MEGA: A Linker for Peptide Thioesterification and Cyclization

Native chemical ligation (NCL) is an important method used for the construction of large polypeptides and the semisynthesis of full-length proteins. In NCL, two peptide fragments — an N-terminal cysteine peptide and a peptide α-thioester — assemble to yield a larger target polypeptide. In this process, the N-terminal cysteine peptide thiol displaces the thiol of the peptide α-thioester, upon which an intramolecular S-to-N acyl shift occurs to produce the N–C peptide bond (Figure 1).

 

Production of N-terminal cysteine peptides is relatively simple, with the fragments readily accessed by standard Fmoc solid-phase peptide synthesis (Fmoc-SPPS). Conversely, production of peptide α-thioesters is challenging and limited by the necessity of harsh reaction conditions and multiple synthetic steps. Recently, however, the Chatterjee laboratory at the University of Washington (Seattle, WA) has introduced an N-mercaptoethoxyglycinamide (MEGA) linker approach to access peptide α-thioesters with ease.¹

 

MEGA Linker Approach to Thioesterification

In Chatterjee’s method of peptide α-thioester synthesis, a C-terminus MEGA-linked peptide undergoes an N-to-S acyl shift under mildly acidic conditions. The resulting rearranged thioester then undergoes nucleophilic attack by a thiol to displace the MEGA linker and yield the desired peptide α-thioester (Figure 2). The resulting peptide α-thioester products can be employed in NCL without further purification.

 

Chatterjee et al. began the synthesis of C-terminus MEGA-linked peptides with the production of MEGA-linked resin. To produce MEGA-linked resin, Rink amide resin is condensed with bromoacetic acid, resulting in bromoacetylated amide resin. Then, displacement of the resin’s bromide ion by S-trityl-protected 2-(aminooxy)ethane diol occurs, yielding the MEGA-linked resin (Figure 3).

Upon completing the synthesis of MEGA-linked resin, the Chatterjee laboratory developed conditions for coupling 17 different amino acids to the MEGA linker’s secondary amine. For each of these initial couplings, respective 4-mer peptides with the sequence AWKX-MEGA were synthesized manually (Figure 3). All 17 of the AWKX-MEGA peptides were produced in good yield and purity, indicating that the N–O bond in MEGA is stable during both synthesis and cleavage.

 

From this point, Chatterjee investigated and optimized thioesterification conditions for each of the AWKX-MEGA peptides. Many factors influenced thioester yield, such as the nature of the C-terminal amino acid, thiol nucleophile, pH, and reaction temperature. The best thioesterification yields were found under mildly acidic conditions (pH 4.0–6.0), with elevated temperatures (37–70 °C), extended reaction times (8–72 h), and with external thiol 2-mercaptoethanesulfonic acid (sodium salt).

Importantly, Chatterjee has demonstrated that the MEGA linker can be used in the production of longer peptides. Chatterjee successfully synthesized the 35-mer peptide p53(1–35) with the Liberty Blue™ automated microwave peptide synthesizer, avoiding N–O bond cleavage entirely and isolating the pure peptide in 22% yield. From this, a 75% yield of MESNa thioester was generated following thioesterification.

Upon demonstrating that MEGA-linked peptide α-thioesters of varying lengths could be synthesized successfully, the Chatterjee laboratory set out to apply the thioesterification to one-pot NCL transformations. Studies began with NCLs between the AWKX-MEGA 4-mer peptides and the N-terminal cysteine peptide, CASW, which proceeded efficiently at room temperature in 1 hour. Next, Chatterjee investigated the use of MEGA in intramolecular NCL, and successfully produced the cyclic forms of CASHEW- and CRDG(D-F)-MEGA.  

To conclude the study, Chatterjee applied the MEGA linker approach in the synthesis of the complex cyclic peptide, Sunflower Trypsin Inhibitor-1 (SFT-1). Upon synthesizing SFT-1’s linear 14-mer sequence on the Liberty Blue™ and subjection to thioesterification conditions, the Chatterjee laboratory isolated SFT-1 in 30% yield, demonstrating the usefulness of the MEGA linker approach for accessing important bioactive peptides.

(1)    Shelton, P. M. M.; Weller, C. E.; Chatterjee, C. J. Am. Chem. Soc. 2017, 139, 3946.