Using enzyme inhibition as a high throughput method to measure the enantiomeric excess of a chiral sulfoxide

[reaction: see text] The enantiomeric excess of methyl p-tolyl sulfoxide can be determined in a high throughput format by measuring its ability to inhibit the alcohol dehydrogenase catalyzed oxidation of ethanol. The two enantiomers of the sulfoxide have very different inhibition constants for the enzyme. Thus, the initial rate of ethanol oxidation in the presence of the sulfoxide is correlated with the sulfoxide enantiomeric excess.     

Dilatometry as a tool to study a new synthesis for calcium hexaluminate

By using a wet chemical route, pure calcium hexaluminate (CA₆) was yielded, significantly lowering the reaction temperature and shortening the synthesis time if compared to usual industrial procedures. owever, dilatometric studies performed on compacts made of the as-prepared powder, just after pre-heating at 450°C, has shown a superposition between sintering shrinkage and expansion related to CA₂ formation, an intermediate phase formed during calcination and phase evolution to CA₆. oupling of such opposite phenomena led to microcracking of the material, mainly if the heating rates (10°C min^-1) were high. However, lower heating rates (1-5°C min^-1) could quite avoid microcracking but also limit densification. This revised version was published online in July 2006 with corrections to the Cover Date.     

Near infrared spectroscopy as a rapid tool to measure volatile aroma compounds in Riesling wine: possibilities and limits

Volatile chemical compounds responsible for the aroma of wine are derived from a number of different biochemical and chemical pathways. These chemical compounds are formed during grape berry metabolism, crushing of the berries, fermentation processes (i.e. yeast and malolactic bacteria) and also from the ageing and storage of wine. Not surprisingly, there are a large number of chemical classes of compounds found in wine which are present at varying concentrations (ng L⁻¹ to mg L⁻¹), exhibit differing potencies, and have a broad range of volatilities and boiling points. The aim of this work was to investigate the potential use of near infrared (NIR) spectroscopy combined with chemometrics as a rapid and low-cost technique to measure volatile compounds in Riesling wines. Samples of commercial Riesling wine were analyzed using an NIR instrument and volatile compounds by gas chromatography (GC) coupled with selected ion monitoring mass spectrometry. Correlation between the NIR and GC data were developed using partial least-squares (PLS) regression with full cross validation (leave one out). Coefficients of determination in cross validation (R ²) and the standard error in cross validation (SECV) were 0.74 (SECV: 313.6 μg L⁻¹) for esters, 0.90 (SECV: 20.9 μg L⁻¹) for monoterpenes and 0.80 (SECV: 1658 μg L⁻¹) for short-chain fatty acids. This study has shown that volatile chemical compounds present in wine can be measured by NIR spectroscopy. Further development with larger data sets will be required to test the predictive ability of the NIR calibration models developed.     

Terahertz time-domain spectroscopy as a novel tool for crystallographic analysis in cellulose: the potentiality of being a new standard for evaluating crystallinity

Cellulose - Given that terahertz (THz) radiation responds to intermolecular forces such as hydrogen bonds, THz time-domain spectroscopy (THz-TDS) has expanded possibilities in cellulose research....     

17O-NMR-spectroscopy as a tool for stereochemical analysis — Application to a diterpene-derivative

Summary Treatment of abietic acid methylester with Hg(OAc)₂/MeOH produces a dimethoxy derivative. The determination of the configuration at C-7 of this product by means of 1D- and 2D-NMR-spectroscopic methods is described. The results are verified by application of¹⁷O-NMR spectroscopy and comparison with well-established stereochemical dependencies of¹⁷O chemical shifts.

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¹⁷O-NMR-Spectroskopie zur stereochemischen Analyse. Anwendung auf ein Diterpen-Derivat

Zusammenfassung Umsetzung von Abietinsäuremethylester mit Hg(OAc)₂/MeOH führt zu einem Dimethoxyderivat. Die Bestimmung der Konfiguration am Zentrum C-7 erfolgt durch Anwendung von 1D- und 2D-NMR-Methoden. Die Resultate konnten durch¹⁷O-NMR-Spektroskopie aufgrund der bekannten stereochemischen Verschiebungsabhängigkeit bestätigt werden.

     

Microcalorimetry as a possible tool for phenanthrene toxicity evaluation to eukaryotic cells

Microcalorimetry and measurement of culture turbidity using a Bioscreen C Analyzer System were applied to study the toxic effect of phenanthrene on Cunninghamella elegans IM 1785/21Gp spore germination. The results of C. elegans spore incubation in Bioscreen C microbiology reader showed the inhibition of spore germination by 70% (with 25 mg l⁻¹ of phenanthrene) and total inhibition of the fungus growth with a higher content of the xenobiotic (50-100 mg l⁻¹). The microcalorimetric technique showed to be useful for the estimation of metabolic activity of C. elegans spores in growth medium up to xenobiotic concentrations of 90 mg l⁻¹. These data corresponded with the microscopic observations. The obtained results showed that the microcalorimetry method could be a valuable supplement in the study on the mechanism of PAHs detoxification by fungi.     

Reflectometric interference spectroscopy (RIfS) as a new tool to measure in the complex matrix milk at low analyte concentration

Measurements in complex matrices like milk still present a challenge in biosensor development. This is especially important when using a label-free detection method or when measuring low analyte concentrations. The direct optical method reflectometric interference spectroscopy (RIfS) was used for investigating matrix effects in immunoassay development. Furthermore, approaches to reduce these effects have been established. As a model system, the hormone testosterone has been chosen because this immunoassay has been well characterized in buffer. In a first step, the immunoassay for the detection of testosterone in buffer was improved beyond former published results. Therefore, the sensor surface was optimized, resulting in a fivefold lower limit of detection (70.2 ng L⁻¹) and limit of quantification (130.0 ng L⁻¹). Additionally, the assay time could be reduced to 15 min. Consequently, we used this improved assay to investigate matrix effects of whole pasteurized bovine milk. To minimize these effects, the surface chemistry was adapted and a suitable evaluation method was established, reducing the effects of Tyndall scattering and nonspecific binding to the sensor surface. These improvements allow for very reliable quantitative measurements in milk. The assay developed required no sample pretreatment and allowed for the regeneration of the sensor surface so that calibration could be performed on one chip. The calibration in milk (3.5% fat) resulted in a limit of detection of 94.4 ng L⁻¹ and a limit of quantification of 229.3 ng L⁻¹. Furthermore, recovery rates between 70% and 120% could be obtained. Thus, for the first time, an analyte in the matrix milk was successfully quantified with RIfS at low concentrations.     

Use of a magnetic suspension microbalance to measure organic vapor sorption for evaluating the impact of polymer converting process

A gravimetric analyzer consisting of a magnetic suspension balance was set up to study the sorption kinetics of benzaldehyde in various forms of polypropylene (PP), including resin, sheet and thermoformed container. Instrument noise was determined throughout the experiments over a maximum of 4000 min or until equilibrium was reached. The sorption of benzaldehyde by PP resin exhibited Fickian sorption kinetics at low vapor activities (0.1–0.5). However, at high vapor activities (0.7 and 0.9), a two-stage sorption involving Fickian diffusion during the initial sorption, followed by a protracted polymer relaxation, which enhanced the sorption capacity, was observed. The converting process of PP resin into sheets and thermoformed containers had a significant impact on the benzaldehyde sorption profile. Benzaldehyde exhibited a much higher solubility in extruded sheet and thermoformed container than in the resin.     

Chemical shifts as a novel measure of interactions between two binding sites of symmetric dialkyldimethylammonium bromides to α-cyclodextrin

Complex formation of α-cyclodextrin (α-CD) with decyltrimethylammonium (DeTAB), N,N-dioctyldimethylammonium (DOAB), and N,N-didecyldimethylammonium bromides (DDeAB) was investigated by proton NMR spectroscopy. Analysis of chemical shifts yielded macroscopic 1:1 and 1:2 binding constants (K₁ and K₂) and chemical shift differences (Δδ_SD and Δδ_SD2) for the 1:1 and 1:2 complexes of DeTAB, DOAB, and DDeAB with α-CD. The K₁ and K₂ values of DDeAB were quantitatively explained on the basis of the assumption that the microscopic 1:1 binding constant of DDeAB is identical to the observed K₁ value of DeTAB. The K₂ value of DDeAB was also explained in terms of its observed K₁ value and the independent binding of two alkyl chains. Furthermore, the Δδ_SD and Δδ_SD2 values for protons of DDeAB and α-CD were quantitatively explained on the basis of the assumption that the geometry of the decyl group of DDeAB in an α-CD cavity is identical to that of DeTAB. The Δδ_SD value was also explicable on the basis of the same geometric assumption and the observed Δδ_SD2 value for this system. Similar results were obtained for the 1:1 and 1:2 DOAB-α-CD complexes.     

Use of capillary electrophoresis as a versatile tool to measure interaction constants between a KDR‐binding PEGylated lipopeptide and pegylated phospholipid micelles

In the frame of our molecular imaging activities, a PEGylated lipopeptide has been developed as a specific ligand for the human vascular endothelial growth factor receptor 2, which is considered as one of the important molecular marker of angiogenesis. In this study, the potential of affinity capillary electrophoresis (ACE) is evaluated to measure the interactions of an active PEGylated lipopeptide, its hydrolysis product and its precursor consisting of a peptide structure with different micelles including Brij‐35, Tween‐20, and pegylated phospholipids. Given the amphiphilic structure of the PEGylated lipopeptide, a MEKC method allowing the simultaneous separation of the compounds of interest was set up, using low percentages of acetonitrile. Analytes were resolved using a BGE consisting of 100 mM borate buffer pH 9.0, 1 mM Brij, and 25% acetonitrile. Optimized conditions were then used to perform ACE experiments. The affinity constants of the analytes with the micelles were calculated on the basis of their mobility decrease when surfactant concentration increased in the electrolyte. The use of different linearization models to estimate affinity constants was discussed and comparison of different surfactants was reported. PEGylated lipopeptide interacted more strongly with pegylated phospholipid micelles than with Brij‐35 or Tween‐20. Moreover, it is likely that the chemical structure of the compounds, and particularly the lipidic part of the molecules, significantly affects the interaction with micelles. In conclusion, the ACE method can be readily applied to investigate interactions of our targeting lipopeptides with various micelles currently used for the preparation of pharmaceutical vehicles.     

Acetylcholinesterase immobilized on modified magnetic beads as a tool for screening a compound library

Acetylcholinesterase (AChE) from Electrophorus electricus was immobilized on the surface of amino-modified magnetic beads (AChE-MB), and its activity evaluated by the quantification of acetylcholine hydrolysis. A reference mixture composed of AChE binders (galanthamine and a probe coumarin, K _i = 0.031 ± 0.010 μM) and non-binders (ketamine and propranolol) was used to probe the fishing assay. The performance of the bioconjugation assay was demonstrated with a library of 12 reference coumarins from which two ligands were directly identified by LC-MS/MS in a single assay, demonstrating the usefulness of this approach.

A bioconjugate-screening assay with AChE-modified magnetic beads was developed to direct identification of AChE binders, in mixtures, by LC-MS/MS. A reference mixture of twelve coumarins was used and, the two ligands were identified.

     

Polyisocyanides as a new alignment medium to measure residual dipolar couplings for small organic molecules

Polyisocycanides were found to give anisotropic molecular alignment in the magnetic field and are useful to measure residual dipolar couplings (RDCs) from analytes, e.g. strychnine. They show less quadrupolar splitting of the deuterated solvent signal compared with other liquid crystal systems such as Poly-γ-benzyl-L-glutamate (PBLG) and hence less undesired line broadening.     

Electrophoretically mediated microanalysis with partial filling technique and indirect or direct detection as a tool for inhibition studies of enzymatic reaction

The inhibition of the model enzyme, haloalkane dehalogenase from Sphingomonas paucimobilis, was investigated by a combination of electrophoretically mediated microanalysis with a partial filling technique, followed by indirect or direct detection. In this setup, part of the capillary is filled with a buffer suitable for the enzymatic reaction (20 mM glycine buffer, pH 8.6) whereas the rest of the capillary is filled with the background electrolyte optimal for separation of substrates and products. Two different background electrolytes and corresponding detection approaches were used to show the versatility of the developed method. The inhibition effect of 1,2-dichloroethane on the dehalogenation of brominated substrate 1-bromobutane was studied by means of 10 mM chromate - 0.1 mM cetyltrimethylammonium bromide (pH 9.2) in combination with indirect detection or 20 mM beta-alanine - hydrochloric acid (pH 3.5) in combination with direct detection. The method was used to estimate the inhibition constant K(I) (0.44 mM by indirect detection and 0.63 mM by of direct detection) and to determine the inhibition type. Compared to spectrophotometric and other discontinuous assays, the method is rapid, can be automated, and requires only small amount of reagents that is especially important in the case of enzymes and inhibitors.     

An integration strategy to measure enzyme activities for detecting irreversible inhibitors with dimethoate on butyrylcholinesterase as a model

An integration strategy was investigated to measure initial rates of horse butyrylcholinesterase (BChE) at 50.0?µmol?L^?1 butyrylthiocholine (BTCh) for detecting irreversible inhibitors as pollutants in environment and foods with dimethoate as a model. In this integration strategy: (a) if BTCh consumption within 5.0?min was >60%, BChE initial rates were derived from maximal reaction rates, estimated by an improved integrated method, according to Michaelies-Menten kinetics at 47.0?µmol?L^?1 BTCh and Michaelis-Menten constant at 94.0?µmol?L^?1; (b) or else initial rates were determined by the classical initial rate method. Thus, the differences in BChE initial rates without and after dimethoate treatment indexed final dimethoate contents in reaction mixtures to treat BChE. Results supported that this integration strategy determined BChE activities with a linear range about two magnitudes and an upper limit about twice that by the classical initial rate method alone at 2.0?mmol?L^?1 BTCh. The coefficient of variation with this integration strategy was below 5%. The difference in BChE initial rates before and after dimethoate treatment was proportional to final dimethoate contents in reaction mixtures. By enzymatic analyses, the molar contents of dimethoate extracted from polluted cabbages were consistent with the summed molar contents of dimethoate and dimethoxon by gas-chromatography. Therefore, this integration strategy was effective to detect irreversible inhibitors as pollutants in environment and foods.     

Thermogravimetry as a Reliable tool to Estimate the Density of Silanols on a Silica Gel Surface

A method was developed to estimate the density of the silanol groups attached to silica gel surfaces from thermogravimetric data. Depending on the mechanism of condensation of silanol groups during heating, after removing physically adsorbed water, the results obtained ranged from 4.3 to 6.7 OH nm^-2 for a series of silicas. The data are in good agreement with those obtained by other techniques. Thermogravimetry proved to be a reliable tool for this kind of study, with the great advantage of being a simple, quick and inexpensive method requiring only a few mg of the silica sample. This revised version was published online in July 2006 with corrections to the Cover Date.     

Vibrational spectroscopy as a tool for examination to the secondary structure of metal-labeled trastuzumab immunoconjugates

Infrared and Raman spectroscopy are effective techniques that allow collecting information about secondary structure of proteins, including antibodies. Trastuzumab, antibody used in our study was in a freeze-dried form, conjugated with different bifunctional chelators and linked with the stable isotopes of lutetium and yttrium. The characterization of the final immunoconjugates showed no significant changes in the structure demonstrated by the presence of the amide bands characteristic for a α-helices and β-sheets structures. These methods could be applied during the production of the antibody freeze-dried kit formulations for the labeling with the radioactive isotopes.