Engineering cytochrome P450 BM3 of Bacillus megaterium for terminal oxidation of palmitic acid.

Directed evolution via iterative cycles of random and targeted mutagenesis was applied to the P450 domain of the subterminal fatty acid hydroxylase CYP102A1 of Bacillus megaterium to shift its regioselectivity towards the terminal position of palmitic acid. A powerful and versatile high throughput assay based on LC-MS allowed the simultaneous detection of primary and secondary oxidation products, which was instrumental for identifying variants with a strong preference for the terminal oxidation of palmitic acid. The best variants identified acquired up to 11 amino acid alterations. Substitutions at F87, I263, and A328, relatively close to the bound substrate based on available crystallographic information contributed significantly to the altered regioselectivity. However, non-obvious residues much more distant from the bound substrate showed surprising strong contributions to the increased selectivity for the terminal position of palmitic acid.

Identification of (S,S)-γ-glutamyl-(cis-S-1-propenyl)thioglycine, a naturally occurring norcysteine derivative, from the Chinese vegetable Toona sinensis.

Extracts of Toona sinensis shoots were studied to identify the precursors of volatile sulfur-containing flavor molecules. T. sinensis was found to contain new compounds (S,S)-γ-glutamyl-(cis-S-1-propenyl)thioglycine, 1, (S,S)-γ-glutamyl-(trans-S-1-propenyl)thioglycine, 2, and γ-glutamyl-(cis-S-1-propenyl)-cysteine, 3. The structures of these compounds were determined by interpretation of multistage mass spectrometric (MS(n)), 1D, and 2D NMR data. The absolute configuration of 1 was established by comparison of experimental with computed infrared and vibrational circular dichroism spectra. Because of the flexibility of the molecule and the novelty of the structure, the configuration was further confirmed by X-ray crystallography. Compounds 1 and 2 are the first examples of norcysteine-containing metabolites reported from nature. They may release thiols via cleavage of the amide bond by proteases, followed by spontaneous decomposition of the resulting unstable alk(en)yl norcysteine moiety.

Development of an analytical approach for identification and quantification of 5-α-androst-16-en-3-one in human milk.

A method was developed for the quantification of 5-α-androst-16-en-3-one in human breast milk based on application of a stable isotope dilution assay using 5α-androst-16-en-3-one-6, 6-d(2). The procedure includes extraction of the human milk by hexane with subsequent clean-up of the obtained extract by gel permeation and silica gel column chromatography. The extracted samples were analyzed by gas chromatography-mass spectrometry. Using this method 5-α-androst-16-en-3-one could be identified and for the first time quantified in a concentration range of 26-155 ng/kg in human milk.

Complexation/encapsulation of green tea polyphenols in mixed calcium carbonate and phosphate micro-particles.

We used a double-jet mixer to encapsulate water-soluble polyphenols, green tea extract (GTE), with calcium-based inorganic materials. The device mixed calcium chloride solutions with a solution of carbonate and phosphate in the presence of a GTE solution, and formed micro-particles which capture the GTE molecules. The micro-particles were analysed by liquid chromatography coupled to tandem mass spectroscopy to determine the encapsulation yield and loading of the different GTE components. We established correlations between (1) the efficiency of the GTE encapsulation and the composition of the mixed anion solutions and (2) the protonation degree of the ions and the molar ratio of calcium cations and carbonate/phosphate anions. An optimal and reproducible GTE loading of about 40% with an encapsulation yield of 65% was observed for a carbonate/phosphate molar composition of 4 : 1. In addition, our experimental results showed that the process is selective and favours the encapsulation of gallated species which form stronger complexes with calcium cations.

Release kinetics of actives from chewing gums into saliva monitored by direct analysis in real time mass spectrometry.

Direct analysis in real time mass spectrometry (DART-MS) was used to monitor the release kinetics of a taste-refreshing compound from chewing gums into the saliva of subjects. A new DART-MS sample probe was designed which was about four times more sensitive than the current benchmark probe. This decreased the impact of the dilution of the saliva samples that was required to minimize ion suppression effects and make quantitative analyses without an internal standard possible. The new probe was also about three times more reproducible, which allowed quantitative measurements to be conducted manually without requiring the enhanced precision provided by an automatic sample positioner. The accuracy of analyses performed by DART-MS was verified by comparing the results obtained from saliva samples analyzed both by DART-MS and by a more classical liquid chromatography/mass spectrometry (LC/MS) method. This investigation showed good agreement between the two techniques. DART-MS could then be used to objectively demonstrate the efficiency of a granular carbohydrate-based delivery system to boost for a few minutes the release of a lipophilic flavor raw material with a high octanol/water partition coefficient, cyclohexanecarboxamide, N-ethyl-5-methyl-2-(1-methylethyl) (WS-3), from chewing gum into saliva.

Real-time monitoring of fragrance release from cotton towels by low thermal mass gas chromatography using a longitudinally modulating cryogenic system for headspace sampling and injection.

An innovative headspace sampling and injection system for gas chromatography was designed using a longitudinally modulating cryogenic system mounted around the sampling loop of a two-position loop injector. The setup was hyphenated to a fast low thermal mass gas chromatograph, allowing transient concentrations of semivolatile analytes to be monitored in real time with a time resolution of 4.5 min. The performance of the instrument, and in particular its cryotrapping efficiency, was characterized using a mixture of long-chain alkanes, methyl esters, ethyl esters, and alcohols of different volatilities. The device was found to be ideally suited to the analysis of semivolatile compounds with boiling points ranging between 190 and 320 degrees C, which are typical for a majority of perfumery raw materials. The new instrument was successfully used to monitor the release of eight odorant compounds from cotton towels to which fabric softener had been applied that alternatively contained the fragrance in free form or in microencapsulated form. The analytical results, unprecedented in their level of precision and time resolution for such an application, evidenced the major impact of microencapsulation technology on the kinetics of fragrance release during the drying of the towels and on the triggering of additional fragrance release by applying mechanical stress to the fabric to rupture the microcapsule walls.

Parameters influencing the release of tertiary alcohols from the surface of "spherical" dendrimers and "linear" stylomers by neighbouring-group-assisted hydrolysis of 2-carbamoylbenzoates.

The influence of structural and physico-chemical parameters on the release of a volatile tertiary alcohol (2-methyl-1-phenyl-2-propanol) by neighbouring-group-assisted cyclisation of 2-carbamoylbenzoates at neutral pH was investigated by comparing the covalent-bond cleavage from the surface of linear, comblike poly(propylene imine) "stylomers" and their corresponding spherical, globular dendrimers. Determination of the kinetic rate constants for the stepwise intramolecular cyclisation of the 2-carbamoylbenzoate moiety by using HPLC showed that the polarity of the conjugates, and thus their solubility in the aqueous reaction medium, has a stronger influence on the rates of hydrolysis than the size (generation) or shape (linear or spherical) of the macromolecules. Furthermore, structural modifications in close proximity to the release unit, such as the presence of functionalities with catalytic activity, have a strong impact on the release efficiency of the active molecules. An understanding of the physico-chemical parameters determining the local environment of the covalent-bond cleavage site is therefore an important prerequisite to transfer the characteristics of small molecules to larger structures such as oligomers and polymers and thus to design efficient macromolecular conjugates for the controlled delivery of bioactive compounds.

Direct mass spectrometric analysis of flavors and fragrances in real applications using DART.

DART (Direct Analysis in Real Time) is an innovative technology to analyze complex solid samples at atmospheric pressure and ground potential by simply placing them between a DART ion source and a mass spectrometer. The analytes are ionized by a gun of neutral metastable species. The first examples of the application of DART to the analysis of flavor and fragrance raw materials in real, complex applications are reported here. A remarkably high potential of the technique is demonstrated. DART was applied to semi-quantitative analyses of perfumery raw materials deposited on smelling strips. In optimal cases, limits of detection around 100 pg were achieved. DART also allowed the assessment of the deposition and release of fragrance on surfaces such as fabric and hair. Finally, DART permitted the screening of twelve chewing gum samples for the possible presence of taste-refreshing compounds in less than 30 min.

Universal two-dimensional HPLC technique for the chemical analysis of complex surfactant mixtures.

Two-dimensional HPLC was applied for the first time to the analysis of complex surfactant mixtures. In the first dimension, ion chromatographic-type separations were performed on a diol column eluted by an acetonitrile-water (0.1% trifluoroacetic acid) gradient. Using this new technique, cationic and amphoteric surfactants were not retained at all, nonionic surfactants exhibited a weak and essentially unspecific retention, and anionic surfactants were retained mainly according to their functional group. Rather than detecting the analytes immediately after this first separation, successive fractions were automatically and quantitatively transferred to parallel C2 (dimethyl) and C4 (butyl) reversed-phase columns using an innovative setup. The second dimension of the separation then took place, by which the analytes were separated according to their hydrophobicity. Surfactants from all four classes, cationic, amphoteric, nonionic, and anionic, were separated simultaneously in single 54-min two-dimensional HPLC runs. The suitability of the method for quantitative measurements was demonstrated.