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A new method for the introduction of several substituents in 1-hydroxyimidazole after the imidazole ring was formed is presented. The new approach was based on deprotonation which was followed by reaction with an electrophile. Debenzylation yielded 2-substituted 1-hydroxyimidazoles which could be deoxygenated to generate 2-substituted imidazoles. Results indicate that the new method is seemingly better than the previous approaches used for the preparation of 1-(benzyloxy)imidazole.

The synthesis of oligosaccharide mimicking the O-antigen motif of Shigella flexineri serotypes 1b and 3a is described, which is used for developing multivalent carbohydrate-based vaccines. The variants are used for preparing glucosylated building blocks and they are converted into acceptors and potential donors compatible with chemical elongation toward oligosaccharide fragments of the O-antigens of the two targeted serotypes.

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 0-antigen motif of Shigella flexneriserotypes 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 α-L-rhamnopyranoside or a tremendously enhanced one toward allyl 2-acetamido-2-deoxy-α-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 α-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. [ABSTRACT FROM AUTHOR]

Two new pale yellow metabolites have been isolated from commercially available Chinese food additive Red Monascus Pigment and from Monascus ruber culture broth. They were isolated by successive TLC and semipreparative HPLC. Their structural characterization was elucidated by a variety of spectroscopic techniques (UV, IR, NMR) and mass spectrometry. These two new metabolites present numerous similarities with monascorubrin and rubropunctatin, differing in their structure only by the absence of the lactone ring. High-resolution mass spectrometry indicated the molecular formulas C(20)H(26)O(4) and C(22)H(30)O(4). The new compounds, named monarubrin and rubropunctin, contain a propenyl group on a pyrone ring, an alkyl side chain, but no gamma-lactone ring. The new metabolites have the property of producing strong blue fluorescence at 340 nm.

The distribution and diversity of natural exopolysaccharides (EPS) produced from sucrose by thirty heterofermentative lactic acid bacteria strains from French traditional sourdoughs was investigated. The EPS production was found to be related to glucansucrase and fructansucrase extracellular activities. Depending on the strain, soluble and/or cell-associated glycansucrases were secreted. Structural characterization of the polymers by 1H and 13C NMR spectroscopy analysis further demonstrated a high diversity of EPS structures. Notably, we detected strains that synthesize glucans showing amazing variations in the amount of alpha-(1-->2), alpha-(1-->3) and alpha-(1-->6) linkages. The representation of Leuconostoc strains which produce putative alternan polymers and alpha-(1-->2) branched polymers was particularly high. The existence of glucan- and fructansucrase encoding genes was also confirmed by PCR detection. Sourdough was thus demonstrated to be a very attractive biotope for the isolation of lactic acid bacteria producing novel polymers which could find interesting applications such as texturing agent or prebiotics.

A number of 1-hydroxyazole derivatives were synthesized as bioisosteres of (S)-glutamic acid (Glu) and as analogues of the AMPA receptor agonist (R,S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA, 3b). All compounds were subjected to in vitro pharmacological studies, including a series of Glu receptor binding assays, uptake studies on native as well as cloned Glu uptake systems, and the electrophysiological rat cortical slice model. Compounds 7a,b, analogues of AMPA bearing a 1-hydroxy-5-pyrazolyl moiety as the distal carboxylic functionality, showed only moderate affinity for [3H]AMPA receptor binding sites (IC(50) = 2.7 +/- 0.4 microM and IC(50) = 2.6 +/- 0.6 microM, respectively), correlating with electrophysiological data from the rat cortical wedge model (EC(50) = 280 +/- 48 microM and EC(50) = 586 +/- 41 microM, respectively). 1-Hydroxy-1,2,3-triazol-5-yl analogues of AMPA, compounds 8a,b, showed high affinity for [3H]AMPA receptor binding sites (IC(50) = 0.15 +/- 0.03 microM and IC(50) = 0.13 +/- 0.02 microM, respectively). Electrophysiological data showed that compound 8a was devoid of activity in the rat cortical wedge model (EC(50) > 1000 microM), whereas the corresponding 4-methyl analogue 8b was a potent AMPA receptor agonist (EC(50) = 15 +/- 2 microM). In accordance with this disparity, compound 8a was found to inhibit synaptosomal [3H]D-aspartic acid uptake (IC(50) = 93 +/- 25 microM), as well as excitatory amino acid transporters (EAATs) EAAT1 (IC(50) = 100 +/- 30 microM) and EAAT2 (IC(50) = 300 +/- 80 microM). By contrast, compound 8b showed no appreciable affinity for Glu uptake sites, neither synaptosomal nor cloned. Compounds 9a-c and 10a,b, possessing 1-hydroxyimidazole as the terminal acidic function, were devoid of activity in all of the systems tested. Protolytic properties of compounds 7a,b, 8b, and 9b were determined by titration, and a correlation between the pK(a) values and the activity at AMPA receptors was apparent. Optimized structures of all the synthesized ligands were fitted to the known crystal structure of an AMPA-GluR2 construct. Where substantial reduction or abolition of affinity at AMPA receptors was observed, this could be rationalized on the basis of the ability of the ligand to fit the construct. The results presented in this article point to the utility of 1-hydroxypyrazole and 1,2,3-hydroxytriazole as bioisosteres of carboxylic acids at Glu receptors and transporters. None of the compounds showed significant activity at metabotropic Glu receptors.