A Facile N-Mercaptoethoxyglycinamide (MEGA) Linker Approach to Peptide Thioesterification and Cyclization

Abstract

The C-terminal selective electrophilic activation of polypeptides is essential for site-specific peptide modification and conjugation techniques such as Native Chemical Ligation (NCL). Peptide C-terminal α-thioesters are particularly valuable precursors for NCL, due to their hydrolytic stability in aqueous buffers and reactivity toward thiol nucleophiles. The synthesis of peptide α-thioesters, however, requires harsh acidic conditions or complex chemical manipulations, which ultimately limits their functional group compatibility and broad utility. Herein, we report a readily accessible N-mercaptoethoxyglycinamide (MEGA) solid-phase linker for the facile synthesis of latent peptide α-thioesters. Incubating peptide-MEGA sequences with 2-mercaptoethanesulfonic acid at mildly acidic pH yielded α-thioesters that were directly used in NCL without purification. The MEGA linker yielded robust access to peptide α-thioesters ranging in length from 4 to 35 amino acids, and greatly simplified the synthesis of cyclic peptides. Finally, the high utility of MEGA was demonstrated by the one-pot synthesis of a functional analog of the Sunflower Trypsin Inhibitor 1.

Exploring chemoselective S-to-N acyl transfer reactions in synthesis and chemical biology

“…Recently Chatterjee and co-workers have reported the application of an N -mercaptoethoxyglycinamide (MEGA) solid-phase linker for the facile synthesis of peptide α-thioesters. Thioester formation occurs from the N -oxyamide via an N -to- S acyl shift through a 6-membered cyclic intermediate 48 . Several other efficient methods for thioester synthesis have been developed.…”

Expedient on-resin modification of a peptide C-terminus through a benzotriazole linker

“… 23 Recently, SFTI-1 analogues were successfully prepared by Chatterjee and coworkers. 24 However, the cyclization required 24 h at 50 °C. An isolated yield of 35% was obtained after chromatographic purification.…”

Solid-Phase Synthesis of Head to Side-Chain Tyr-Cyclodepsipeptides Through a Cyclative Cleavage From Fmoc-MeDbz/MeNbz-resins

“…In this regard, the thioester should be formed after elongation of the peptide. Although several linkers have been developed for this purpose (Camarero et al, 2004;Mulder et al, 2014;Kawakami et al, 2016;Morisaki et al, 2016;Olivier et al, 2017;Rao and Liu, 2017;Shelton et al, 2017;Flood et al, 2018), the 3-(Fmoc-amino)-4-(methylamino) benzoic acid (Fmoc-MeDbz, the acronym derives from N-methyl-diaminobenzoic acid) and/or its non-methylated analog developed by Blanco-Canosa and Dawson are optimal (Blanco-Canosa and Dawson, 2008;Blanco-Canosa et al, 2015) Fmoc-MeDbz is anchored to an amino resin. After removal of the Fmoc group, peptide elongation proceeds smoothly using normal coupling reagents (the electronic deactivation of the aromatic ring by the growing peptide chain and the presence of the Me in the second amino group impede the development of a second peptide chain on the N-Me-amino).…”