Synthesis and biological evaluation of novel C-aryl d-glucofuranosides as sodium-dependent glucose co-transporter 2 inhibitors.

Novel C-aryl-d-glucofuranosides were synthesized and evaluated for their capacity to inhibit human sodium-dependent glucose co-transporter 2 (hSGLT2) and hSGLT1. Compound 21q demonstrated the best in vitro inhibitory activity against SGLT2 in this series (EC50=0.62µM).

Design of alpha-transglucosidases of controlled specificity for programmed chemoenzymatic synthesis of antigenic oligosaccharides.

Combined with chemical synthesis, the use of biocatalysts holds great potential to open the way to novel molecular diversity. We report in vitro chemoenzymatic pathways that, for the first time, take advantage of enzyme engineering to produce complex microbial cell-surface oligosaccharides and circumvent the chemical boundaries of glycochemistry. Glycoenzymes were designed to act on nonnatural conveniently protected substrates to produce intermediates compatible with a programmed chemical elongation. The study was focused on the synthesis of oligosaccharides mimicking the O-antigen motif of Shigella flexneri serotypes 1b and 3a, which could be used for the development of multivalent carbohydrate-based vaccines. A semirational engineering approach was successfully applied to amylosucrase, a transglucosidase that uses a low cost sucrose substrate as a glucosyl donor. The main difficulty was to retain the enzyme specificity toward sucrose, while creating a new catalytic function to render the enzyme able to regiospecifically glucosylate protected nonnatural acceptors. A structurally guided library of 133 mutants was generated from which several mutants with either completely new specificity toward methyl alpha-l-rhamnopyranoside or a tremendously enhanced one toward allyl 2-acetamido-2-deoxy-alpha-d-glucopyranoside acceptors were isolated. The best variants were used to synthesize glucosylated building blocks. They were then converted into acceptors and potential donors compatible with chemical elongation toward oligosaccharide fragments of the O-antigens of the two targeted serotypes. This is the first report of a successful engineering of an alpha-transglycosidase acceptor binding site that led to new specificities. It demonstrates the potential of appropriate combinations of a planned chemoenzymatic pathway and enzyme engineering in glycochemistry.

Enzymatic transformations in supersaturated substrate solutions: I. A general study with glycosidases.

The results of an initial study of enzymatic catalysis in metastable supersaturated solutions of carbohydrates are presented. It has been shown that such solutions, formed in the presence of small amounts of water and alcohol as plasticizers, are sufficiently stable under ambient conditions to enable enzymatic transformations of substrates. A partial phase diagram for a system consisting of glucose, water, and (poly)ethylene glycol was constructed to identify the regions which are most suitable for biotransformations. It was confirmed that the glass transition in this system occurred below the reaction temperature at any given composition of the constituent components. Several glycosidases were found to be catalytically active in this medium and the activity of beta-glucosidase from almond was determined at several compositions of the reaction mixture and related to the corresponding regions of the phase diagram. The synthetic utility of the system was illustrated by glucosylation of several alpha,omega-alkyldiols, short-chain polyethylene glycols, and hydroxyalkyl and glyceryl monoacrylates.

[Synthesis and properties of carrier-fixed enzymes. VII. Linking of glucoamylase to dialdehyde cellulose, carboxymethylcellulose hydrazide and carboxymethylcellulose azide]. / Synthese und Eigenschaften trägerfixierten Enzyme. VII. Fixierung von Glukoamylase an Dialdehydzellulose. Karboxymethylzellulosehydrazid und Karboxymethylzelluloseazid.

Glucoamylase (alpha-1,4-glucan glucohydrolase, EC 3.2.1.3) has been covalently linked to dialdehyde cellulose resulting in an immobilized enzyme containing 0.98% protein and an activity of 4.5 mg of the native enzyme per g of matrix, i.e. 46% relative activity. The complex lost its activity in continuous and batch hydrolysis of starch at 55 degrees C down to a limit of 18% of its original value. In contrast, the activity of the complex did not change when working at a temperature of 25 degrees C. Glucoamylase-carboxymethylcellulose complexes synthesized via carboxymethylcellulose hydrazide and azide, in contrast to MAEDA und SUZUKI [1], showed only an activity of 1 mg of the native enzyme per g of matrix. We did not succeed in coupling periodate-oxidized glucoamylase to carboxymethylcellulose hydrazide because the enzyme used lost nearly all of its activity already during periodate oxidation.