Akihiro Ishiwata

The difructose dianhydride I synthase/hydrolase catalyzes the reversible transformation of inulobiose into difructose dianhydride I, a component of caramel. The proposed reaction mechanism of such a transformation consists of a glycosylation step followed by a cyclization to yield the dianhydride from the disaccharide. Here, we make use of extensive hybrid QM/MM metadynamics studies to shed light on the molecular basis of it. Our simulations show that the global reaction for the transformation of inulobiose by is slightly exergonic in good agreement with the experimental data. Furthermore, we observe that the glycosylation step is the rate‐limiting step. Interestingly, our work shows that the –1 sugar of inulobiose changes from an initial E conformation into a E puckering via the conformational pathway E E / T E E . Our calculations highlight that in the cyclization step, the three residues E85, K147, and N226 are essential for the rotation of the +1 sugar in the substrate to facilitate the intramolecular attack of oxygen O1' to the anomeric carbon. Prompted by these results, we expressed and assayed the N226A variant. Enzyme activity data confirm an important role for N226 during the cyclization step but show that it is dispensable for the hydrolysis step.

The intracellular polymerization of synthetic macromolecules presents a unique method for modifying cell behavior, enabling real‐time imaging, and enhancing therapeutic effectiveness. However, most current strategies rely on external catalysts or non‐physiological triggers, often lacking target‐cell specificity. Here, we report a cancer‐selective intracellular cyclodimerization cascade polymerization driven solely by endogenous acrolein, an oncometabolite overproduced in malignant cells. Acrolein plays dual roles as a polymerization initiator through imine formation and as a structural component of the resulting polymer. We designed a meta ‐phenylene‐bis(2‐aminoethanol) monomer that incorporates an aggregation‐induced emission (AIE)‐active tetraphenylethylene unit. In acrolein‐rich cancer cells, condensation between the aminoethanol groups and acrolein generates imines, which then undergo a spontaneous, catalyst‐free cyclodimerization cascade, yielding eight‐membered 1,5‐diazacyclooctane polymers that embed native acrolein. This polymerization triggers robust AIE fluorescence “turn‐on”, facilitating high‐contrast imaging of malignant cells with minimal background in healthy cells. We demonstrated selective fluorescence in multiple cancer cell lines and applied this platform to freshly resected human breast tumor samples, showing its utility for rapid intraoperative assessment. This strategy establishes a versatile toolkit for precision diagnostics, engineered intracellular materials, and next‐generation therapeutics.

The intracellular polymerization of synthetic macromolecules presents a unique method for modifying cell behavior, enabling real‐time imaging, and enhancing therapeutic effectiveness. However, most current strategies rely on external catalysts or non‐physiological triggers, often lacking target‐cell specificity. Here, we report a cancer‐selective intracellular cyclodimerization cascade polymerization driven solely by endogenous acrolein, an oncometabolite overproduced in malignant cells. Acrolein plays dual roles as a polymerization initiator through imine formation and as a structural component of the resulting polymer. We designed a meta ‐phenylene‐bis(2‐aminoethanol) monomer that incorporates an aggregation‐induced emission (AIE)‐active tetraphenylethylene unit. In acrolein‐rich cancer cells, condensation between the aminoethanol groups and acrolein generates imines, which then undergo a spontaneous, catalyst‐free cyclodimerization cascade, yielding eight‐membered 1,5‐diazacyclooctane polymers that embed native acrolein. This polymerization triggers robust AIE fluorescence “turn‐on”, facilitating high‐contrast imaging of malignant cells with minimal background in healthy cells. We demonstrated selective fluorescence in multiple cancer cell lines and applied this platform to freshly resected human breast tumor samples, showing its utility for rapid intraoperative assessment. This strategy establishes a versatile toolkit for precision diagnostics, engineered intracellular materials, and next‐generation therapeutics.

The difructose dianhydride I synthase/hydrolase αFFase1 catalyzes the reversible transformation of inulobiose into difructose dianhydride I, a compound present in differ ent caramel compositions. The proposed reaction mechanism of such a transformation consists of a glycosylation step followed by a cyclization to yield the dianhydride from the disaccharide. In response to the absence of mechanistical details for the reaction, we made use of extensive hybrid QM/MM metadynamics studies to shed light on the molecular basis of it. Our simulations show that the global reaction for the transfor mation of inulobiose by αFFase1 is slightly exothermic (∆G0 =–1.3 kcal mol−1) in good agreement with the experimental data. Furthermore, we observe that the glyco sylation step is the rate-limiting step of the reaction with an energy barrier of 12.3 kcal mol−1 compared to 8.7 kcal mol-−1 for the cyclization step. Interestingly, our work shows that the–1 sugar of inulobiose changes from an initial E5 conformation into a 4E puckering via the conformational pathway E5 → E5/4T5 → E3 → 4E. Our calculations highlight that in the cyclization step, the three residues E85, K147, and N226 are es sential for the rotation of the +1 sugar in the substrate to facilitate the intramolecular attack of oxygen O1’ to the anomeric carbon. Prompted by these results, we expressed and assayed the N226A αFFase1 variant. Kinetic data confirm an important role for N226 during the cyclization step but are irrelevant for the hydrolysis step.

Anomer-retaining glycoside hydrolases (GHs) generally catalyze a double displacement reaction via a covalent intermediate. However, neutron crystallography of glycoside ligand-bound states has not been performed. In this study, we investigated β-glucosidase Td2F2 from GH family 1 as a model enzyme for anomer-retaining GHs. We determined joint X-ray/neutron structures of Td2F2 in ligand-free form, covalent intermediate with a 2-deoxy-2-fluoro glucoside (2F-Glc) inhibitor, and glucose product complex using hydrogen/deuterium-exchanged crystals at room temperature, with neutron diffraction resolutions of 1.80–1.70 Å. Extensive hydrogen bonds recognizing the hydroxy groups of 2F-Glc were identified, along with the positions of deuterium atoms. The acid/base catalyst residue Glu166 was anchored by a hydrogen bond network pivoted by Asn293. Tyr295 forms a hydrogen bond with the catalytic nucleophile residue Glu352 in the ligand-free and glucose complex forms, while the active center undergoes significant reorganization, including side chain displacements of Glu352 and Tyr295, as well as the incorporation of a water molecule. An alternative conformation of Tyr295 was observed in the 2F-Glc structure at room temperature, suggesting its role in positioning the nucleophilic water during the deglycosylation step. Steady-state and pre-steady-state kinetic analyses of Y295F mutant supported the functional involvement of Tyr295 in both glycosylation and deglycosylation steps. The tyrosine hydrogen bonded to the nucleophile is also conserved in many other anomer-retaining GH families, underscoring its importance in catalysis. Based on the deuterium/hydrogen positions determined from neutron structures, we proposed a detailed reaction mechanism for Td2F2.

Anomer-retaining glycoside hydrolases (GHs) generally catalyze a double displacement reaction via a covalent intermediate; however, neutron crystallography of glycoside ligand-bound states has not been performed. In this study, we investigated β-glucosidase Td2F2 from GH family 1 as a model enzyme for anomer-retaining GHs. We determined joint X-ray/neutron structures of Td2F2 in ligand-free form, covalent intermediate with a 2-deoxy-2-fluoro glucoside (2F-Glc) inhibitor, and glucose product complex using hydrogen/deuterium-exchanged crystals at room temperature, with neutron diffraction resolutions of 1.80–1.70 Å. Extensive hydrogen bonds recognizing the hydroxy groups of 2F-Glc were identified, along with the positions of deuterium atoms. The acid/base catalyst residue Glu166 was anchored by a hydrogen bond network pivoted by Asn293. Tyr295 forms a hydrogen bond with the catalytic nucleophile residue Glu352 in the ligand-free and glucose complex forms, while the active center undergoes significant reorganization, including side chain displacements of Glu352 and Tyr295, as well as the incorporation of a water molecule. An alternative conformation of Tyr295 was observed in the 2F-Glc structure at room temperature, suggesting its role in positioning the nucleophilic water during the deglycosylation step. The tyrosine hydrogen bonded to the nucleophile is also conserved in many other anomer-retaining GH families, underscoring its importance in catalysis. Based on the deuterium/hydrogen positions determined from neutron structures, we proposed a detailed reaction mechanism for Td2F2. Significance Statement Glycoside hydrolases perform diverse functions in organisms, with over 180 known enzyme families. Although hydrogen bonds and proton transfer play important roles in enzymatic reactions, hydrogen atoms are generally invisible in macromolecular X-ray crystallography. In anomer-retaining glycoside hydrolases, general acid/base catalysis and the formation and hydrolysis of a covalent glycosyl-enzyme intermediate have been postulated. Here, we report neutron crystal structures of a β-glucosidase, where hydrogen and deuterium atoms were visualized at high resolution. An intricate hydrogen-bonding network and remarkable remodeling at the catalytic center were observed during covalent intermediate formation, revealing a detailed catalytic mechanism. The enzyme belongs to glycoside hydrolase family 1 and represents numerous enzymes employing the anomer-retaining mechanism.

An efficient and versatile glycosylation methodology is crucial for the systematic synthesis of oligosaccharides and glycoconjugates. A direct intermolecular and an indirect intramolecular methodology have been developed, and the former can be applied to the synthesis of medium-to-long-chain glycans like that of nucleotides and peptides. The development of a generally applicable approach for the stereoselective construction of glycosidic bonds remains a major challenge, especially for the synthesis of 1,2-cis glycosides such as β-mannosides, β-L-rhamnosides, and β-D-arabinofuranosides with equatorial glycosidic bonds as well as α-D-glucosides with axial ones. This review introduces the direct formation of cis-glycosides using ZnI₂-mediated cis-glycosylations of various constrained glycosyl donors, as well as the recent advances in the development of stereoselective cis-glycosylations.