Michel Therisod

Ionic liquids as a new reaction medium for oxidase–peroxidase-catalyzed sulfoxidation

Abstract The possibility of the application of oxidase and peroxidase as catalysts in ionic liquids is demonstrated in the chemo- and stereoselective oxidation of sulfides. The high operational stability of these enzymes in ionic liquids is reported. The substrate of glucose oxidase (glucose) and the substrate of peroxidase (sulfide) are perfectly soluble.

Glycolytic and Non-glycolytic Functions of Mycobacterium tuberculosis Fructose-1,6-bisphosphate Aldolase, an Essential Enzyme Produced by Replicating and Non-replicating Bacilli

Background: New drugs active against persistent Mycobacterium tuberculosis are needed. Results: The fructose-1,6-bisphosphate aldolase (FBA-tb) is essential for growth of M. tuberculosis, is expressed by replicating and non-replicating bacilli, and displays plasminogen binding activity. Conclusion: FBA-tb is an essential TB enzyme that might also play a role in host/pathogen interactions. Significance: FBA-tb shows potential as a novel anti-TB therapeutic target. The search for antituberculosis drugs active against persistent bacilli has led to our interest in metallodependent class II fructose-1,6-bisphosphate aldolase (FBA-tb), a key enzyme of gluconeogenesis absent from mammalian cells. Knock-out experiments at the fba-tb locus indicated that this gene is required for the growth of Mycobacterium tuberculosis on gluconeogenetic substrates and in glucose-containing medium. Surface labeling and enzymatic activity measurements revealed that this enzyme was exported to the cell surface of M. tuberculosis and produced under various axenic growth conditions including oxygen depletion and hence by non-replicating bacilli. Importantly, FBA-tb was also produced in vivo in the lungs of infected guinea pigs and mice. FBA-tb bound human plasmin(ogen) and protected FBA-tb-bound plasmin from regulation by α2-antiplasmin, suggestive of an involvement of this enzyme in host/pathogen interactions. The crystal structures of FBA-tb in the native form and in complex with a hydroxamate substrate analog were determined to 2.35- and 1.9-Å resolution, respectively. Whereas inhibitor attachment had no effect on the plasminogen binding activity of FBA-tb, it competed with the natural substrate of the enzyme, fructose 1,6-bisphosphate, and substantiated a previously unknown reaction mechanism associated with metallodependent aldolases involving recruitment of the catalytic zinc ion by the substrate upon active site binding. Altogether, our results highlight the potential of FBA-tb as a novel therapeutic target against both replicating and non-replicating bacilli.

The biosynthesis of thiamine. Syntheses of [1,1,1,5-2H4]-1-deoxy-D-threo-2-pentulose and incorporation of this sugar in biosynthesis of thiazole by Escherichia coli cells

Non-growing, washed cells of Escherichia coli, depressed for the synthesis of thiamine, were incubated in the presence of [1,1,1,5-2H4]-1-deoxy-D-threo-2-pentulose (9) in a medium containing the pyrimidine moiety of thiamine, L-tyrosine, and glucose. The thiamine thus biosynthesized was extracted and cleaved to give 5-(2-hydroxyethyl)-4-methylthiazole (HET) which was examined as the trifluoroacetate derivative by electron-impact mass spectrometry. The distribution of the label in the fragments indicated that the pentulose (9) was a precursor of the C5-chain of HET without C–C bond cleavage. Several routes to 1-deoxypentuloses are described. Condensation of 2,4-O-benzylidene-D-[4-2H1]threose (23) with trideuteriomethylmagnesium iodide gave the protected 1-deoxypentitols (24) and (25). Brominolysis of the mixed dibutylstannylidenes then afforded [1,1,1,5-2H4]-3,5-O-benzylidene-1-deoxy-D-threo-2-pentulose (26), which was converted into the free sugar (9) by acidic hydrolysis. 1-Deoxy-D-erythro-2-pentulose was prepared in similar manner. Condensation of 2-([2H3]-methyl)-1,3-dithian with 2,3-O-isopropylidene-D-glyceraldehyde, followed by a C-3 epimerization step also led, after deprotection, to a mixture of [1,1,1-2H3]-1-deoxy-D-erythro- and [1,1,1-2H3]-1-deoxy-D-threo-2-pentulose, (5) and (6).

Rational Design, Synthesis, and Evaluation of New Selective Inhibitors of Microbial Class II (Zinc Dependent) Fructose Bis-phosphate Aldolases.

We report the synthesis and biochemical evaluation of several selective inhibitors of class II (zinc dependent) fructose bis-phosphate aldolases (Fba). The products were designed as transition-state analogues of the catalyzed reaction, structurally related to the substrate fructose bis-phosphate (or sedoheptulose bis-phosphate) and based on an N-substituted hydroxamic acid, as a chelator of the zinc ion present in active site. The compounds synthesized were tested on class II Fbas from various pathogenic microorganisms and, by comparison, on a mammalian class I Fba. The best inhibitor shows K(i) against class II Fbas from various pathogens in the nM range, with very high selectivity (up to 10(5)). Structural analyses of inhibitors in complex with aldolases rationalize and corroborate the enzymatic kinetics results. These inhibitors represent lead compounds for the preparation of new synthetic antibiotics, notably for tuberculosis prophylaxis.

Synthesis and Biochemical Evaluation of Selective Inhibitors of Class II Fructose Bisphosphate Aldolases: Towards New Synthetic Antibiotics.

We report the synthesis and biochemical evaluation of selective inhibitors of class II (zinc-dependent) fructose bisphosphate aldolases. The most active compound is a simplified analogue of fructose bisphosphate, bearing a well-positioned metal chelating group. It is a powerful and highly selective competitive inhibitor of isolated class II aldolases. We report crystallographic studies of this inhibitor bound in the active site of the Helicobacter pylori enzyme. The compound also shows activity against Mycobacterium tuberculosis isolates.

The origin of the carbon chain in the thiazole moiety of thiamine in Escherichia coli: Incorporation of deuterated 1-deoxy-D-threo-2-pentulose

Abstract Non growing washed cells of Escherichia coli, derepressed for the biosynthesis of thiamine, have been incubated in the presence of glucose and either 1-deoxy- D -threo-2-pentulose 1 or 1-deoxy- D -erythro-2-pentulose 2 trideuterated on the methyl group. The incorporation of deuterium into the thiazole moiety of thiamine was measured by mass spectrometry. The label of the threo-compound was found in more than 40% of the thiazole biosynthesized in its presence; the label of the erythro-compound in less than 5%. Hence it is likely that the carbon chain of 1-deoxy- D -threo-2-pentulose is the precursor of the five carbons chain of the thiazole moiety of the thiamine molecule in E. coli.

Enantioselective synthesis of sulfoxides catalysed by an oxidase–peroxidase bienzymatic system

Abstract We have devised a new bienzymatic oxidase–peroxidase system in which hydrogen peroxide is produced from air by a d -amino acid oxidase. The generated hydrogen peroxide is instantly used for asymmetric oxygenation of sulphides by a peroxidase. In the absence of peroxidase no spontaneous oxidation is observed, indicating that hydrogen peroxide production by the d -amino acid oxidase is fully controlled. Arylmethyl sulfoxides can be synthesised on a preparative scale with high yields and good enantiomeric excess.

Stereocontrol by a Pair of Epimeric Sugar-Derived Ligands of the Coordination Sphere of Copper(II) Complexes

A pair of novel C3-epimeric sugar-derived ligands (glycoligands) with a neutral N4O donor set was synthesized. Copper(II) complexes of both ligands were obtained and characterized by X-ray crystallography. Cyclic voltammetry, electron paramagnetic resonance, and UV-vis spectroscopies showed similar electronic properties. Mirror-image CD spectra were obtained for the Cu(II) d-d band, indicating an enantiomeric character of the coordination sphere, which has been rationalized structurally. This example shows the possible predetermination of stereochemistry for complexes by ligands based on a glycoscaffold.

Facile solvent-free oxidation of thiols mediated by mineral supports

Abstract Oxidation of thiols dispersed without solvent on a weakly basic mineral support was easily performed by heating in air.

Oxidation of chlorophenols catalyzed by Coprinus cinereus peroxidase with in situ production of hydrogen peroxide

Degradation of 2,6‐dichlorophenol (2,6‐DCP) was accomplished by oxidation catalyzed by Coprinus cinereus peroxidase. Immobilization of the enzyme in a polyacrylamide matrix enhanced DCP oxidation. Hydrogen peroxide, peroxidaseapos;s natural substrate, was produced enzymatically in situ to avoid peroxidase inactivation by its too high concentration. In the case of larger scale utilization, the method would also avoid direct handling of this hazardous reagent.