岡本 亮

General strategies for synthesizing sulfated oligosaccharides employ the protection of sulfate groups for glycosylation or post-sulfation after the synthesis of oligosaccharides. However, the chemical behavior of free sulfate groups in glycosylation reactions has not been thoroughly studied. We examined several glycosyl donors with free sulfate groups, but neither glycosyl imidates nor thioglycosides achieved products. Conversely, activating 6-sulfated GalNAc donors with either a 2-NTroc group or 2-O-acetyl group using an ortho-hexynylbenzoate group at the anomeric position yielded β-glycosides in good yield. These results indicate that glycosylations with a free sulfate group can be performed and that the neighboring group participation of 2-NHTroc and 2-O-acetyl groups works even with an unprotected sulfate group at 6-position. Ab initio calculations supported the formation of acyloxionium-cation via 2-O-acyl participation. Additionally, glycosylation reactions under various counter cations of the sulfate group, such as Na+, Li+, K+, Ba2+ and pyridinium were examined. Results showed that sodium and lithium salt donors yielded the products in good yield. These results were also supported by ab initio calculations. Practical glycosylation reactions between disaccharide donor-acceptor pairs with free sulfate groups successfully yielded the sulfated tetrasaccharides. This study also discusses how a sodium salt acts as a protecting group during glycosylation.

A bioinspired semisynthesis of human‐interleukin‐6 bearing N‐glycan at Asn143 (143glycosyl‐IL‐6) was performed by intentional glycosylation effects and protein folding chemistry for regioselective peptide‐backbone activation. 143Glycosyl‐IL‐6 is a genetically coded cytokine, but isolation was difficult owing to a tiny amount. IL6‐polypeptide (1‐141‐position) with an intentionally inserted cysteine at 142‐position was expressed in E. coli. The expressed polypeptide was treated with a chemical folding process to make a specific helices bundle conformation through native two‐disulfide bonds (43–49 and 72–82). Utilizing the successfully formed free‐142‐cysteine, sequential conversions using cyanation of 142‐cysteine, hydrazinolysis, and thioesterification created a long polypeptide (1–141)‐thioester. However, the resultant polypeptide‐thioester caused considerable aggregation owing to a highly hydrophobic peptide sequence. After the reduction of two‐disulfide bonds of polypeptide (1–141)‐thioester, an unprecedented hydrophilic N‐glycan tag was inserted at the resultant cysteine thiols. The N‐glycan tags greatly stabilized polypeptide‐thioester. The subsequent native chemical ligation and desulfurization successfully gave a whole 143glycosyl‐IL‐6 polypeptide (183‐amino acids). Removal of four N‐glycan tags and immediate one‐pot in vitro folding protocol efficiently produced the folded 143glycosyl‐IL‐6. The folded 143glycosyl‐IL‐6 exhibited potent cell proliferation activity. The combined studies with molecular dynamics simulation, semisynthesis, and bioassays predict the bioactive conformation of latent 143glycosyl‐IL‐6.

Protein ubiquitination is involved in nearly all biological processes in Eukaryotes. To gain precise insights into the function of ubiquitination in these processes, researchers frequently employ ubiquitinated protein probes with well-defined structures. While chemical protein synthesis has afforded a variety of ubiquitinated protein probes, there remains a demand for efficient synthesis methods for complex probes, such as ubiquitinated glycoproteins and ubiquitinated cysteine-containing proteins. In this study, we introduce a new method to obtain ubiquitinated proteins through isopeptide bond formation mediated by δ-selenolysine residues. We synthesized δ-selenolysine derivatives in both L- and D-forms starting from DL-δ-hydroxy-DL-lysine, accomplished by substituting the δ-mesylate with KSeCN and by enzymatic optical resolution with L- and D-aminoacylase. We synthesized ubiquitin (46–76)-α-hydrazide with a δ-seleno-L-lysine residue at position 48, as well as ubiquitin (46–76)-α-thioester, using solid-phase peptide synthesis. Subsequently, the δ-selenolysine-mediated ligation of these peptides, followed by one-pot deselenization, provided the desired isopeptide-linked ubiquitin peptide. The new δ-selenolysine-mediated isopeptide bond formation offers an alternative method to obtain complex ubiquitin- and ubiquitin-like probes with multiple post-translational modifications. These probes hold promise for advancing our understanding of ubiquitin biology.

To uncover how cells distinguish between misfolded and correctly-folded glycoproteins, homogeneous misfolded glycoproteins are needed as a probe for analysis of their structure and chemical characteristic nature. In this study, we have synthesized misfolded glycosyl interleukin-8 (IL-8) by combining E. coli expression and chemical synthesis to improve the synthetic efficiency. In order to prepare N-terminal peptide-thioester segment (1-33), we prepared an E. coli expressed peptide and then activated the C-terminal Cys by using an intramolecular N-to-S acyl shift reaction, followed by trans-thioesterification of the Cys-thioester with an external bis(2-sulfanylethyl)amine (SEA). The glycopeptide segment (34-49) was prepared by solid phase peptide synthesis and the C-terminal peptide (50-72) was prepared in E. coli. These peptide and glycopeptide segments were successfully coupled by sequential native chemical ligation. To obtain homogeneous misfolded glycoproteins by shuffling the disulfide bond pattern, folding conditions were optimized to maximize the yield of individual homogeneous misfolded glycoproteins.

Serine protease inhibitor Kazal type 13 (SPINK13) is a secreted protein that has been recently studied as a therapeutic drug and an interesting biomarker for cancer cells. Although SPINK13 has a consensus sequence (Pro-Asn-Val-Thr) for N-glycosylation, the existence of N-glycosylation and its functions are still unclear. In addition to this, the preparation of glycosylated SPINK 13 has not been examined by both the cell expression method and chemical synthesis. Herein we report the chemical synthesis of the scarce N-glycosylated form of SPINK13 by a rapid synthetic method combined with the chemical glycan insertion strategy and a fast-flow SPPS method. Glycosylated asparagine thioacid was designed to chemoselectively be inserted between two peptide segments where is the sterically bulky Pro-Asn(N-glycan)-Val junction by two coupling reactions which consist of diacyl disulfide coupling (DDC) and thioacid capture ligation (TCL). This insertion strategy successfully afforded the full-length polypeptide of SPINK13 within two steps from glycosylated asparagine thioacid. Because the two peptides used for this synthesis were prepared by a fast-flow SPPS, the total synthetic time of glycoprotein was considerably shortened. This synthetic concept enables us to repetitively synthesize a target glycoprotein easily. Folding experiments afforded well-folded structure confirmed by CD and disulfide bond map. Invasion assays of glycosylated SPINK13 and non-glycosylated SPINK13 with pancreatic cancer cells showed that non-glycosylated SPINK-13 was more potent than that of glycosylated SPINK13.

Antifreeze glycoprotein (AFGP), which inhibits the freezing of water, is highly O-glycosylated with a disaccharide, d-Galβ1-3-d-GalNAcα (GalGalNAc). To elucidate the function of the sugar residues for antifreeze activity at the molecular level, we conducted a total chemical synthesis of partially sugar deleted AFGP derivatives, and unnatural forms of AFGPs incorporating glucose (Glc)-type sugars instead of galactose (Gal)-type sugars. These elaborated AFGP derivatives demonstrated that the stereochemistry of each sugar residue on AFGPs precisely correlates with the antifreeze activity. A hydrogen-deuterium exchange experiment using synthetic AFGPs revealed a different dynamic behavior of water around sugar residues depending on the sugar structures. These results indicate that sugar residues on AFGP form a unique dynamic water phase that disturbs the absorbance of water molecules onto the ice surface, thereby inhibiting freezing.

Peptide-aminothiazoline enabled the sequential peptide ligation in one-pot manner and demonstrated convergent synthesis of a circular protein and homogeneous glycoproteins.

N-acetylglucosaminyltransferase-V (GnT-V or MGAT5) catalyzes the formation of an N-glycan β1,6-GlcNAc branch on selective target proteins in the Golgi apparatus and is involved in cancer malignancy and autoimmune disease etiology. Several three-dimensional structures of GnT-V were recently solved, and the recognition mechanism of the oligosaccharide substrate was clarified. However, it is still unclear how GnT-V selectively acts on glycoprotein substrates. In this study, we focused on an uncharacterized domain at the N-terminal side of the luminal region (N domain) of GnT-V, which was previously identified in a crystal structure, and aimed to reveal its role in GnT-V action. Using lectin blotting and fluorescence assisted cell sorting analysis, we found that a GnT-VΔN mutant lacking the N domain showed impaired biosynthetic activity in cells, indicating that the N domain is required for efficient glycosylation. To clarify this mechanism, we measured the in vitro activity of purified GnT-VΔN toward various kinds of substrates (oligosaccharide, glycohexapeptide, and glycoprotein) using HPLC and a UDP-Glo assay. Surprisingly, GnT-VΔN showed substantially reduced activity toward the glycoprotein substrates, whereas it almost fully maintained its activity toward the oligosaccharides and the glycopeptide substrates. Finally, docking models of GnT-V with substrate glycoproteins suggested that the N domain could interact with the substrate polypeptide directly. Our findings suggest that the N domain of GnT-V plays a critical role in the recognition of glycoprotein substrates, providing new insights into the mechanism of substrate-selective biosynthesis of N-glycans.

This paper reports the optimized methods that can accelerate the semisynthesis of homogeneous glycoproteins based on recombinant expression and chemical conversion.

Degradation of misfolded glycoproteins by the ubiquitin-proteasome system (UPS) is a very important process for protein homeostasis. To demonstrate the accessibility toward a ubiquitinated glycoprotein probe for the study of glycoprotein degradation by UPS, we synthesized ubiquitinated glycoprotein CC motif chemokine 1 (CCL1) bearing a high-mannose-type N-glycan, starting from six peptide segments. A native isopeptide linkage was constructed using δ-thiolysine (thioLys)-mediated chemical ligation. CCL1 glycopeptide with a high-mannose-type N-glycan as well as a δ-thioLys residue was synthesized chemically. The chemical ligation between δ-thioLys-containing glycopeptide and ubiquitin-α-thioester successfully yielded a ubiquitinated glycopeptide with a native isopeptide bond after desulfurization, even in the presence of a large N-glycan. In vitro folding experiments under reduced and redox conditions gave the desired two types of ubiquitinated glycosylated CCL1s, consisting of unfolded CCL1 and folded ubiquitin, and the folded form of both CCL1 as well as ubiquitin. We achieved the chemical synthesis of a complex protein molecule that contains not only the two major post-translational modifications, ubiquitination and glycosylation, but also controlled folding states of ubiquitin and CCL1. These chemical probes could have useful applications in the study of complex ubiquitin biology and glycobiology.

The benzylidene acetal group is one of the most important protecting groups not only in carbohydrate chemistry but also in general organic chemistry. In the case of 4,6-O-benzylidene glycosides, we previously found that the stereochemistry at 4-position altered the reaction rate constant for hydrolysis of benzylidene acetal group. However, a detail of the acceleration or deceleration factor was still unclear. In this work, the hydrolysis reaction of benzylidene acetal group was analyzed using the Arrhenius and Eyring plot to obtain individual parameters for glucosides (Glc), mannosides (Man), and galactosides (Gal). The Arrhenius and Eyring plot indicated that the pre-exponential factor (A) and ΔS⧧ were critical for the smallest reaction rate constant of Gal among nonacetylated substrates. On the other hand, both Ea/ΔH⧧ and A/ΔS⧧ were influential for the smallest reaction rate constant of Gal among diacetylated substrates. All parameters obtained suggested that the rate constant for hydrolysis reaction was regulated by protonation and hydration steps along with solvation. The obtained parameters support wide use of benzylidene acetal group as orthogonal protection of cis- and trans-fused bicyclic systems through the fast hydrolysis of the trans-fused benzylidene acetal group.

Glycosylation is an accepted strategy to improve the therapeutic value of peptide and protein drugs. Insulin and its analogues are life-saving drugs for all type I and 30% of type II diabetic patients. However, they can readily form fibrils which is a significant problem especially for their use in insulin pumps. Because of the solubilizing and hydration effects of sugars, it was thought that glycosylation of insulin could inhibit fibril formation and lead to a more stable formulation. Since enzymatic glycosylation results in heterogeneous products, we developed a novel chemical strategy to produce a homogeneous glycoinsulin (disialo-glycoinsulin) in excellent yield (∼60%). It showed a near-native binding affinity for insulin receptors A and B in vitro and high glucose-lowering effects in vivo, irrespective of the route of administration (s.c. vs i.p.). The glycoinsulin retained insulin-like helical structure and exhibited improved stability in human serum. Importantly, our disialo-glycoinsulin analogue does not form fibrils at both high concentration and temperature. Therefore, it is an excellent candidate for clinical use in insulin pumps.