state B and subsequent nucleophilic attack from nearby tyrosine residues results in a sialic acid-sialidase complex C. This transient complex then undergoes a hydrolysis reaction via backside attack to produce sialic acid, which retains the original stereochemistry, completing the catalytic cycle. The F-containing monosaccharide derivative 2 is also recognized as a substrate by sialidase, but the F-atom at C-3 acts an electronegative group that destabilizes the cationic intermediate or transition state, relatively stabilizes the sialidase-sialic acid complex, and retards further hydrolysis, leading to irreversible inhibition of sialidase activity. Therefore, compound 2 acts as an MBI. However, the F-atom at C-3 also slows down the first step, the cleavage reaction of the substituent at C-2, so a good leaving group is necessary at this position. Based on this putative mechanism, the introduction of an F-atom at the C-3 position of sialoglycan, such as sialylgalactose disaccharide, should obtain a sialoglycan analogue that would be exo-sialidase-resistant due to the lack of leaving group ability. Namely, these molecules would not act as MBIs in the same manner as monosaccharide derivatives. Thus, we needed to develop a new molecular design strategy for exo-sialidase MBIs with disaccharide or oligosaccharide-type substrate structures.Finally, we designed a novel type of MBIs for exo-sialidases. The concept is very simple, and just involves the introduction of an exomethylene group next to the anomeric position, i.e., 3-exoSia-containing pseudo-glycans 3 (Figure 2B). If 3 act as a substrate for exo-sialidase, the glycan can probably be cleaved Figure 2.A) Mechanism of sialic acid cleavage reaction by exo-sialidase and structure of MBI (2)B) a designed MBI (3) and expected mechanism of covalent bond formation with exo-sialidase by 3.through a similar transition state E to provide the complex D. If this hydrolysis reaction proceeds, this molecule is just a substrate. However, the putative cationic transition state E has electrophilicity not only at the anomeric position but also at the terminal of the exomethylene group. If an appropriate nucleophilic amino acid is present, it could form a covalent bond at the exomethylene group, leading to the more stable complex F and causing irreversible sialidase inhibition.Synthesis of 3-exoSia-containing pseudo-glycans was challenging. Eventually, we succeeded in the synthesis of 2,6- or 2,3-sialylgalactose derivatives (4 and 5, Figure 3A) with exomethylene functionality by employing the H-bond-mediated aglycon delivery strategy developed by Prof. Demichenko’s group, together with glycosylation using an Au-catalytic system, as developed by Prof. Yu’s group. We also prepared the BODIPY-labeled molecule 6 (Figure 3B) based on the 2,6-sialylgalactose analogue.We first confirmed that the BODIPY-labeled probe was actually covalently bound the active site of the sialidase, as we expected. Namely, treatment of the BODIPY probe 6 with exo-sialidase NanI (from C. welchii) followed by SDS-PAGE resulted in the generation of a sialidase band on the gel in a concentration- and time-dependent manner, as determined by fluorescence detection. The corresponding sialidase band was remarkably diminished in the presence of a large excess of the competitive inhibitor DANA (7, Figure 3A). Furthermore, covalent bond 123To be published at a later date.
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