Native Chemical Ligation–Photodesulfurization in Flow

Abstract

Native chemical ligation (NCL) combined with desulfurization chemistry has revolutionized the way in which large polypeptides and proteins are accessed by chemical synthesis. Herein, we outline the use of flow chemistry for the ligation-based assembly of polypeptides. We also describe the development of a novel photodesulfurization transformation that, when coupled with flow NCL, enables efficient access to native polypeptides on time scales up to 2 orders of magnitude faster than current batch NCL-desulfurization methods. The power of the new ligation-photodesulfurization flow platform is showcased through the rapid synthesis of the 36 residue clinically approved HIV entry inhibitor enfuvirtide and the peptide diagnostic agent somatorelin.

Challenges and Perspectives in Chemical Synthesis of Highly Hydrophobic Peptides

“…All presented methods and protocols are based on traditional "batch" chemistry but a novel approach toward continuousflow peptides synthesis (Mijalis et al, 2017) or ligation and desulfurization (Chisholm et al, 2018). They presented an in-line flow-based ligation and desulfurization protocol and presented synthesis of enfuvirtide (HIV drug) and the diagnostic agent somatorelin.…”

Peptide Synthesis Utilizing Micro‐flow Technology

“…[44] A2 0mm aqueouss olution of peptide 69,g uanidine hydrochloride (Gdn·HCl), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and tris{(2-carboxyethyl)phosphine} (TCEP) was introduced into the micro-flow reactor via inlet A. A1 0mm aqueous solution of peptide 70,G dn·HCl, HEPES, and TCEP was introduced into the microflow reactor from inlet B. NCL was performed in the first heating tube (37 8C, 3min for 69 a;1 1min for 69 b). [44] A2 0mm aqueouss olution of peptide 69,g uanidine hydrochloride (Gdn·HCl), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and tris{(2-carboxyethyl)phosphine} (TCEP) was introduced into the micro-flow reactor via inlet A. A1 0mm aqueous solution of peptide 70,G dn·HCl, HEPES, and TCEP was introduced into the microflow reactor from inlet B. NCL was performed in the first heating tube (37 8C, 3min for 69 a;1 1min for 69 b).…”

“…Am icro-flow reactor was used to accomplish native chemical ligation( NCL) and photochemical desulfurization of cysteine side chains (Scheme 17). [44] A2 0mm aqueouss olution of peptide 69,g uanidine hydrochloride (Gdn·HCl), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and tris{(2-carboxyethyl)phosphine} (TCEP) was introduced into the micro-flow reactor via inlet A. A1 0mm aqueous solution of peptide 70,G dn·HCl, HEPES, and TCEP was introduced into the microflow reactor from inlet B. NCL was performed in the first heating tube (37 8C, 3min for 69 a;1 1min for 69 b). An intermedi- ate, 71,c ontained two molecules of 69 because ad ouble amount of 69 was used compared with 70.Asolution of glutathione (GSH) was introduced into the reactor via inlet C. Heating at 37 8Cf or 1t o6min in the second heatingt ube afforded at hiol intermediate 72.S ubsequent photochemical desulfurization afforded the desired peptide 73.T he use of am icroflow photochemical reactor accelerated the desulfurization, and the reactionr equired only 1min.…”

Peptide‐Chain Elongation Using Unprotected Amino Acids in a Micro‐Flow Reactor

“…We have also developed efficient synthetic approaches for peptides using protected amino acids . In addition, a limited number of micro‐flow syntheses of peptides using unprotected amino acids or peptides have been reported, although those approaches require premodification of substrates or the use of a Cys residue . We recently reported a highly efficient amino acid N ‐carboxyanhydride synthesis based on the rapid mixing (<0.1 s) of an MeCN/water biphasic system using micro‐flow technology .…”

“…[24] In addition, al imited number of micro-flow syntheses of peptides using unprotected amino acids or peptides have been reported, although those approaches require premodification of substrates [25] or the use of aC ys residue. [26] We recently reported ah ighly efficient amino acid N-carboxyanhydride synthesis based on the rapid mixing [27] (< 0.1 s) of an MeCN/water biphasic system using micro-flow technology. [28] Here, we reportm ixed carbonic anhydride-based amidation with unprotecteda minoa cids using micro-flow technology (Scheme1 (IV)).…”