Coumarin-based, switchable fluorescent substrates for enzymatic bacterial detection.

Enzymatically-switchable fluorescent substrates, such as the commercially available 4-methyl umbelliferones (4-MU) are used as standard indicators of enzymatic activity for the detection of various microorganisms and pathogens. However, a major disadvantage of 4-MU is its relatively high pKa leading to only partial dissociation of the fluorescent anion under the conditions where the enzymes are most effective (pH 6-6.5). Here we present a method for new, enzymatically-switchable, fluorescent substrates with improved photo-physico/chemical properties. The lead derivative, 4-AAU, shows excellent solubility in aqueous media (0.81 mg/mL) when compared to 4-MU (0.16 mg/mL), significantly improved quantum yield and wider dynamic range of its fluorescence properties. The corresponding bacterial substrate ß-4-AAUG showed superior selectivity in the detection of clinically relevant amounts of E. coli, Enterococcus and K. pneumonia (1 CFU). The fluorescence intensity of ß-4-AAUG was almost 5 times higher than that of the standard, the detection was possible in reasonably short time (∼ 2.5 h) and with excellent sensitivity.

Novel 4-Methylumbelliferone Amide Derivatives: Synthesis, Characterization and Pesticidal Activities.

A series of novel 4-methylumbelliferone amide derivatives were designed, synthesized and characterized by ¹H NMR, 13C NMR and HR-ESI-MS. The structures of compounds 4bd and 4be (compounds named by authors) were further confirmed by X-ray single crystal diffraction. The acaricidal, herbicidal and antifungal activities of the synthesized compounds were assayed for their potential use as pesticide. The results indicated that compounds 4bi, 4ac and 4bd were strong acaricidals against Tetranychus cinnabarinus, with 72h corrected mortalities of greater than 80% at 1000 mg/L. Meanwhile, compounds 4bh and 4bf exhibit the strongest inhibition against the taproot development of Digitaria sanguinalis and Chenopodium glaucum, and were even more potent than the commercial herbicide Acetochlor against D. sanguinalis. In addition, compounds 4bk, 4bh and 4bp showed the highest antifungal activity against the mycelium growth of Valsa mali, which makes them more effective than commercial fungicide Carbendazim.

4-Methylumbelliferones Analogues as Anticancer Agents: Synthesis and in Cell Pharmacological Studies.

BACKGROUND: Cancer is one of the most serious clinical problems worldwide, and considerable efforts have been devoted to discovering therapeutic agents with novel modes of action. Natural and synthetic coumarin derivatives have attracted intense research interest due to their diverse structural features and remarkable array of biological properties. OBJECTIVE: In the present study, we synthesized a series of 4-MU derivatives containing urea-piperazine and thioureapiperazine moieties and evaluated their antitumor activities to find efficacy antitumor drugs. METHOD: Cell proliferation, apoptosis, cell cycle, the generation of reactive oxygen species and calcium were measured using MTT assay and flow cytometry, respectively. The expression of apoptosis- and proliferation-related proteins was determined by western blotting. The effect of 4l on apoptosis-related mRNA expression in NCI-H460 cells was detected by RT-PCR. RESULTS: Most of the target compounds exhibited potential anticancer activities against tested cancer cells but had low cytotoxicity to normal cells. Compound 4l inhibited the growth and proliferation of NCI-H460 cells and resulted in apoptosis. Successive studies conducted with 4l in NCI-H460 cells demonstrated that this compound induced the intracellular reactive oxygen species generation and calcium overload, suppressed nuclear factor-κB (NF-κB) activity and regulated anti- and pro-apoptotic proteins. In addition, compound 4l effectively arrested NCI-H460 cells in G2 phase and altered the cell cycle regulatory proteins especially cyclin B1. CONCLUSION: Compound 4l exerts significant anticancer effects on NCI-H460 cells in vitro through targeting of mitochondria-dependent apoptotic pathway. These results indicate that the strategy for rational design of 4-MU derivatives may identify potential anticancer agents.

Synthesis and evaluation of a series of 6-chloro-4-methylumbelliferyl glycosides as fluorogenic reagents for screening metagenomic libraries for glycosidase activity.

Screening of large enzyme libraries such as those derived from metagenomic sources requires sensitive substrates. Fluorogenic glycosides typically offer the best sensitivity but typically must be used in a stopped format to generate good signal. Use of fluorescent phenols of pKa < 7, such as halogenated coumarins, allows direct screening at neutral pH. The synthesis and characterisation of a set of nine different glycosides of 6-chloro-4-methylumbelliferone are described. The use of these substrates in a pooled format for screening of expressed metagenomic libraries yielded a "hit rate" of 1 in 60. Hits were then readily deconvoluted with the individual substrates in a single plate to identify specific activities within each clone. The use of such a collection of substrates greatly accelerates the screening process.

An Improved Helferich Method for the α/ß-Stereoselective Synthesis of 4-Methylumbelliferyl Glycosides for the Detection of Microorganisms.

An improved Helferich method is presented. It involves the glycosylation of 4-methyl-umbelliferone with glycosyl acetates in the presence of boron trifluoride etherate combined with triethylamine, pyridine, or 4-dimethylaminopyridine under mild conditions, followed by deprotection to give fluorogenic 4-methylumbelliferyl glycoside substrates. Due to the use of base, the glycosylation reaction proceeds more easily, is uncommonly α- or ß-stereoselective, and affords the corresponding products in moderate to excellent yields (51%-94%) under appropriate conditions.

Chemoenzymatic Synthesis of a Type 2 Blood Group A Tetrasaccharide and Development of High-throughput Assays Enables a Platform for Screening Blood Group Antigen-cleaving Enzymes.

A facile enzymatic synthesis of the methylumbelliferyl ß-glycoside of the type 2 A blood group tetrasaccharide in good yields is reported. Using this compound, we developed highly sensitive fluorescence-based high-throughput assays for both endo-ß-galactosidase and α-N-acetylgalactosaminidase activity specific for the oligosaccharide structure of the blood group A antigen. We further demonstrate the potential to use this assay to screen the expressed gene products of metagenomic libraries in the search for efficient blood group antigen-cleaving enzymes.

Synthesis, structural studies and biological activity of new Cu(II) complexes with acetyl derivatives of 7-hydroxy-4-methylcoumarin.

The new Cu(II) complexes with 6-acetyl-7-hydroxy-4-methylcoumarin (HL1) and 8-acetyl-7-hydroxy-4-methylcoumarin (HL2) have been obtained by the electrochemical method. The density functional theory calculations and X-ray absorption spectroscopy techniques have been used to geometrically describe a series of new compounds. The studies have been focused on the coordination mode of the hydroxy ligands to the metallic centre. The complexes, Cu(HL1)2 and Cu(HL2)2⋅0.5H2O, have flat square geometry with oxygen atoms in the first coordination sphere. Two bidentate anionic coumarins are bonded to the metal cation via the acetyl and deprotonated hydroxyl O atoms. Biological activity, including microbiological and cytotoxic, has been evaluated and found to be enhanced in comparison with the parent ligands. Moreover, the Cu(II) complex with 8-acetyl-7-hydroxy-4-methylcoumarin shows similar antifungal activity as commercially used fluconazole.

Design and synthesis of 4'-O-alkyl-chitobiosyl-4-methylumbelliferone as human chitinase fluorogenic substrates.

The synthesis of three fluorogenic chitobiosyl derivatives, modified at the non-reducing 4'-OH with, either a methyl, an isopropyl or a cyclohexylmethyl substituent, is described. The 4'-capped 4-methylumbelliferyl chitobiosides are hydrolysed by the human chitinase CHIT1 following Michaelis-Menten kinetics and in contrast to unmodified chitobiosyl-4-methylumbelliferone do not undergo transglycosylation. The compounds are also relatively poor hexosaminidase substrates and thus provide useful alternatives to 4'-deoxychitobiosyl-4-methylumbelliferone, previously reported by us as fluorogenic substrate to monitor CHIT1 activity as a marker for Gaucher disease state.

Photo responsive monoolein cubic phase containing coumarin-Tween 20 conjugates.

Photo-responsive monoolein (MO) cubic phase was developed by incorporating coumarin-Tween 20 conjugate in the cubic phase. 7-chlorocarbonylmethoxycoumarin was obtained from 7-hydroxycoumarin through three-step reactions with the yield of 19.8% and it was conjugated to the head group of Tween 20. The molar ratio of the coumarin derivative/Tween 20 in the conjugate was about 1/1 on ¹H NMR spectrum. The cubic phase was prepared by melting the mixture of MO/conjugate (100/0.88, w/w) and hydrating the molten mixture with 5(6)-carboxyfluorescein (CF) solution. UV irradiation (254 nm and/or 365 nm) for 3 h resulted in 1.27% to 2.69% reduction in the double bond of MO but the cubic phase was stable in terms of its integrity under the UV irradiation. The release of CF from coumarin-Tween 20 conjugate-incorporated cubic phase was somewhat suppressed by being subjected to the UV irradiation. The head groups of coumarin-Tween 20 conjugate will be cross-linked so the diffusion in the water channel will be suppressed.

Synthesis and structural characterisation of selective non-carbohydrate-based inhibitors of bacterial sialidases.

The major human pathogen Streptococcus pneumoniae plays a key role in several disease states including septicaemia, meningitis and community-acquired pneumonia. Although vaccines against S. pneumoniae are available as prophylactics, there remains a need to identify and characterise novel chemical entities that can treat the diseases caused by this pathogen. S. pneumoniae expresses three sialidases, enzymes that cleave sialic acid from carbohydrate-based surface molecules. Two of these enzymes, NanA and NanB, have been implicated in the pathogenesis of S. pneumoniae and are considered to be validated drug targets. Here we report our studies on the synthesis and structural characterisation of novel NanB-selective inhibitors that are inspired by the ß-amino-sulfonic acid family of buffers.

Transglycosylation specificity of Acremonium sp. α-rhamnosyl-ß-glucosidase and its application to the synthesis of the new fluorogenic substrate 4-methylumbelliferyl-rutinoside.

Transglycosylation potential of the fungal diglycosidase α-rhamnosyl-ß-glucosidase was explored. The biocatalyst was shown to have broad acceptor specificity toward aliphatic and aromatic alcohols. This feature allowed the synthesis of the diglycoconjugated fluorogenic substrate 4-methylumbelliferyl-rutinoside. The synthesis was performed in one step from the corresponding aglycone, 4-methylumbelliferone, and hesperidin as rutinose donor. 4-Methylumbelliferyl-rutinoside was produced in an agitated reactor using the immobilized biocatalyst with a 16% yield regarding the sugar acceptor. The compound was purified by solvent extraction and silica gel chromatography. MALDI-TOF/TOF data recorded for the [M+Na](+) ions correlated with the theoretical monoisotopic mass (calcd [M+Na](+): 507.44 m/z; obs. [M+Na](+): 507.465 m/z). 4-Methylumbelliferyl-rutinoside differs from 4-methylumbelliferyl-glucoside in the rhamnosyl substitution at the C-6 of glucose, and this property brings about the possibility to explore in nature the occurrence of endo-ß-glucosidases by zymographic analysis.

A 4-methylumbelliferone-based fluorescent probe for the sensitive detection of captopril.

A highly sensitive fluorogenic probe for captopril, 4-methylumbelliferyl-2, 4-dinitrobenzenesulfonate (4-MUDNBS), was designed and synthesized. 4-MUDNBS is a nonfluorescent compound and was synthesized via the one-step reaction of 4-methylumbelliferone (4-MU) with 2,4-dinitrobenzenesulfonyl chloride. Upon mixing with captopril in basic solution, the 2,4-dinitrobenzenesulfonyl group of 4-MUDNBS was efficiently removed and highly fluorescent 4-MU was released, hence leading to the dramatic fluorescence increase of the reaction solution. The fluorescence intensity is linear with captopril concentration in the range 3.0-500 ng mL(-1) with a detection limit of 2.2 ng mL(-1) (3sigma). The effect of substituents on the benzenesulfonyl moiety of the probe is discussed, and the presence of electronegative groups is favorable for the thiolate-induced cleavage reaction. The proposed method has been successfully applied to the captopril determination in pharmaceutical preparations.

[A short path to synthesis of 4-deoxy-4-fluoroglucosaminides: methylumbelliferyl N-acetyl-4-deoxy-4-fluoro-beta-D-glucosaminide].

A convenient method of synthesis of 1,6-anhydro-4-deoxy-2-O-tosyl-4-fluoro-beta-D-glucopyranose by fusion of 1,6;3,4-dianhydro-2-O-tosyl-beta-D-galactopyranose with 2,4,6-trimethylpyridinium fluoride was found. By successive action of ammonia, methyl trifluoroacetate, and acetic anhydride, the resulting compound was transformed into 1,6-anhydro-3-O-acetyl-2,4-dideoxy-2-trifluoroacetamido-4-fluoro-beta-D-glucopyranose, which was converted into 3,6-di-O-acetyl-2,4-dideoxy-2-trifluoroacetamido-4-fluoro-alpha-D-glucopyranosyl fluoride by the reaction with HF/Py. The resulting fluoride was further used as a glycosyl donor in the synthesis of methylumbelliferyl N-acetyl-4-deoxy-4-fluoro-beta-D-glucosaminide.

Synthesis and antimycobacterial activity of 7-O-substituted-4-methyl-2H-2-chromenone derivatives vs Mycobacterium tuberculosis.

A series of 7-O-alkoxy-4-methylumbelliferone derivatives were prepared using a convenient one step synthesis. Additionally the bromo- and azido derivatives 7-O-(4-bromobutoxy)-, 7-O-(6-bromohexyloxy)- and 7-O-(6-azidohexyloxy)-4-methylumbelliferone derivatives were prepared. In vitro evaluation of antimycobacterial activity determined % inhibition and MIC vs M. tuberculosis H37Rv with toxicity (IC50) assessed in VERO cells. The coumarins with longer alkyl chains (nonyl and decyl) showed the optimum inhibitory activity in this series (MIC 3.13 microg/mL) and IC50 > 10 microg/mL.

Highly sensitive active-site titration of lipase in microscale culture media using fluorescent organophosphorus ester.

The fluorescent organophosphorus esters, diethyl 4-methylumbelliferyl phosphate (1), ethyl hexyl 4-methylumbelliferyl phosphate (2) and ethyl 4-methylumbelliferyl heptylphosphonate (3) have been synthesized and evaluated as a sensitive active-site titrant of lipase. The phosphorus esters 1, 2 and 3 inactivated the lipase from Pseudomonas aeruginosa (LPL-312) with a second-order rate constant for enzyme inactivation (k(on)) of 1.8, 32 and 5600 s(-1) M(-1), respectively. The long-chain phosphonate 3 turned out to be the most potent inactivator of the lipase to release a stoichiometric amount of highly fluorescent 4-methylumbelliferone (4MU) as a leaving group. By using the phosphate 3 as an active-site titrant, the low concentration (4.5 nM) of the active lipase was titrated successfully. The highly sensitive active-site titration with 3 enabled the direct determination of the concentration of the active lipase expressed in a microscale culture medium. Although the expression level differed significantly from one culture to another, the titrated concentration of the active lipase was proportional to the apparent activity for all the independent cultures. The molecular activity calculated for the expressed lipase was found to be the same as that of the purified lipase. The present active-site titration method is widely applicable to the biocatalytic engineering of lipases such as directed evolution, site-directed mutagenesis, chemical modification and immobilization.

Chemical synthesis of 4-trifluoromethylumbelliferyl-alpha-D-N-acetylneuraminic acid glycoside and its use for the fluorometric detection of poorly expressed natural and recombinant sialidases.

When compared to bacterial or viral sialidases, eukaryotic sialidases are expressed at lower levels and frequently show poor specific activities. The identification and characterization of sialidases from eukaryotes have been slowed down due to the limited sensitivity of available sialidase substrates. Therefore, we chemically synthesized a fluorogenic compound, 4-trifluoromethylumbelliferyl-alpha-D-N-acetylneuraminic acid (CF3MU-Neu5Ac), and tested its use as a substrate for eight different sialidases, including enzymes from viral, bacterial, and eukaryotic sources. Kinetic analysis revealed CF3MU-Neu5Ac to be a very sensitive sialidase substrate. Furthermore, this substance proves to be perfectly suitable for the in vivo examination of sialidases and for the detection of recombinant sialidase by means of expression cloning.

[Synthesis of beta-maltosides, derivatives of p-nitrophenol, 2-chloro-4-nitrophenol, and 4-methylumbelliferone, and their use as substrates for determining alpha-glucosidase activity]. / Sintez beta-mal'tozidov, proizvodnykh p-nitrofenola, 2-khlor-4-nitrofenola i 4-metilumbelliferonea, i ikh ispol'zovanie v kachestve substratov dlia opredeleniia aktivnosti alpha-gliukozidazy.

Synthesis of beta-maltosides, p-nitrophenyl-beta-D-maltoside and 4-methylumbelliferyl-beta-D-maltoside, based on interaction of hepta-acetate-beta-D-maltosyl fluoride with the corresponding trimethylsilyl ethers of p-nitrophenol and 4-methylumbelliferone is described. 2-Chloro-4-nitrophenyl-beta-D-maltoside was synthesized by interaction of hepta-acetate-alpha-D-maltosyl bromide with 2-chloro-4-nitrophenol in two phase system using phase transfer catalyst. The method of assay of neutral alpha-glucosidase from human kidney and urine using synthesized beta-maltosides (p-nitrophenyl-beta-D-maltoside, 2-chloro-4-nitrophenyl-beta-D-maltoside and 4-methylumbelliferyl-beta-D-maltoside) as substrates and beta-glucosidase as an auxiliary enzyme is proposed. The method is simple, convenient and 10-fold more sensitive than the commonly used alpha-glucosidase assay procedure with the corresponding synthetic alpha-glucosides, p-nitrophenyl-alpha-D-glucoside and 4-methylumbelliferyl-alpha-D-glucoside. A modification of the method, with p-nitrophenyl-beta-D-maltoside as substrate, was applied to the semi-automatic assay of urinary alpha-glucosidase in 96-well microtitre plates.

A specific chromophoric substrate for activity assays of 1,3-1,4-beta-D-glucan 4-glucanohydrolases.

The synthesis of 4-methylumbelliferyl 3-beta-O-cellobiosyl-beta-D-glucopyranoside (3a) and its use as specific substrate to monitor enzyme activity of 1,3-1,4-beta-D-glucan 4-glucanohydrolases are described. The chromophoric substrate 3a is prepared by a chemoenzymatic approach starting from barley grain, whose beta-D-glucan polysaccharide is degraded down to a tri- and tetrasaccharide by an extracellular extract of recombinant E. coli expressing and secreting Bacillus licheniformis 1,3-1,4-beta-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.

A fluorometric assay of ceramide glycanase with 4-methylumbelliferyl beta-D-lactoside derivatives.

4-Methylumbelliferyl 6'-O-benzyl-beta-D-lactoside (6'Bn-MU-Lac) and some related compounds were synthesized via different selective reactions including phase-transfer glycosylation. Their suitability as substrates for a fluorometric assay of ceramide glycanase (CGase) was evaluated. Among others, the 6'Bn-MU-Lac, which is resistant to exogalactosidase, was found to be a suitable substrate for routine assay of the CGase activity. For American leech CGase, the K(m) value is 0.232 mM at pH 5.