides in vitro and in vivo (cells).

References:

Ynamides as Racemization-Free Coupling Reagents for Amide and Peptide Synthesis; L. Hu, S. Xu, Z. Zhao, Y. Yang, Z. Peng, M. Yang, C. Wang, J. Zhao; J. Am. Chem. Soc. 2016; 138(40): 13135-13138. https://doi.org/10.1021/jacs.6b07230

Ynamide Coupling Reagents: Origin and Advances; L. Hu, J. Zhao; Acc. Chem. Res. 2024; 57(6): 855-869. https://doi.org/10.1021/acs.accounts.3c00743

Ynamide Coupling Reagent for the Chemical Cross-Linking of Proteins in Live Cells; S. Li, C. Zhu, Q. Zhao, Z. M. Zhang, P. Sun, Z. Li; ACS Chem Biol. 2023; 18(6): 1405-1415. https://doi.org/10.1021/acschembio.3c00149

Ynamides: A Modern Functional Group for the New Millennium; K. A. DeKorver, H. Li, A. G. Lohse, R. Hayashi, Z. Lu, Y. Zhang, R. P. Hsung; Chem. Rev. 2010; 110(9): 5064-5106. https://doi.org/10.1021/cr100003s

Exploiting Remarkable Reactivities of Ynamides: Opportunities in Designing Catalytic Enantioselective Reactions; J. Luo, G.-S. Chen, S.-J. Chen, J.-S. Yu, Z.-D. Li, Y. L. Liu; ACS Catal. 2020; 10(23): 13978-13992. https://doi.org/10.1021/acscatal.0c04180

Recent advances in asymmetric synthesis of chiral amides and peptides: racemization-free coupling reagents; Y. Guo, M. Wang, Y. Gao, G. Liu; Org. Biomol. Chem. 2024; 22(22): 4420-4425. https://doi.org/10.1039/d4ob00563e

The anionic chemistry of ynamides: A review; G. Evano, B. Michelet, C. Zhang; C. R. Chimie 2017, 20(6): 648-664. https://doi.org/10.1016/j.crci.2016.12.002

Ynamide-Mediated Peptide Bond Formation: Mechanistic Study and Synthetic Applications; S. Xu, D. Jiang, Z. Peng, L. Hu, T. Liu, L. Zhao, J. Zhao; Angew. Chem. Int. Ed. 2022; 61(46): e202212247. https://doi.org/10.1002/anie.202212247

Inverse Peptide Synthesis Using Transient Protected Amino Acids; T. Liu, Z. Peng, M. Lai, L. Hu, J. Zhao; J. Am. Chem. Soc. 2024; 146(6): 4270-4280. https://doi.org/10.1021/jacs.4c00314

Solid-Phase Peptide Synthesis in the Reverse (N → C) Direction; N. Thieriet, F. Guibe, F. Albericio; Org. Lett. 2000; 2(13): 1815-1817. https://doi.org/10.1021/ol0058341