Characterization of a thiourea derivative that targets viral transactivators of cytomegalovirus and herpes simplex virus type 1.

Although currently available antivirals against certain herpesviruses are effective, the development of resistance during long-term use has necessitated the search for seed compounds that work against novel target molecules. In this report, we identified a thiourea derivative compound, 147B3, that inhibits the infection of human cytomegalovirus (HCMV) in fibroblasts and herpes simplex virus type 1 (HSV-1) in Vero cells at a 50% effective concentration of 0.5 µM and 1.9 µM, respectively. Characterization of the compound provided the following clues regarding its mode of action. 1) Time-of-addition and block-release assays showed that 147B3 behaved similarly to ganciclovir. 2) 147B3 reduced the expression of early and late but not immediate-early gene products and the accumulation of viral genomic DNA in both HCMV-infected and HSV-1-infected cells. 3) 147B3 inhibited the HCMV IE2-dependent activation of viral early gene promoters. 4) Four HSV-1 clones resistant to 147B3 were isolated and next-generation sequencing analysis of their genome DNA revealed that all of them had a mutation(s) in the infected cell protein 4 (ICP4) gene, which encodes a viral transcriptional factor. 5) Although 147B3 did not reduce the amount of ICP4 in an immunoblotting analysis, it changed the localization of the ICP4 from the speckles in the nuclei to diffused dots in the cytoplasm. 6) 147B3 did not affect the localization of promyelocytic leukemia (PML) bodies. Our findings suggest that 147B3 targets viral transactivators, potentially through their interaction with factors required for the viral gene expression system.

Chiral Thioureas-Preparation and Significance in Asymmetric Synthesis and Medicinal Chemistry.

For almost 20 years, thioureas have been experiencing a renaissance of interest with the emerged development of asymmetric organocatalysts. Due to their relatively high acidity and strong hydrogen bond donor capability, they differ significantly from ureas and offer, appropriately modified, great potential as organocatalysts, chelators, drug candidates, etc. The review focuses on the family of chiral thioureas, presenting an overview of the current state of knowledge on their synthesis and selected applications in stereoselective synthesis and drug development.

Enzyme-Functionalized Piezoresistive Hydrogel Biosensors for the Detection of Urea.

Urea is used in a wide variety of industrial applications such as the production of fertilizers. Furthermore, urea as a metabolic product is an important indicator in biomedical diagnostics. For these applications, reliable urea sensors are essential. In this work, we present a novel hydrogel-based biosensor for the detection of urea. The hydrolysis of urea by the enzyme urease leads to an alkaline pH change, which is detected with a pH-sensitive poly(acrylic acid-co-dimethylaminoethyl methacrylate) hydrogel. For this purpose, the enzyme is physically entrapped during polymerization. This enzyme-hydrogel system shows a large sensitivity in the range from 1 mmol/L up to 20 mmol/L urea with a high long-term stability over at least eight weeks. Furthermore, this urea-sensitive hydrogel is highly selective to urea in comparison to similar species like thiourea or N-methylurea. For sensory applications, the swelling pressure of this hydrogel system is transformed via a piezoresistive pressure sensor into a measurable output voltage. In this way, the basic principle of hydrogel-based piezoresistive urea biosensors was demonstrated.

Poly(glyceryl monomethacrylate-co-ethylene glycol dimethacrylate) monolithic columns with incorporated bare and surface modified gluconamide fumed silica nanoparticles for hydrophilic interaction capillary electrochromatography.

This research article presents the preparation and characterization of monolithic capillary columns with incorporated bare fumed silica nanoparticles (FSNPs) and surface coated gluconamide FSNPs and their subsequent use in hydrophilic interaction capillary electrochromatography (HI-CEC) of small relatively polar solutes. The monolithic support was based on the in situ polymerization of glyceryl monomethacrylate (GMM) and ethylene glycol dimethacrylate (EDMA) yielding the poly(GMM-co-EDMA) monolith for the incorporation of bare and gluconamide-FNSPs. The poly(GMM-co-EDMA) monolith functioned as a true "support" for both types of polar FSNPs "stationary phases". In other words, monolithic capillary columns with "FSNPs stationary phases" were obtained in the sense that the contribution of the monolith proper to solute' retention was at its minimum. The gluconamide-FSNPs were obtained by reacting the FSNPs with the polar organosilane N-(3-triethoxysilylpropyl)gluconamide either by a pre- or on-column approach yielding p-gluconamide-FSNPs or o-gluconamide-FSNPs, respectively. While the p-gluconamide-FSNPs was coated by an oligosiloxane gluconamide layer as revealed by thermogravimetric analysis, the o-gluconamide-FSNPs are thought to be covered with a monomeric layer of gluconamide ligands as was manifested by the higher plate number obtained on the latter than on the former gluconamide-FSNPs incorporated monolithic columns. In the on-column modification process of FSNPs, the reaction was performed in a closed system whereby atmospheric water vapor are not available to cause the polymerization of the trifunctional organosilane N-(3-triethoxysilylpropyl)gluconamide. Also, the fact that the o-gluconamide-FSNPs incorporated monoliths were made from bare-FSNPs incorporated monoliths may indicate that the bare FSNPs were better dispersed into the monolithic matrix than the p-gluconamide-FSNPs, a condition that might have further contributed to the lower plate count obtained on p-gluconamide- than o-gluconamide-FSNPs incorporated monolithic columns. Overall, o-gluconamide-FSNPs stationary phases and to a lesser extent bare-FSNPs stationary phases proved useful in HI-CEC of small polar solutes, including DMF, formamide, thiourea, some phenols and nucleobases.

Thiourea sensor development based on hydrothermally prepared CMO nanoparticles for environmental safety.

Low-dimensional cobalt oxide codoped manganese oxide nanoparticles (CMO NPs; dia. ~ 25.6nm) were synthesized using the hydrothermal method in alkaline phase. The optical, morphological, and structural properties of CMO NPs were characterized in details using FT-IR, UV/vis., FESEM, XEDS, XPS, TEM, and XRD techniques. Glassy carbon electrode (GCE) was fabricated with a thin-layer of CMO NPs by conducting coating binders for the development of selective and sensitive thiourea (TU) sensors. Electrochemical responses along with higher sensitivity, large-dynamic-range, and long-term stability towards TU were performed by electrochemical I-V approach. The calibration curve was found linear over a wide linear dynamic range (LDR) of TU concentration. From the gradient of the calibration plot, limit of detection (LOD), and sensitivity were calculated as 12.0±0.05pM and 3.3772nAnM-1cm-2 respectively. It is an organized route for the development of chemical sensor based on very low-dimensional CMO NPs/GCE using electrochemical reduction phenomena. As far as we know, this report is the maiden publication on highly sensitive TU sensor based on the CMO NPs/GCE. This method could be a pioneer developer in TU sensitive chemical sensor development using doped NPs in the simple I-V method for the important sensor applications with useful doped materials coupled nano-technological systems for environmental safety.

Rapid detection of economic adulterants in fresh milk by liquid chromatography-tandem mass spectrometry.

A method to aid in the detection of the economically driven adulteration of fresh milk with a range of small, nitrogen containing compounds, including melamine, ammeline, ammelide, cyanuric acid, allantoin, thiourea, urea, biuret, triuret, semicarbazide, aminotriazine, 3- and 4-aminotriazole, cyanamide, dicyandiamide, guanidine, choline, hydroxyproline, nitrate, and a range of amino acids, has been developed. (15)N2-Urea is used as an internal standard. The adulteration of milk with exogenous urea has previously been difficult to detect because of the variation in the naturally occurring levels of urea in milk. However, by monitoring the contaminants biuret and triuret, which comprise up to 1% of synthetic urea, the adulteration of milk with urea-based fertilizer can be detected. We estimate that to be economically viable, adulteration of the order of 90-4000ppm of the above adulterants would need to be added to fresh milk. For most of the compounds, an arbitrary detection threshold of 2ppm is therefore more than sufficient. For biuret, a lower detection threshold, better than 0.5ppm, is desirable and the sensitivity for biuret and triuret can be improved by the post-column addition of lithium to create lithium adducts under electrospray ionisation. Sample handling involves a two-step solvent precipitation method that is deployed in a 96-well plate format, and the hydrophilic interaction liquid chromatography uses a rapid gradient (1.2min). Three separate injections, to detect the positively charged compounds, the negatively charged compounds and amino acids and finally the lithium adducts, are used. This rapid and qualitative survey method may be deployed as a second tier screening method to quickly reduce sample numbers indicated as irregular by an FTIR based screening system, and to direct analysis to appropriate quantification methods.

Myrosinase hydrolysates of Brassica oleraceae L. var. italica reduce the risk of colon cancer.

By means of liquid chromatography-electrospray ionization (LC-ESI) mass spectrometry two glucosinolates, glucoiberin and 3-hydroxy,4(α-L-rhamnopyranosyloxy) benzyl glucosinolate, were identified in the aqueous extract of Brassica oleraceae L var. italica. Further, two compounds were isolated after enzymatic hydrolysis of the aqueous extract by myrosinase, one of them was identified as 4-vinyl-3-pyrazolidinone. The second compound (sulphoraphane) 1-isothiocyanate-4-methyl-sulphinyl butane, converted to the most stable form of thiourea (sulphoraphane thiourea). The crude extract (80% alcohol extract) of broccoli florets was examined for cytotoxic activity against different human cancer cell lines, it showed good inhibition of colon cancer (IC(50) 3.88 µg/mL). On the other hand each of the successive extracts (petroleum ether, chloroform, ethyl acetate and ethanol) showed no significant cytotoxic activity. When myrosinase hydrolysate was tested for cytotoxic activity on the colon cancer cell line it showed very high activity - 95% lethality up to 0.78 µg/mL.

A novel ionic liquid monolithic column and its separation properties in capillary electrochromatography.

A novel ionic liquid (IL) monolithic capillary column was successfully prepared by thermal free radical copolymerization of IL (1-vinyl-3-octylimidazolium chloride, ViOcIm(+)Cl(-)) together with lauryl methacrylate (LMA) as the binary functional monomers and ethylene dimethacrylate (EDMA) as the cross-linker in binary porogen. The proportion of monomers, porogens and cross-linker in the polymerization mixture was optimized in detail. The resulting IL-monolithic column could not only generate a stable reversed electroosmotic flow (EOF) in a wide pH range (2.0-12.0), but also effectively eliminate the wall adsorption of the basic analytes. The obtained IL-monolithic columns were examined by scanning electron microscopy (SEM) and Fourier transform infrared (FT-IR). These results indicated that the IL-monolithic capillary column possessed good pore properties, mechanical stability and permeability. The column performance was also evaluated by separating different kinds of compounds, such as alkylbenzenes, thiourea and its analogues, and amino acids. The lowest plate height of ~6.8 µm was obtained, which corresponded to column efficiency (theoretical plates, N) of ~147,000 plates m(-1) for thiourea. ILs, as a new type of functional monomer, present a promising option in the fabrication of the organic polymer-based monolithic columns in CEC.

Magnetically immobilized frits for the preparation of packed columns used in capillary electrochromatography.

Fabrication of porous frits to retain stationary phases is a critical issue in column preparation for capillary electrochromatography (CEC). In this work, porous frits were prepared by applying an external magnetic field to magnetically responsive particles placed inside a fused-silica capillary. Three batches of uniform magnetite spheres with particle diameters of 0.3, 0.4, and 0.6 microm and saturation magnetization values of 73.03, 74.41, and 77.83 emu/g, respectively, were used as frit particles and octadecyl- and phenyl-bonded silica gels were packed successfully into frit-containing capillaries. The performance of the resulting magnetically immobilized frits and packed columns was evaluated. The electroosmotic mobilities in capillaries containing outlet frit only were found to be reduced by 2-4% whereas the plate heights of an unretained marker increased by 30-50% as compared to those in open capillaries. These variations are believed to be associated with the inhomogeneities of the packed structure of the frits. The magnetically immobilized frits showed adequate mechanical strength to withstand the flow drag force, allowing separation in capillaries packed with 5-mum stationary phases up to 10-15 cm, thus rendering column efficiency and reproducibility comparable with those obtained with sintered frits. Taken together, retaining frits made of uniform magnetite particles serves as a viable alternative to sintered frits for column preparation, which offers several distinct advantages such as ease of preparation, improved durability as compared to sintered frits where the removal of the polyimide coating makes the packed column susceptible to breakage, and use of large-bore capillaries for semipreparative separations.

Asymmetric generation of fluorine-containing quaternary carbons adjacent to tertiary stereocenters: uses of fluorinated methines as nucleophiles.

Organocatalytic asymmetric Michael reactions of fluorinated nucleophiles with nitroolefins catalyzed by Cinchona alkaloid-derived thiourea catalysts generated the desired Michael products containing vicinal fluorinated quaternary and tertiary chiral centers with exceptional enantioselectivity.

Discovery of a non-peptidic inhibitor of west nile virus NS3 protease by high-throughput docking.

BACKGROUND: The non-structural 3 protease (NS3pro) is an essential flaviviral enzyme and therefore one of the most promising targets for drug development against West Nile virus (WNV) and dengue infections. METHODOLOGY: In this work, a small-molecule inhibitor of the WNV NS3pro has been identified by automatic fragment-based docking of about 12000 compounds and testing by nuclear magnetic resonance (NMR) spectroscopy of only 22 molecules. Specific binding of the inhibitor into the active site of NS3pro and its binding mode are confirmed by 15N-HSQC NMR spectra. The inhibitory activity is further validated by an enzymatic assay and a tryptophan fluorescence quenching assay. CONCLUSION: The inhibitor [4-(carbamimidoylsulfanylmethyl)-2,5-dimethylphenyl]-methylsulfanylmethanimidamide has a good ratio of binding affinity versus molecular weight (ligand efficiency of 0.33 kcal/mol per non-hydrogen atom), and thus has good potential as lead compound for further development to combat West Nile virus infections.

Anion exchange silica monolith for capillary liquid chromatography.

An anion exchange monolithic silica capillary column was prepared by surface modification of a hybrid monolithic silica capillary column prepared from a mixture of tetramethoxysilane (TMOS) and methyltrimethoxysilane (MTMS). The surface modification was carried out by on-column copolymerization of N-[3-(dimethylamino)propyl]acrylamide methyl chloride-quaternary salt (DMAPAA-Q) with 3-methacryloxypropyl moieties bonded as an anchor to the silica surface to form a strong anion exchange stationary phase. The columns were examined for their performance in liquid chromatography (LC) and capillary electrochromatography (CEC) separations of common anions. The ions were separated using 50 mM phosphate buffer at pH 6.6. Evaluation by LC produced an average of 30,000 theoretical plates (33 cm column length) for the inorganic anions and nucleotides. Evaluation by CEC, using the same buffer, produced enhanced chromatographic performance of up to ca. 90,000 theoretical plates and a theoretical plate height of ca. 4 mum. Although reduced efficiency was observed for inorganic anions that were retained a long time, the results of this study highlight the potential utility of the DMAPAA-Q stationary phase for anion separations.

Non-enzymatic reduction of a 1,2,4-thiadiazolium derivative.

It was discovered that 2,3-bis-(2-methoxy-phenyl)-5-phenylamino-[1,2,4]-thiadiazolium bromide (1), a 1,2,4-thiadiazolium derivative, could be reduced to the corresponding imidoylthiourea, 1-[(2-methoxy-phenyl)-(2-methoxy-phenylimino)-methyl]-3-phenyl-thiourea (3), by some biologically interesting reducing reagents including glutathione, cysteine, and ascorbic acid. The reduction also occurred in Sprague-Dawley rat and Yorkshire swine plasma, suggesting that thiol containing biological molecules existing in the plasma are mainly responsible for this reaction. A facile method for preparation of 3 from 1 was established by using 2-thioethanol as reaction reagent as well as solvent. The structure of 3 was fully characterized using nuclear magnetic resonance (NMR) and mass spectrometry with electrospray ionization source (ESI-MS). Those new findings could shed light on the development of 1,2,4-thiadiazolium derivatives for their potential pharmaceutical applications.

Efficiency of methacrylate monolithic columns in reversed-phase liquid chromatographic separations.

Butyl-methacrylate-based porous monoliths were prepared inside fused-silica capillaries as reversed-phase separation media for liquid chromatography (LC) and capillary electrochromatography (CEC). During our previous research on methacrylate-based monoliths for reversed-phase separations, we noticed that a separation efficiency of up to 300,000 plates/m can easily be obtained in the CEC mode for unretained compounds. However, the efficiencies for retained compounds were much lower in reversed-phase systems, especially in pressure-driven LC. In this work methacrylate-based columns were prepared and characterized in terms of efficiency and retention in reversed-phase (pressure-driven) LC and in CEC. Much attention has been paid to the mass-transfer mechanism in the stationary phase. Factors that affect the plate heights for specific compounds have been investigated. A possible explanation for the relatively low separation efficiency of retained compounds and suggestions to improve molecular mass transfer are provided.

Magnetically immobilized beds for capillary electrochromatography.

Fritless packed beds comprised of magnetically responsive octadecylsilane bonded silica particles have been constructed for reversed-phase electrochromatography. The magnetic particles were immobilized in the capillary by applying an external magnetic field transverse to the direction of electroosmotic flow. Being subjected to the interplay of fluid dragging and magnetic forces, the initial loosely packed particle assembly was compacted into a uniform packing structure. The magnetically immobilized beds obtained were used as stationary phases for separation of neutral compounds, with retention behavior and column efficiency similar to those of slurry-packed columns. The results suggest that the magnetic attraction approach to fritless column packing may be used for construction of advanced chip-based chromatography, especially in complex architectures comprising curved and intersecting channels.

Preparation and evaluation of C18-bonded 1-microm silica particles for pressurized capillary electrochromatography.

Nonporous silica spheres (1 microm) were synthesized and bonded with octadecylsilane functionality. These stationary phase particles were packed electrokinetically into fused-silica capillaries with 100 microm id for a length of 20 cm, which was evaluated by using pressurized CEC (pCEC). The efficiency of the C18 RP column was characterized through the theoretical plates of thiourea, benzyl alcohol, toluene, styrene, and naphthalene. The effects of experimental parameters such as the applied voltage, sample size, pump flow rate, pH value and the concentration of the buffer solution, and the content of methanol in the mobile phase, on-column efficiency were evaluated. Column efficiency as high as 200 000 theoretical plates per meter for naphthalene was obtained with the optimal condition of 70% v/v methanol and 30% v/v of 10 mmol/L phosphate buffer (pH 7.8) at an applied voltage of 10 kV and a supplementary pressure of 500 psi.

[Transfer of solutes in capillary electrochromatography with mixed-mode stationary phase].

In capillary electrochromatography with the mixed-mode stationary phase, while the transfer of solutes could be controlled by the contributions of chromatography (reversed-phase and ion exchange), the transfer of charged solutes could be influenced by electrophoresis transfer. According to the principle of ion independent transfer and several kinds of interaction between solute, mobile phase, and stationary phase, an theoretical expression was derived to describe the relationship among the apparent transfer velocity of solute, the transfer velocities in various forms and the diversified interactions. The studies focused on the influence of various parameters (e.g., pH, organic modifier content, stationary phase proportion) and the transfer characters of various solutes in the mixed-mode capillary electroosmotic chromatography (CEC). These results demonstrate that electroosmotic flow in the mixed stationary phase electrochromatography are kept high and steady within wide ranges of pH and organic modifier content. The separation was affected by the pH through the change of the solutes conformation. The influence of neutral solutes by organic modifier complies with the rule of conventional reversed-phase CEC. The separation of charged solutes was influenced evidently by the compete reagents which could improve the shape of the peak, but it was not so important as in the conventional ion exchange chromatography due to the adjustment by the function of electrophoresis.

[Investigation of separation mechanism for neutral solutes on cyano column in capillary electrochromatography].

The separation mechanism for neutral solutes on a cyano column in capillary electrochromatography (CEC) was investigated and the effects of components of mobile phases and the kinds of buffers on retention of samples were studied. In comparing the separation behavior of samples on CN column with reversed-phase ODS column and normal phase SI column, the peak order was different on CN column under different experimental conditions, and the characteristics of CN column having both reversed and normal phase mechanisms were obvious. The migration velocities for neutral solutes of different polarity on CN column were very different by the effects of two mechanisms under different operating conditions. It is easy to change peak order and to adjust selectivity.

Characteristics of particle-loaded monolithic sol-gel columns for capillary electrochromatography. I. Structural, electrical and band-broadening properties.

Particle-loaded (3 microm, C18) monolithic sol-gel columns have been prepared and selected characteristics measured. They have a surprisingly high permeability, allowing their operation in the microLC mode at pressures as low as 69 kPa where their efficiency is about 50000 plates per meter and the CEC mode where efficiency is at least 106000 plates per meter. These columns can withstand over 13.8 MPa pressure without compression or movement within the 75 microm capillary. Field strengths in the packed segments are approximately 50% greater than those in the open segments, due to the higher resistivity of the particle-laden regions. There is a relatively rapid loss of efficiency with increasing linear velocity in both the CEC and microLC modes, which may be due to a tortuosity effect in the inter- and intra-particulate voids. Chromatographic behavior is characteristic of conventional C18 particles, indicating that analytes have significant access to the surface within the pores of the immobilized bonded phase.

Evaluation of 1.5 microM reversed phase nonporous silica in packed capillary electrochromatography and application in pharmaceutical analysis.

Reversed-phase nonporous silica (RP-NPS) of 1.5 microm dp is employed to demonstrate rapid and efficient separations in packed capillary electrochromatography (CEC). Two methods for packing capillaries and two techniques to manufacture frits used to hold the packing in place are evaluated for their effect upon separation performance using polyaromatic hydrocarbons (PAHs) and polar neutral pharmaceutical compounds. Attention is given to conditioning of the packed capillaries for high efficiency separations without necessity for sodium dodecyl sulfate (SDS). Separation conditions for the nonporous materials were modified from those previously determined on porous reversed-phase silica. Feasibility for method development and validation of a parent pharmaceutical compound and related impurities in the range of 0.1-120% of a 5 mg/mL concentration was assessed and reported. An approach to improving detection sensitivity through use of large-bore capillaries is briefly discussed.