Synthesis, nitric oxide release, and α-glucosidase inhibition of nitric oxide donating apigenin and chrysin derivatives.

α-Glucosidase (AG) play crucial roles in the digestion of carbohydrates. Inhibitors of α-glucosidase (AGIs) are promising candidates for the development of anti-diabetic drugs. Here, five series of apigenin and chrysin nitric oxide (NO)-donating derivatives were synthesised and evaluated for their AG inhibitory activity and NO releasing capacity in vitro. Except for 9a-c, twelve compounds showed remarkable inhibitory activity against α-glucosidase, with potency being better than that of acarbose and 1-deoxynojirimycin. All organic nitrate derivatives released low concentrations of NO in the presence of l-cysteine. Structure activity relationship studies indicated that 5-OH, hydrophobic coupling chain, and carbonyl groups of the coupling chain could enhance the inhibitory activity. Apigenin and chrysin derivatives therefore represents a new class of promising compounds that can inhibit α-glucosidase activity and supply moderate NO for preventing the development of diabetic complications.

Probing the intestinal α-glucosidase enzyme specificities of starch-digesting maltase-glucoamylase and sucrase-isomaltase: synthesis and inhibitory properties of 3'- and 5'-maltose-extended de-O-sulfonated ponkoranol.

The synthesis and glucosidase inhibitory activities of two C-3'- and C-5'-ß-maltose-extended analogues of the naturally occurring sulfonium-ion inhibitor, de-O-sulfonated ponkoranol, are described. The compounds are designed to test the specificity towards four intestinal glycoside hydrolase family 31 (GH31) enzyme activities, responsible for the hydrolysis of terminal starch products and sugars into glucose, in humans. The target sulfonium-ion compounds were synthesized by means of nucleophilic attack of benzyl protected 1,4-anhydro-4-thio-D-arabinitol at the C-6 position of 6-O-trifluoromethanesulfonyl trisaccharides as alkylating agents. The alkylating agents were synthesized from D-glucose by glycosylation at C-4 or C-2 with maltosyl trichloroacetimidate. Deprotection of the coupled products by using a two-step sequence, followed by reduction afforded the final compounds. Evaluation of the target compounds for inhibition of the four glucosidase activities indicated that selective inhibition of one enzyme over the others is possible.

Convergent synthesis of homogeneous Glc1Man9GlcNAc2-protein and derivatives as ligands of molecular chaperones in protein quality control.

A detailed understanding of the molecular mechanism of chaperone-assisted protein quality control is often hampered by the lack of well-defined homogeneous glycoprotein probes. We describe here a highly convergent chemoenzymatic synthesis of the monoglucosylated glycoforms of bovine ribonuclease (RNase) as specific ligands of lectin-like chaperones calnexin (CNX) and calreticulin (CRT) that are known to recognize the monoglucosylated high-mannose oligosaccharide component of glycoproteins in protein folding. The synthesis of a selectively modified glycoform Gal(1)Glc(1)Man(9)GlcNAc(2)-RNase was accomplished by chemical synthesis of a large N-glycan oxazoline and its subsequent enzymatic ligation to GlcNAc-RNase under the catalysis of a glycosynthase. Selective removal of the terminal galactose by a ß-galactosidase gave the Glc(1)Man(9)GlcNAc(2)-RNase glycoform in excellent yield. CD spectroscopic analysis and RNA-hydrolyzing assay indicated that the synthetic RNase glycoforms maintained essentially the same global conformations and were fully active as the natural bovine ribonuclease B. SPR binding studies revealed that the Glc(1)Man(9)GlcNAc(2)-RNase had high affinity to lectin CRT, while the synthetic Man(9)GlcNAc(2)-RNase glycoform and natural RNase B did not show CRT-binding activity. These results confirmed the essential role of the glucose moiety in the chaperone molecular recognition. Interestingly, the galactose-masked glycoform Gal(1)Glc(1)Man(9)GlcNAc(2)-RNase also showed significant affinity to lectin CRT, suggesting that a galactose ß-1,4-linked to the key glucose moiety does not significantly block the lectin binding. These synthetic homogeneous glycoprotein probes should be valuable for a detailed mechanistic study on how molecular chaperones work in concert to distinguish between misfolded and folded glycoproteins in the protein quality control cycle.

Sulfatase-catalyzed assembly of regioselectively O-sulfonated p-nitrophenyl alpha-D-gluco- and alpha-D-mannopyranosides.

A chemoenzymic methodology is extended to the library synthesis of regioselectively O-sulfonated pNP D-gluco and D-mannopyranosides. The method involves the sequential reactions of chemical O-sulfonation and sulfatase-catalyzed O-desulfonation. pNP 2,6-di-O-sulfo-alpha-D-glucopyranoside and pNP 3,6-di-O-sulfo-alpha-D-mannopyranoside were obtained as sodium salts using chemical methods by way of dibutylstannylene acetals or tributylstannyl ethers. They were then applied to enzyme reactions using three molluscan enzymes (snail, limpet, and abalone). The sulfatase reactions cleaved a sulfate group at the secondary O-2 or O-3 position to yield the corresponding pNP 6-O-sulfo sugars. Neither pNP 6-O-sulfo-alpha-D-glucopyranoside nor 6-O-sulfo-alpha-D-mannopyranoside became the enzyme substrate. Evidently, the molluscan sulfatases have a tendency to cleave the secondary O-sulfo group with assistance from the 6-O-sulfo group.

Production of intracellular gamma-glucosidase in brewery waste by: saccharomyces carlsbergensis

Saccharomyces carlsbergensis strain ATCC 9080 was grown on acid hidrolyzed brewery waste. The sustrate contains 22 mg/ml of total carbohydrates of which approximately 60% represents reducing sugars such as maltose and glucose. Optimal conditions for substrate hydrolysis were 1.5% w/v sulfuric acid, 20 minutes and temperature of 110 grados C. Maximum enzyme production was obtained in a medium containing 0.05% w/v yeast extract at 35 grado C and at an initial pH of 6. Addition of nitrogen sources was unnecessary. Optimal conditions for gamma-glucosidase extraction from yeast included pH 7.5, 30 grados C, and 10 minutes of sonication. Maximum hydrolysis of PNPG occured at pH 7.5, temperature between 30 grados C and 25 minutes of incubation. Michaelis-Menten constants and Vmax were 1 mM, 0.56 mol/min/mg of protein on P-nitrophenly-gamma-D-glucophyranoside and 5 mV, 5 mol/min/mg of protein on maltose respectively