Synthesis of N4-aryl-β-d-glucopyranosylcytosines: a methodology study

A number of leaving groups, including arylsulfonates, triazoles, 3-nitrotriazoles, and tetrazoles, have been studied for the substitution reaction by aryl and alkyl amines at the 4-position of β-d-glucopyranosyluracils. Examination of the stability, ease of purification and reactivity in the substitution reaction led to a number of optimized conditions with the most convenient involving substitution of triazole derivatives under microwave conditions in the presence of silica gel. Under these conditions, a number of N4-aryl-substituted β-d-glucopyranosylcytosines were prepared as potential inhibitors of glycogen phosphorylase, a molecular target for type-2 diabetes mellitus

EstDZ3:a new esterolytic enzyme exhibiting remarkable thermostability

Lipolytic enzymes that retain high levels of catalytic activity when exposed to a variety of denaturing conditions are of high importance for a number of biotechnological applications. In this study, we aimed to identify new lipolytic enzymes, which are highly resistant to prolonged exposure at elevated temperatures. To achieve this, we searched for genes encoding for such proteins in the genomes of a microbial consortium residing in a hot spring located in China. After performing a functional genomic screening on a bacterium of the genus Dictyoglomus, which was isolated from this hot spring after in situ enrichment, we identified a new esterolytic enzyme, termed EstDZ3. Detailed biochemical characterization of the recombinant enzyme, revealed that it constitutes a slightly alkalophilic and highly active esterase against esters of fatty acids with short to medium chain lengths. Importantly, EstDZ3 exhibits remarkable thermostability, as it retained high levels of catalytic activity after exposure to temperatures as high as 95 oC for several hours. Interestingly, EstDZ3 was found to have very little similarity to previously characterized esterolytic enzymes. Computational modelling of the three-dimensional structure of this new enzyme predicted that it exhibits a typical α/β hydrolase fold, which seems to include a subdomain insertion. This insertion is similar to the one present in its closest homologue of known function and structure, the cinnamoyl esterase Lj0536 from Lactobacillus johnsonii. As it was found in the case of Lj0536, this structural feature is expected to be an important determinant of the catalytic properties of EstDZ3. The high levels of esterolytic activity of EstDZ3, combined with its remarkable thermostability and good stability against a wide range of metal ions, organic solvents, and other denaturing agents, render this new enzyme a candidate biocatalyst for high-temperature biotechnological applications

EstDZ3: A New Esterolytic Enzyme Exhibiting Remarkable Thermostability

Lipolytic enzymes that retain high levels of catalytic activity when exposed to a variety of denaturing conditions are of high importance for a number of biotechnological applications. In this study, we aimed to identify new lipolytic enzymes, which are highly resistant to prolonged exposure at elevated temperatures. To achieve this, we searched for genes encoding for such proteins in the genomes of a microbial consortium residing in a hot spring located in China. After performing a functional genomic screening on a bacterium of the genus Dictyoglomus, which was isolated from this hot spring after in situ enrichment, we identified a new esterolytic enzyme, termed EstDZ3. Detailed biochemical characterization of the recombinant enzyme, revealed that it constitutes a slightly alkalophilic and highly active esterase against esters of fatty acids with short to medium chain lengths. Importantly, EstDZ3 exhibits remarkable thermostability, as it retained high levels of catalytic activity after exposure to temperatures as high as 95 oC for several hours. Interestingly, EstDZ3 was found to have very little similarity to previously characterized esterolytic enzymes. Computational modelling of the three-dimensional structure of this new enzyme predicted that it exhibits a typical α/β hydrolase fold, which seems to include a subdomain insertion. This insertion is similar to the one present in its closest homologue of known function and structure, the cinnamoyl esterase Lj0536 from Lactobacillus johnsonii. As it was found in the case of Lj0536, this structural feature is expected to be an important determinant of the catalytic properties of EstDZ3. The high levels of esterolytic activity of EstDZ3, combined with its remarkable thermostability and good stability against a wide range of metal ions, organic solvents, and other denaturing agents, render this new enzyme a candidate biocatalyst for high-temperature biotechnological applications

Glucose-derived spiro-isoxazolines are anti-hyperglycemic agents against type 2 diabetes through glycogen phosphorylase inhibition

International audienceGlycogen phosphorylase (GP) is a target for the treatment of hyperglycaemia in the context of type 2 diabetes. This enzyme is responsible for the depolymerization of glycogen into glucose thereby affecting the levels of glucose in the blood stream. Twelve new d-glucopyranosylidene-spiro-isoxazolines have been prepared from O-peracylated exo-D-glucals by regio- and stereoselective 1,3-dipolar cycloaddition of nitrile oxides generated in situ by treatment of the corresponding oximes with bleach. This mild and direct procedure appeared to be applicable to a broad range of substrates. The corresponding O-unprotected spiro-isoxazolines were evaluated as glycogen phosphorylase (GP) inhibitors and exhibited IC50 values ranging from 1 to 800 μM. Selected inhibitors were further evaluated in vitro using rat and human hepatocytes and exhibited significant inhibitory properties in the primary cell culture. Interestingly, when tested with human hepatocytes, the tetra-O-acetylated spiro-isoxazoline bearing a 2-naphthyl residue showed a much lower IC50 value (2.5 μM), compared to that of the O-unprotected analog (19.95 μM). The most promising compounds were investigated in Zucker fa/fa rat model in acute and sub-chronic assays and decreased hepatic glucose production, which is known to be elevated in type 2 diabetes. This indicates that glucose-based spiro-isoxazolines can be considered as anti-hyperglycemic agents in the context of type 2 diabetes

{Anomeric Spironucleosides of β\beta-d-Glucopyranosyl Uracil as Potential Inhibitors of Glycogen Phosphorylase}

In the case of type 2 diabetes, inhibitors of glycogen phosphorylase (GP) may prevent unwanted glycogenolysis under high glucose conditions and thus aim at the reduction of excessive glucose production by the liver. Anomeric spironucleosides, such as hydantocidin, present a rich synthetic chemistry and important biological function (e.g., inhibition of GP). For this study, the Su{\'}rez radical methodology was successfully applied to synthesize the first example of a 1,6-dioxa-4-azaspiro[4.5]decane system, not previously constructed via a radical pathway, starting from 6-hydroxymethyl-$\beta$-d-glucopyranosyluracil. It was shown that, in the rigid pyranosyl conformation, the required [1,5]-radical translocation was a minor process. The stereochemistry of the spirocycles obtained was unequivocally determined based on the chemical shifts of key sugar protons in the 1H-NMR spectra. The two spirocycles were found to be modest inhibitors of RMGPb

{Synthesis of N4-aryl-β\beta-d-glucopyranosylcytosines: a methodology study}

{\textcopyright} 2015 Elsevier Ltd. All rights reserved.A number of leaving groups, including arylsulfonates, triazoles, 3-nitrotriazoles, and tetrazoles, have been studied for the substitution reaction by aryl and alkyl amines at the 4-position of $\beta$-D-glucopyranosyluracils. Examination of the stability, ease of purification and reactivity in the substitution reaction led to a number of optimized conditions with the most convenient involving substitution of triazole derivatives under microwave conditions in the presence of silica gel. Under these conditions, a number of N4-aryl-substituted $\beta$-D-glucopyranosylcytosines were prepared as potential inhibitors of glycogen phosphorylase, a molecular target for type-2 diabetes mellitus