Our laboratory is focusing on the development of pseudo-glycans, which we define as glycan or glycoconjugate analogues with very small structural modifications that enhance the original function of the parent molecule in cellulo/in vivo or result in the acquisition of a new function. One of our important achievements is the development of metabolically-stable analogues of glycolipid ganglioside GM3. We designed analogues in which the O-glycosidic linkage of sialic acid is replaced by a C-glycosidic linkage, so that the compounds cannot be degraded by glycohydrolases (or glycoside hydrolases, GH). We developed a CHF-glycoside of sialic acid, which has an F atom at the C-glycoside bond, in order to control the flexible glycan conformation as well as to mimic the properties of the original O-glycoside. Synthesis of a series of GM3 analogues with C-glycoside linkages demonstrated that CHF-linked analogues showed higher biological activity than the corresponding CF2- or CH2-linked analogues or native GM1). We also found that the (S)-CHF-linked 1 was more effective than the (R)-CHF-linked analogue, benefiting from conformational regulation by the F-atom (Figure 1). Based on these findings, we are now trying to develop GH-resistant analogues of other glycans and glycoconjugates, using our own direct C-glycosylation methodologies2-4).Cells maintain their biological activities through a variety of Figure 1. Development of (S)-CHF-linked GM3 analogue (1) as an example of a pseudo-glycanorganic chemical reactions. Previous studies have provided insight into the production and degradation (metabolism) of various biological substances, but glycoconjugates present particular difficulty in functional analysis due to their structural diversity. One of the enzymes responsible for the degradation of complex carbohydrates is GH, which not only recycles monosaccharides from glycans, but also induces changes in glycoconjugate function that contribute to intracellular signal transduction. In most cases, identification of specific substrates for a certain glycoconjugate has been performed by preparing purified GH and examining whether prepared target substrates are degraded in vitro, but the results of studies at the enzyme level alone are often insufficient to provide an understanding of biological phenomena in cells. However, a method to analyze "which enzyme" performs "degradation of specific glycans or glycoconjugates" at the cellular level remains unestablished. It is difficult to probe this simple degradation process, which is fundamental to biological activity, using existing technology, and indeed, most biochemistry textbooks say little about glycan degradation by GH.Under these circumstances, we envisioned that pseudo-glycans would be useful to probe the degradation of specific glycans or glycoconjugates at the cellular level. In order to realize this idea, we simply need to synthesize pseudo-glycan molecules that can form covalent bonds with GH only after cleavage by GH. Molecules that do this are called mechanism-based inhibitors (MBIs) or suicide substrates. Here, we use exo-sialidases, which cleave sialic acid located on the non-reducing ends in glycoconjugates, as an example. Most MBIs for exo-sialidases possess a sialic acid monosaccharide structure 2 (Figure 2A) with F-atoms at the C2 and C3 positions. We considered that the mechanism of their hydrolytic cleavage by exo-sialidases would be as follows (Figure 2A). The terminal sialic acid has a typical 2C5 conformation, which is recognized by sialidases as a substrate, giving the Michaelis complex A. At this stage, the substituent at the anomeric position to be cleaved is considered to adopt a pseudo-axial orientation by changing to 4S2 conformation. Cleavage of the O-glycoside bond occurs through a 4H5-type transition 122To be published at a later date.Development of novel mechanism-based inhibitors for glycohydrolasesGo HiraiGraduate School of Pharmaceutical Sciences, Kyushu University
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