Glycoprotein Semisynthesis by Chemical Insertion of Glycosyl Asparagine Using a Bifunctional Thioacid-Mediated Strategy

Abstract

Glycosylation is a major modification of secreted and cell surface proteins, and the resultant glycans show considerable heterogeneity in their structures. To understand the biological processes arising from each glycoform, the preparation of homogeneous glycoproteins is essential for extensive biological experiments. To establish a more robust and rapid synthetic route for the synthesis of homogeneous glycoproteins, we studied several key reactions based on amino thioacids. We found that diacyl disulfide coupling (DDC) formed with glycosyl asparagine thioacid and peptide thioacid yielded glycopeptides. This efficient coupling reaction enabled us to develop a new glycoprotein synthesis method, such as the bifunctional thioacid-mediated strategy, which can couple two peptides with the N-and C-termini of glycosyl asparagine thioacid. Previous glycoprotein synthesis methods required valuable glycosyl asparagine in the early stage and subsequent multiple glycoprotein synthesis routes, whereas the developed concept can generate glycoproteins within a few steps from peptide and glycosyl asparagine thioacid. Herein, we report the characterization of the DDC of amino thioacids and the efficient ability of glycosyl asparagine thioacid to be used for robust glycoprotein semisynthesis.

Protecting Group-Minimum, Practical N-to-C Peptide Synthesis

“…To realize N-to-C peptide synthesis, we employed the peptide thiocarboxylic acid (PTC) platform (Figure 1-b). 33 Amide bond formation using PTC under various conditions has been reported, [34][35][36][37][38][39][40][41][42][43] but PTCs have never been used in iterative N-to-C peptide synthesis. Based on our previous development of a general, one-step PTC synthesis from peptides using a catalytic diacetyl sulfide (Ac2S) and potassium thioacetate (AcSK), 44 we envisioned the scheme shown in Figure 1-b.…”

Total synthesis of interleukin-2 via a tunable backbone modification strategy

“…Many proteins of biological interests have been chemically synthesized and used to correlate the protein structure to function. [14][15][16][17][18][19][20][21][22][23][24] In particular, protein chemical synthesis provides a unique means to generate proteins with tailor-made modications, which are difficult or impossible for expression systems. [25][26][27][28][29][30][31][32][33][34][35][36] Despite these achievements in protein chemical synthesis, the synthesis of proteins or peptides with aggregation-prone properties remains a challenging task and requires case by case analysis and study.…”

Rapid Chemical Synthesis of Serine Protease Inhibitor Kazal‐type 13 (SPINK13) Glycoform by a Combined Method with Glycan Insertion Strategy and Fast‐Flow Fmoc SPPS**

“…To overcome this situation, recently we developed the highly efficient convergent strategy for glycoprotein synthesis. [6] The method employs chemical glycan insertion strategy, that can couple two peptides with N-and C-terminus of glycosyl asparagine thioacid 1 (Figure 1-A). The first coupling is diacyl disulfide coupling (DDC) [6] between a peptide thioacid 2 and glycosyl asparagine thioacid 1 to afford glycopeptide 3.…”