Synthesis Of 4-methylumbelliferyl Research Articles

Practical Synthesis of the Fluorogenic Enzyme Substrate 4-Methylumbelliferyl α-l-Idopyranosiduronic Acid

A practical and concise synthesis of 4-methylumbelliferyl α-l-idopyranosiduronic acid, a fluorogenic enzyme substrate diagnostic for α-l-iduronidase, was accomplished. It features successive radical bromination and radical reduction of easily accessible methyl 4-methyl­umbelliferyl-2,3,4-tri-O-acetyl-β-d-glucouronate in four steps with 28% overall yield.

Synthesis of 4-methylumbelliferyl α-d-mannopyranosyl-(1→6)-β-d-mannopyranoside and development of a coupled fluorescent assay for GH125 exo-α-1,6-mannosidases

Certain bacterial pathogens possess a repertoire of carbohydrate processing enzymes that process host N-linked glycans and many of these enzymes are required for full virulence of harmful human pathogens such as Clostridium perfringens and Streptococcus pneumoniae. One bacterial carbohydrate processing enzyme that has been studied is the pneumococcal virulence factor SpGH125 from S. pneumoniae and its homologue, CpGH125, from C. perfringens. These exo-α-1,6-mannosidases from glycoside hydrolase family 125 show poor activity toward aryl α-mannopyranosides. To circumvent this problem, we describe a convenient synthesis of the fluorogenic disaccharide substrate 4-methylumbelliferone α-d-mannopyranosyl-(1→6)-β-d-mannopyranoside. We show this substrate can be used in a coupled fluorescent assay by using β-mannosidases from either Cellulomonas fimi or Helix pomatia as the coupling enzyme. We find that this disaccharide substrate is processed much more efficiently than aryl α-mannopyranosides by CpGH125, most likely because inclusion of the second mannose residue makes this substrate more like the natural host glycan substrates of this enzyme, which enables it to bind better. Using this sensitive coupled assay, the detailed characterization of these metal-independent exo-α-mannosidases GH125 enzymes should be possible, as should screening chemical libraries for inhibitors of these virulence factors.

Concise synthesis of 4-methylumbelliferyl-penta- N-acetylchitopentaoside and its inhibition effect on chitinase

The synthesis of 4-methylumbelliferyl (UMB)-penta- N-acetylchitopentaoside 4 and its inhibition effect on chitinase are described. The fluorophore-assisted carbohydrate electrophoresis (FACE) analysis showed that the partially N-acetylated chitooligosaccharide (COS) mixture mainly contained glucosamine (GlcN) and oligomers [(GlcN) n , n = 2–7]. The peracetylated COSs [(GlcNAc) n , n = 1–7] were synthesized by treating the partially N-acetylated COS mixture with Ac 2O–NaOAc. The peracetylated chitopentaoside 1 was obtained by isolation of peracetylated COS mixture. The peracetylated UMB chitopentaoside 3 was synthesized by treating compound 1 with 4-methylumbelliferone and a Lewis acid (SnCl 4) catalyst. NaOMe in dry methanol was used for deacetylation of the blocked derivative, to give the target compound 4 in an overall yield of 32%. In binding chitinase assay, it indicates that compound 4 is much more stable than the corresponding penta- N-acetylchitopentaose 2.

Purification and some properties of an Aspergillus niger beta-apiosidase from an enzyme preparation hydrolyzing aroma precursors.

A beta-apiosidase was isolated and purified to electrophoretic homogeneity from an enzyme preparation, Klerzyme 200, through ammonium sulfate precipitation, gel filtration chromatography, ion-exchange chromatography, and HPLC on ion-exchange and size exclusion columns. The purification of the enzyme was aided by the synthesis of 4-methylumbelliferyl beta-D-apiofuranoside for the specific detection of activity on electrophoresis gels. The molecular mass estimated by SDS-PAGE was 120 kDa. The optimum activity of the beta-apiosidase was found at pH 5 and 40 degrees C. The K(m) and V(max) for p-nitrophenyl beta-D-apiofuranoside were 4.2 mM and 2460 nkat/mg of protein, respectively. The enzyme was not inhibited by glucose and ethanol. This enzyme hydrolyzed the intersugar linkages of apiofuranosylglucosides, aroma precursors from grape.

A specific chromophoric substrate for activity assays of 1,3-1,4-β- d-glucan 4-glucanohydrolases

The synthesis of 4-methylumbelliferyl 3-β- O-cellobiosyl-β- d-glucopyranoside ( 3a) and its use as specific substrate to monitor enzyme activity of 1,3-1,4-β- d-glucan 4-glucanohydrolases are described. The chromophoric substrate 3a is prepared by a chemoenzymatic approach starting from barley grain, whose β- d-glucan polysaccharide is degraded down to a tri- ( 1) and tetrasaccharide ( 2) by an extracellular extract of recombinant E. coli expressing and secreting Bacillus licheniformis 1,3-1,4-β-glucanase. The trisaccharide 1 is further chemically transformed into the title compound. Its use as substrate for an enzyme activity assay, the specificity of cleavage, and kinetic parameters are reported. As it undergoes a single glycosidic bond hydrolysis with release of 4-methylumbelliferone, direct UV monitoring of the reaction provides a sensitive kinetic assay of the enzyme action.

Synthesis of 4-methylumbelliferyl glycosides for the detection of α- and β-D-galactopyranosaminidases

Reaction at room temperature of either 3,4,6-tri-O-acetyl-2-azido-2-deoxy-α- or -β-D-galactopyranosyl chloride with a twofold excess of 4-methylumbelliferone and silver trifluoromethanesulfonate in dichloromethane containing an equimolar amount of.sym-collidine yielded 4-methylumbelliferyl tri-O-acetyl-2-azido-2-deoxy-α-D-galactopyranoside in 33% yield. The β anomer was formed in 20 and 10% yields, respectively. Reduction of the azido group, acetylation followed by de-O-acetylation, provided the desired 4-methylumbelliferyl 2-acetamido-2-deoxy-α- and -β-D-galactopyranosides (N-acetyl-α- and -β-D-galactopyranosaminides). The α-glycoside proved to be an effective substrate for the highly sensitive fluorimetric detection of N-acetyl-α-D-galactopyranosaminidase activity in human tissues and was used to examine the deficiency of this activity, which is the enzymatic defect in Schindler disease. Keywords: synthesis of 4-methylumbelliferyl N-acetyl-α- and -β-D-galactopyranosaminides, N-acetyl-α-D-galactosaminidase deficiency (Schindler disease).

Synthesis of 4-methylumbelliferyl 1,2- cis-glycosides

Condensation of 4-methylumbelliferone (7-hydroxy-4-methylcoumarin) with 1,2,3,4,6-penta- O-acetyl-β- d-glucopyranose ( 1) and 1,2,3,4,6-penta- O-acetyl-β- d-galactopyranose ( 9) in the presence of zinc chloride gave, respectively, a mixture of α-( 2) and β- d-glucopyranosides ( 3) in the ratio of 2:3, and a mixture of α- d-galactopyranoside ( 10), β- d-galactofuranoside, and β- d-galactopyranoside ( 11) in the ratio of 10:17:23. The proportion of 1,2- cis-glycosides could be slightly increased by treating 1 and 9 with the O-trimethylsilyl derivative of 4-methylumbelliferone in the presence of stannic chloride. Condensations of the sodium salt of 4-methylumbelliferone with 2,3,4,6-tetra- O-acetyl-β- d-glucopyranosyl chloride, 2,3,4,6-tetra- O-acetyl-β- d-galactopyranosyl chloride, and 2,3,4,6-tetra- O-acetyl-α- d-mannopyranosyl bromide in hexamethylphosphoric triamide gave, respectively, pure 2, pure 10, and a mixture of α- and β- d-mannopyranosides in the ratio of 1:3, with much better yields. O-Deacetylation of 2, 3, 10, and 11, followed by platinum-catalyzed oxidation, afforded the corresponding 4-methylumbelliferyl d-glycopyranosiduronic acids.