Hydrolysis Of Glycosidic Linkages Research Articles

Isomerization of 6-O-substituted glucose and fructose under neutral pH conditions and subsequent β-elimination reactions

Isomaltose (6-O-α-d-glucopyranosyl-d-glucose) and isomaltulose (palatinose; 6-O-β-d-glucopyranosyl-d-fructose) were heated to 90 °C in 100 mM sodium phosphate buffer (pH 7.5). Aldose–ketose isomerization between isomaltose, isomaltulose, and epi-isomaltulose was observed in the early stage of the reaction, alongside the release of a small amount of glucose. The total concentration of these disaccharides gradually decreased as the heating time increased. However, this decrease did not correlate with the amount of glucose or fructose released, suggesting that the releases of these monosaccharides were not caused by the hydrolysis of glycosidic linkages. A slight decrease in the pH of the reaction solution was attributed to the formation of two organic acids, 6-O-β-d-glucopyranosyl-3-deoxy-d-arabino-hexonic acid (1) and 6-O-β-d-glucopyranosyl-3-deoxy-d-ribo-hexonic acid (2). These compounds were formed from the β-elimination of the hydroxyl group at the C-3 of fructose, leaving a substituted glucose residue at the C-6 position, followed by keto–enol tautomerization and benzilic acid rearrangement. Although approximately 30% of 1 and 2 were degraded after 360 min of heating at 90 °C in 100 mM sodium phosphate, a little release of glucose was observed, indicating no hydrolysis of the glucoside bond at C-6. Besides 1 and 2, time-dependent changes in the NMR spectra of the reaction mixture in water indicated the formation of formic acid and the presence of species possibly resulting from the β-elimination of the hydroxyl group from 3- and 4-ulose. The glucose released by heating isomaltose and isomaltulose may be generated via tautomerizations of keto-enols between the C-4 and C-5 positions and cleavage of 6-O-glycosidic linkage via β-elimination.

α-Galactobiosyl units: thermodynamics and kinetics of their formation by transglycosylations catalysed by the GH36 α-galactosidase from Thermotoga maritima

Broad regioselectivity of α-galactosidase from Thermotoga maritima (TmGal36A) is a limiting factor for application of the enzyme in the directed synthesis of oligogalactosides. However, this property can be used as a convenient tool in studies of thermodynamics of a glycosidic bond. Here, a novel approach to energy difference estimation is suggested. Both transglycosylation and hydrolysis of three types of galactosidic linkages were investigated using total kinetics of formation and hydrolysis of pNP-galactobiosides catalysed by monomeric glycoside hydrolase family 36 α-galactosidase from T. maritima, a retaining exo-acting glycoside hydrolase. We have estimated transition state free energy differences between the 1,2- and 1,3-linkage (ΔΔG‡0 values were equal 5.34±0.85kJ/mol) and between 1,6-linkage and 1,3-linkage (ΔΔG‡0=1.46±0.23kJ/mol) in pNP-galactobiosides over the course of the reaction catalysed by TmGal36A. Using the free energy difference for formation and hydrolysis of glycosidic linkages (ΔΔG‡F−ΔΔG‡H), we found that the 1,2-linkage was 2.93±0.47kJ/mol higher in free energy than the 1,3-linkage, and the 1,6-linkage 4.44±0.71kJ/mol lower.

Detection of a covalent intermediate in the mechanism of action of porcine pancreatic alpha-amylase by using 13C nuclear magnetic resonance.

The catalytic mechanism of porcine pancreatic alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) has been examined by nuclear magnetic resonance (NMR) at subzero temperatures by using [1-13C]maltotetraose as substrate. Spectral summation and difference techniques revealed a broad resonance peak, whose chemical shift, relative signal intensity and time-course appearance corresponded to a beta-carboxyl-acetal ester covalent enzyme-glycosyl intermediate. This evidence supports a double-displacement covalent mechanism for porcine pancreatic alpha-amylase-catalyzed hydrolysis of glycosidic linkages, based on the presence of catalytic aspartic acid residues within the active site of this enzyme.

Mechanism of ozone attack on α‐methyl glucoside and cellulosic materials

AbstractOzone attack on cellulose and related substances was investigated and appears to involve a twofold mechanism. One is a free‐radical chain mechanism involving oxygen in the propagating step. This slow reaction is highly indiscriminate, showing very little, if any, specificity as to the site of attack and resulting in the formation of peroxide, carbonyl, carboxyl, and presumably lactone groups. In the presence of oxygen, kinetic chain lengths of over 100 were measured, but in nitrogen atmosphere the total oxidation of substrate was equivalent to the amount of ozone consumed. The second process appears to be an electrophilic attack which liberates the anomeric carbon of glycosides via an ozone‐catalyzed hydrolysis of glycosidic linkages. This reaction, analogous to a nitronium or chloronium ion‐ or proton‐catalyzed hydrolysis, is postulated since chain degradation of polysaccharides is equally severe in the presence or absence of oxygen, and occurs in buffered solutions or suspensions even in the neutral pH range, and because the main product of ozone attack on α‐methyl glucoside is glucose. Brightness improvement in cellulose involves an electrophilic attack on double bonds and therefore is a direct function of the quantity of ozone used, like chain degradation. It is therefore not surprising that we have found no method of enhancing the specificity of ozone attack on the colored material relative to chain degradation. Because of the topochemical and diffusion‐controlled character of the reaction on cellulose, the use of a different temperature range will cause only a trivial difference in the relative rates of attack.