Extended Sugar-Assisted Glycopeptide Ligations:  Development, Scope, and Applications

Abstract

Recently, we reported the development of sugar-assisted ligation (SAL), a novel peptide ligation method for the synthesis of glycopeptides. After screening a large number of glycoprotein sequences in a glycoprotein database, it became evident that a large proportion (approximately 53%) of O-glycosylation sites contain amino acid residues that will not undergo SAL reactions. To overcome these inherent limitations and broaden the scope of the method we report here the development of an extended SAL method. Glycopeptides containing up to six amino acid extensions N-terminal to the glycosylated residue were shown to facilitate ligation reactions with peptide thioesters, and these products were isolated in good yields. Kinetic analysis was used to show that as glycopeptides were extended by further amino acid residues, ligation reactions became slower. This finding was rationalized by molecular dynamics simulations using AMBER9. These studies suggested a general trend whereby the proximal distance between the reactive sites of the thioester intermediate (the N-terminal amine and the carbonyl carbon of the thioester) increased as glycopeptides were extended, thus slowing down the ligation rate. Each of the extended SAL methods showed broad tolerance to a number of different amino acid combinations at the ligation junction. Re-evaluation of the glycoprotein database suggested that 95% of the O-linked glycosylation sites can now be utilized to facilitate SAL or extended SAL reactions. As such, this method represents an extremely valuable tool for the synthesis of naturally occurring glycopeptides and glycoproteins. To demonstrate the applicability of the method, extended SAL was successfully implemented in the synthesis of the starting unit of the cancer-associated MUC1 glycoprotein.

Methods for the Peptide Synthesis and Analysis

“…Limitation of this hybrid technology is the mandatory use of a Cys residue at the N-terminal of the first fragment, and the synthesis of an appropriate C-terminal thioester in the second fragment, which in some cases provides poor yields [197]. Advances in the field include, for example, the use of conformationally assisted ligation [198], removable auxiliaries [199,200], Staudinger ligation [201][202][203], thiolalkylation [204], desulfurization methods [205][206][207][208], and sugar-assisted ligation (SAL) [209,210]. More interestingly, a side-chain-assisted chemical ligation has been reported lately, with no limits to the assembled amino acids [211].…”

Postligation-Desulfurization: A General Approach for Chemical Protein Synthesis

“…Subsequent metal-based desulfurization reduced both thiols on GalNAc and Cys 37 to afford diptericin 155 in 54% yield (Scheme 39). Payne et al aimed to apply the SAL strategy to the total synthesis of native glycoproteins [96]. However, direct utilization of the SAL method was unsuitable for 75% of glycoproteins, indicated by the screening of over 200 O-linked glycoproteins in O-GlycBase v6.00.…”

Chemoselective Peptide Ligation: A Privileged Tool for Protein Synthesis

“…Final desulfurization conditions (see Section 13.3.3.1) yield the native glycopeptide 65. It was demonstrated that glycopeptides with up to six additional amino acids N-terminal to the glycosylation site were able to undergo SAL [94]. In further studies, a b-thioacetic ester in the C-3 position of a GalNAc residue as the auxiliary 66 can be used as well, which allows a final auxiliary removal with hydrazine (Scheme 13.16B) [95].…”