Alexander Lucia Leonardus Duchateau

Ochrobactrum anthropi NCIMB 40321 : a new biocatalyst with broad-spectrum L-specific amidase activity

Of 125 microorganisms that were able to use α-hydroxy acid amides as sole nitrogen source, Ochrobactrum anthropi NCIMB 40321 was selected for its ability to hydrolyse racemic amides l-selectively. The substrate specificity of whole O. anthropi cells is remarkably wide and ranges from α-H-α-amino-, α-alkyl-α-amino, N-hydroxy-α-amino acid amides to α-hydroxy-acid amides. After 50% conversion, both the l-acids formed and the remaining d-amides were present in >99% enantiomeric excess, and ammonia accumulated in stoichiometric amounts. Using mandelic acid amide as a model substrate, the hydrolysis was optimized. Optimal rates were observed at pH 8.5 at 50°C. At higher temperatures the initial rate was even higher; however, fairly rapid inactivation occurred.

Fast parallel enantiomeric analysis of unmodified amino acids by sensing with fluorescent β-cyclodextrins

Modified cyclodextrins bearing a metal binding site and a dansyl fluorophore 6-deoxy-6-N-(Nα-[(5-dimethylamino-1-naphthalenesulfonyl)aminoethyl]-aminocylamino-β-cyclodextrin, containing L-Phe (S-1), L-PhGly (S-2) or L-Pro (S-3) moieties, were used as enantioselective fluorescence sensors for the discrimination of enantiomers of the amino acids valine and proline, according to a ligand exchange mechanism. The best conditions to perform enantiomeric analyses were studied and a fast protocol using fluorescence quenching by the copper(II)/amino acid complexes in a fluorescence microplate reader was developed, allowing the detection of samples with high enantiomeric excess. Calibration of the fluorescence response as a function of enantiomeric composition was obtained using the Stern–Volmer model; the calibration curves showed good linearity, allowing fast evaluation of enantiomeric excess within 6% error. Two calibration curves and triplicate analyses of six valine samples were performed in two minutes.

On‐Line Monitoring of Chemical Reactions by using Bench‐Top Nuclear Magnetic Resonance Spectroscopy

Real-time nuclear magnetic resonance (NMR) spectroscopy measurements carried out with a bench-top system installed next to the reactor inside the fume hood of the chemistry laboratory are presented. To test the system for on-line monitoring, a transfer hydrogenation reaction was studied by continuously pumping the reaction mixture from the reactor to the magnet and back in a closed loop. In addition to improving the time resolution provided by standard sampling methods, the use of such a flow setup eliminates the need for sample preparation. Owing to the progress in terms of field homogeneity and sensitivity now available with compact NMR spectrometers, small molecules dissolved at concentrations on the order of 1 mmol L(-1) can be characterized in single-scan measurements with 1 Hz resolution. Owing to the reduced field strength of compact low-field systems compared to that of conventional high-field magnets, the overlap in the spectrum of different NMR signals is a typical situation. The data processing required to obtain concentrations in the presence of signal overlap are discussed in detail, methods such as plain integration and line-fitting approaches are compared, and the accuracy of each method is determined. The kinetic rates measured for different catalytic concentrations show good agreement with those obtained with gas chromatography as a reference analytical method. Finally, as the measurements are performed under continuous flow conditions, the experimental setup and the flow parameters are optimized to maximize time resolution and signal-to-noise ratio.

Enantioseparation of amino compounds by derivatization with o-phthalaldehyde and d-3-mercapto-2-methylpropionic acid

Abstract o-Phthalaldehyde in combination with d -3-mercapto-2-methylpropionic acid is described as a chiral reagent for the enantioseparation of amino compounds. The reagent was applied to the optical resolution of α-amino acids, α-alkyl-α-amino acids, α-amino acid amides, α-alkyl-α-amino acid amides, β-amino alcohols and β-alkyl-β-amino alcohols. Separation of the diastereomers was carried out by reversed-phase chromatography and the derivatives were detected fluorimetrically. The diastereoselectivity obtained was compared with that of other commercially available chiral thiols. The rate of the reaction of various amino compounds with o-phthalaldehyde- d -3-mercapto-2-methylpropionic acid was studied, together with the stability of the derivatives obtained. Application of the method to the enantioseparation of chiral monofunctional amines was demonstrated by using α-methylbenzylamine as a model compound. The method described offers good enantioselectivity combined with high sensitivity for various chiral amino compounds derived from chemo-enzymatic processes.

Selection of buffers and of an ion-pairing agent for thermospray liquid chromatographic—mass spectrometric analysis of ionic compounds

Abstract The applicability of ammonium formate, ammonium acetate and ammonium bicarbonate as volatile electrolytes for thermospray liquid chromatographic—mass spectrometric analysis of three decomposition products of α-aspartame is demonstrated. With these buffers, the pH range of the mobile phases for silica-based reversed-phase columns can be covered. With respect to the buffer capacity and ultraviolet transparency, a buffer salt concentration of 50 mM was used in the mobile phase. The retention times of the compounds studied in the volatile buffers were comapared with those obtained in sodium acetate and sodium phosphate buffers and were found to match quite well. For the reversed-phase ion-pair separation of α-aspartame and β-aspartame in combination with thermospray mass spectrometry, a method is described using trifluoroacetate as pairing ion. Liquid chromatographic—mass spectrometric analysis of l -aspartyl- l -phenylalanine shows that the highest signal-to-noise ratio is obtained in ammonium formate. For the compounds studied, no correlation could be found between the signal-to-noise ratio and source temperature, or between signal-to-noise ratio and vaporizer temperature.

Determination of the enantiomers of α-H-α-amino acids, α-alkyl-α-amino acids and the corresponding acid amides by high-performance liquid chromatography

Abstract o-Phthalaldehyde in combination with N-acetyl- l -cysteine is a useful derivatization reagent for the optical resolution of enantiomeric α-H-α-amino acids, α-alkyl-α-amino acids and the corresponding acid amides. By using reversed-phase high-performance liquid chromatography with a mobile phase containing copper(II) acetate and l -proline, the diastereomeric derivatives of the α-amino compounds can be separated under isocratic conditions. The rate of reaction of α-alkyl-α-amino compounds with o-phthalaldehyde-N-acetyl- l -cysteine can be increased by selectively increasing the amount of o-phthalaldehyde in the reaction mixture. When the derivatization parameters were controlled automatically, the derivatization process showed good reproducibility and the method was found to be suitable for quantitative measurements. The method was applied to monitor the enantiomeric purity of α-H-α-amino acids and α-alkyl-α-amino acids obtained by enantioselective hydrolysis of the corresponding acid amides using an aminopeptidase.

Volatile ion-pairing agents for liquid chromatographic—thermospray mass spectrometric determination of amino acids and amino acid amides

Abstract The behaviour of volatile ion-pairing agents in terms of performance of the combined liquid chromatography—mass spectrometry is described. The applicability of heptafluorobutanoic acid, tridecafluoroheptanoic acid and nonadecafluorodecanoic acid as volatile ion-pairing agents is demonstrated by the analysis of mixtures of amino acids and the corresponding amino acid amides. Translation of a non-volatile to a volatile ion-pair system that is suitable for thermospray mass spectrometry is described. Using nonadecafluorodecanoic acid, the retention times of the compounds studied were comparable to those obtained with n -dodecylsulphonic acid. Further, with nonadecafluorodecanoic acid, the repeatability of the tests and the calibration graphs of the compounds investigated were good. The sensitivity of the mass spectrometer towards the compounds of interest was greatly improved by postcolumn addition of trifluoroacetic acid and ionization with gaseous ammonia. Using nonadecafluorodecanoic acid, the method was applied to the analysis of samples from bio-organic synthesis.

Thermospray liquid chromatography/mass spectrometry study of diastereomeric isoindole derivatives of amino acids and amino acid amides.

A thermospray liquid chromatography/mass spectrometry (TSP-LC/MS) method is described for determination of the enantiomeric excess of alpha-amino acids and alpha-amino acid amides as their o-phthalaldehyde/N-acetyl-L-cysteine (OPA/NAC) derivatives. The source temperature is an important factor in optimizing the sensitivity of the TSP-LC/MS analysis, whereas the repeller voltage is of minor importance. On-column mass spectra were acquired for the OPA/NAC derivatives of several alpha-amino acids and alpha-amino acid amides. For the main fragment ions, mass spectra fragmentation pathways are proposed. The applicability of the method is demonstrated by means of the enantiomeric excess determination of valine in a sample from an enzymatic hydrolysis experiment. Using single ion monitoring, the detection limit of D-valine in the presence of excess L-valine is 10 pmol. The present TSP-LC/MS method is useful for validating the results obtained from LC/UV or LC/fluorescence methods for the enantiomeric excess determination of alpha-amino acids and alpha-amino acid amides.

Determination of the enantiomers of α-amino acids and α-amino acid amides by high-performance liquid chromatography with a chiral mobile phase

Abstract A high-performance liquid chromatographic method for the enantiomeric analysis of a mixture of an α-amino acid and the corresponding acid amide is described. Reversed-phase chromatography with copper(II) acetate and N,N-di- n -propyl- l -alanine in the mobile phase are used for the separation. For Val and Val-NH 2 , several parameters affecting retention and enantioselectivity were investigated. The results indicate that by manipulation of pH, ionic strength, temperature, concentration of Cu II , N,N-di- n -propyl- l -alanine and ion-pairing reagent, good control of enantiomeric separation can be achieved. For α-amino acid amides a mechanism is proposed which may account for the retention and enantioselectivity. Examples of enantiomeric analysis of mixtures of α-amino acids and α-amino acid amides with aliphatic, aromatic and polar side-chains are given. The method can be used for the control of the enantiometric purity of α-amino acids and the corresponding acid amides obtained by enantioselective synthesis.

Determination of the Enantiomers of 3-Tert.-Butylamino-1,2-Propanediol by High-Performance Liquid Chromatography Coupled to Evaporative Light Scattering Detection

A method for the separation and quantitation of the enantiomers of 3-tert.-butylamino-1,2-propanediol by high-performance liquid chromatography and evaporative light scattering detection has been developed. Separation of the enantiomers was performed in normal-phase liquid chromatography on a Chiralpak AS chiral stationary phase. The influence of the gas nature, gas pressure and temperature of the drift tube of the evaporative light scattering detector on the detection sensitivity was investigated. The method was validated in terms of linearity, limit of quantitation, accuracy and precision. The enantiomeric excess of (S)-3-tert.-butylamino-1,2-propanediol, used for the industrial synthesis of (S)-timolol, was measured from 0 to 94%.