Osamu Kanie

Oxidative protein folding, which is critical to proteins achieving their functional structures, is catalyzed in cells by protein disulfide isomerase (PDI)an enzyme that couples redox catalysis with the transient capture of folding intermediates to promote native disulfide formation while preventing aggregation. Although PDI improves oxidative folding in both chemically synthesized and recombinantly produced proteins, its use is restricted to homogeneous systems, limiting reusability and operational robustness. Artificial PDI mimics have advanced in vitro folding; however, no system has yet combined sufficient redox activity for native disulfide formation with a folding environment that suppresses aggregation, nor demonstrated true reusability. Here, we introduce a polymer-based “solid chaperone” that realizes PDI-like dual activity on an abiotic surface, achieving what natural PDI cannot: recyclable, HPLC-free oxidative folding without the stability and single-use limitations of enzymes. The covalent immobilization of cyclic diselenide onto polystyrene beads yields a redox-active and hydrophobic interface that transiently captures unfolded proteins, catalyzes both disulfide bond formation and isomerization, and suppresses aggregation even at high substrate concentrations. This solid-phase catalyst outperformed its homogeneous counterpart, producing native peptides and proteins in up to 99% yield and retaining full activity over multiple reuse cycles. These results demonstrate that complex biological folding functions, once confined to fragile enzymes, can be re-engineered into durable polymeric materials. This solid-phase strategy not only enables recyclable oxidative folding but also establishes a paradigm for translating enzymatic behavior into scalable synthetic systems with industrial potential.

Chromogenic and fluorogenic substrates are important for the detection of enzymatic activity. Conjugates of 4‐nitrophenol are among the earliest investigated classes of molecules and have been used in diagnostic applications, including those for type‐2 diabetes. The detection of 4‐nitrophenol under physiological conditions enables real‐time monitoring of glucosidase activity. This becomes possible when the resonance structure of 4‐nitrophenol is altered, making it distinguishable from its precursor chromogenic substrate. One of the surface‐modified silica materials, comprising an acetyl‐protected mannoside along with precipitated byproducts such as acyl‐urea and urea formed during carbodiimide coupling, induces a unique resonance shift in 4‐nitrophenol upon interaction. Raman microscopic analysis can distinguish the phenol‐type band at 1330 cm⁻¹ and the quinone‐type band at 860 cm⁻¹, thereby enabling monitoring of the α‐glucosidase reaction.

Glycoproteins are often considered as drug candidates. However, the regulation of post-translational glycan attachment remains an issue. We hypothesized that replacing the oxygen atom in the glycosidic linkage with sulfur atoms would stabilize the labile linkage against glycosidases, resulting in improved pharmacokinetics. In this study, we focused on the macrophage-activating factor (MAF) carrying O-linked N-acetylgalactosamine (GalNAc) and creating a miniature glycopeptide associated with MAF. A partial structure of MAF with a chemical mutation at three amino acid residues was designed in which threonine was replaced with cysteine (Cys), leading to a thioglycosidically linked GalNAc and a conformationally stable cyclic peptide due to the disulfide bond. GalNAc-Cys was used in solid-phase peptide synthesis, and the desired cyclic glycopeptide was synthesized. In the synthesis of GalNAc-Cys, glycosylation reactions were carried out based on the hard and soft acids and bases concept, where glycosyl trichloroacetimidate and fluoride were successfully used to couple with the thiol group in Cys. GalNAc-Cys was also evaluated as a substrate of α-GalNAc-ase and was shown to resist hydrolysis, supporting our concept. The synthesized cyclic miniature MAF induced LPS-assisted IL-12 production and resisted against α-GalNAc-ase.

The oxidative crosslinking of thiols via a disulfide (SS) bond is a fundamental chemical reaction in the industrial production of protein- and peptide-based formulations. In this study, we developed a new heterogeneous catalyst/mediator, polystyrene-resin-immobilized cyclic telluride [(S)-tetrahydrotellurophen-3-amine] (RSTe), which significantly promotes the oxidation of thiols to disulfides (SS formation). Under solid–liquid biphasic reaction conditions, RSTe exhibited glutathione-peroxidase-like activity in the oxidative SS formation of 15 organic solvent- and water-soluble thiols using a peroxide (H₂O₂ or ^tBuOOH) as an oxidant. Notably, all reactions were completed within 90 min, and RSTe and the peroxide could be removed by filtration and concentration, respectively, thereby affording the target disulfides in sufficient yield and purity without further purification. This reaction system could also be utilized for SS formation in cysteine-containing polypeptide chains, suggesting the potential application of RSTe in oxidative folding, which is an essential process in the production of bioactive proteins.

O-linked α-N-acetylgalactosamine (α-GalNAc) in the Gc protein is essential for macrophage activation; thus, the GalNAc-attached form of Gc protein is called Gc macrophage activating factor (GcMAF). O-linked glycans in Gc proteins from human plasma mainly consist of trisaccharides. GcMAF is produced when glycans on the Gc protein are hydrolyzed by α-Sia-ase and β-Gal-ase, leaving an α-GalNAc. Upon hydrolysis of α-GalNAc present on GcMAF, the protein loses the macrophage-activating effect. In contrast, our synthesized pyrrolidine-type iminocyclitol possessed strong in vitro α-GalNAc-ase inhibitory activity. In this study, we examined the protective effects of iminocyclitol against GcMAF via inhibition of α-GalNAc-ase activity. Detailed mass spectrometric analyses revealed the protective effect of the inhibitor on GcMAF. Furthermore, structural information regarding the glycosylation site and glycan structure was obtained using tandem mass spectrometric (MS/MS) analysis of the glycosylated peptides after tryptic digestion.

Invited for this month's cover is the group of Osamu Kanie and co‐workers at Tokai University. The cover picture shows the interaction of an incoming molecule and a receptor surface as the first process of a molecular sensing mechanism. For this, porous silica particles were decorated with protected carbohydrates to create artificial pocket‐like structures on its surface. More information can be found in the Research Article by O. Kanie and co‐workers.