RESUMEN
The titration of the (S)-enantiomer of omeprazole with the (R)-enantiomer in chloroform-d(1) is monitored by continuous-flow capillary microcoil (1)H NMR spectroscopy employing a microcoil with a detection volume of 1.5 µl. The observed changes of the (1)H NMR chemical shifts indicate the formation of a heterochiral (R,S) dimer of omeprazole via its sulfinyl group and the NH group of the benzimidazole ring.
Asunto(s)
Antiulcerosos/química , Espectroscopía de Resonancia Magnética/instrumentación , Omeprazol/química , Dimerización , EstereoisomerismoRESUMEN
An overview upon modern analytical techniques for the isolation, separation, and structural identification of the essential bioactive carotenoid bixin is given. Isolation from biological matrices is performed by matrix solid phase dispersion (MSPD). The extract is separated with shape-selective C(30 )columns. Structural assignment of the separated compounds is done by online LC-MS and capillary HPLC-NMR.
Asunto(s)
Carotenoides/análisis , Carotenoides/química , Cromatografía Liquida/métodos , Espectroscopía de Resonancia Magnética/métodos , Espectrometría de Masas/métodos , Extractos Vegetales/análisis , Acetona/análisis , Cromatografía Líquida de Alta Presión , Diseño de Equipo , Radicales Libres , Modelos Químicos , Extractos Vegetales/química , Isoformas de ProteínasRESUMEN
The major autolysin of Staphylococcus aureus (AtlA) and of Staphylococcus epidermidis (AtlE) are well-studied enzymes. Here we created an atlA deletion mutant in S. aureus that formed large cell clusters and was biofilm-negative. In electron micrographs, the mutant cells were distinguished by rough outer cell surface. The mutant could be complemented using the atlE gene from S. epidermidis. To study the role of the repetitive sequences of atlE, we expressed in Escherichia coli the amidase domain encoded by the gene, carrying no repeat regions (amiE) or two repeat regions (amiE-R1,2), or the three repeat regions alone (R1,2,3) as N-terminal His-tag fusion proteins. Only slight differences in the cell wall lytic activity between AmiE and AmiE-R1,2 were observed. The repetitive sequences exhibit a good binding affinity to isolated peptidoglycan and might contribute to the targeting of the amidase to the substrate. AmiE and AmiE-R1,2 have a broad substrate specificity as shown by similar activities with peptidoglycan lacking wall teichoic acid, O-acetylation, or both. As the amidase activity of AtlA and AtlE has not been proved biochemically, we used purified AmiE-R1,2 to determine the exact peptidoglycan cleavage site. We provide the first evidence that the amidase indeed cleaves the amide bond between N-acetyl muramic acid and L-alanine.
Asunto(s)
N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , Staphylococcus aureus/metabolismo , Staphylococcus epidermidis/metabolismo , Adhesión Bacteriana , Eliminación de Gen , Genes Bacterianos , Prueba de Complementación Genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hidrólisis , Mutagénesis , N-Acetil Muramoil-L-Alanina Amidasa/genética , Peptidoglicano/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Recombinación Genética , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidad , Staphylococcus epidermidis/genética , Staphylococcus epidermidis/patogenicidad , VirulenciaRESUMEN
Thermal and iodine-catalyzed photochemical (Z/E)-isomerization of deoxylutein II [(3R,6'R)-3-hydroxy-3',4'-didehydro-beta,gamma-carotene, anhydrolutein I] (2), the dehydration product of lutein [(3R,3'R,6'R)-beta,epsilon-carotene-3,3'-diol] (4), yielded multi-component mixtures of (Z)-isomers. By I(2)-catalyzed photoisomerization, (9Z)-2, (9'Z)-2, (13Z)-2, (13'Z)-2 and (15Z)-2 are generated as main products. In addition, this thermodynamic-equilibrium mixture contains traces of (9Z,9'Z)-2 and other (di-Z)-isomers in minor concentrations. Thirteen isomers are chromatographically separated and detected on-line by UV-vis and mass spectrometry. (all-E)-Deoxylutein II (2) and six of its (Z)-configured isomers are separated by capillary HPLC (acetone-d(6)/D(2)O = 85:15) and detected on-line by (1)H NMR spectroscopy in a microprobe. With the microprobe and the active detection volume of 1.5 microl, it is possible to perform structure elucidation with very small amounts available for various (Z)-isomers of deoxylutein II (2) in the isomerization mixture.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Luteína/análogos & derivados , Resonancia Magnética Nuclear Biomolecular/métodos , Cromatografía Líquida de Alta Presión/instrumentación , Isomerismo , Luteína/química , Resonancia Magnética Nuclear Biomolecular/instrumentación , Sistemas en Línea , FotoquímicaRESUMEN
HPLC atmospheric pressure chemical ionization (APCI)/MS, GC MS, HPLC diode array detection (DAD), and NMR were used for the identification of astaxanthin and astaxanthin fatty acid esters in krill (Euphausia superba Dana). Matrix solid phase dispersion was applied for the extraction of the carotenoids. This gentle and expeditious extraction technique for solid and viscous samples leads to distinct higher enrichment rates than the conventional liquid-liquid extraction. The chromatographic separation was achieved employing a C30 RP column that allows the separation of shape-constrained geometrical isomers. A methanol/tert-butylmethyl ether/water gradient was applied. (all-E) Astaxanthin and the geometrical isomers were identified by HPLC APCI/MS, by coelution with isomerized authentical standard, by UV spectroscopy (DAD), and three isomers were unambiguously assigned by microcoil NMR spectroscopy. In this method, microcoils are transversally aligned to the magnetic field and have an increased sensitivity compared to the conventional double-saddle Helmholtz coils, thus enabling the measurement on small samples. The carotenol fatty acid esters were saponified enzymatically with Lipase type VII from Candida rugosa. The fatty acids were detected by GC MS after transesterification, but also without previous derivatization by HPLC APCI/MS. C14:0, C16:0, C16:1, C18:1, C20:0, C20:5, and C22:6 were found in astaxanthin monoesters and in astaxanthin diesters. (all-E) Astaxanthin was identified as the main isomer in six fatty acid ester fractions by NMR. Quantitation was carried out by the method of internal standard. (13-cis) Astaxanthin (70 microg/g), 542 microg/g (all-E) astaxanthin, 36 microg/g unidentified astaxanthin isomer, 62 microg/g (9-cis) astaxanthin, and 7842 microg/g astaxanthin fatty acid esters were found.
Asunto(s)
Euphausiacea/química , Animales , Carotenoides/aislamiento & purificación , Cromatografía Líquida de Alta Presión/métodos , Ácidos Grasos/análisis , Espectroscopía de Resonancia Magnética , Espectrometría de Masas/métodos , Xantófilas/aislamiento & purificaciónRESUMEN
The development of miniaturized hyphenated systems such as capillary high-performance liquid chromatography--and nuclear magnetic resonance spectroscopy (HPLC-NMR) remains challenging in the field of structure elucidation. In combination with a highly specific sample preparation technique, matrix solid-phase dispersion (MSPD), and a highly selective C30 reverse phase HPLC-NMR enables the identification of small amounts of natural compounds. Here, the investigation of five carotenoids in a standard solution and two carotenoids from a spinach sample demonstrate the potential of this new development. The separation of the carotenoids is performed with self-packed fused-silica capillaries with a binary solvent gradient consisting of acetone and water. The miniaturized system allows the use of fully deuterated solvents for on-line HPLC-NMR coupling. The 1H NMR spectra of the various carotenoids obtained in stopped-flow mode gave a high signal-to-noise ratio with a sample amount in the low nanogram range. All necessary parameters for structure elucidation such as multiplet structure, coupling constants and integration values can be detected unambiguously.
Asunto(s)
Carotenoides/aislamiento & purificación , Cromatografía Líquida de Alta Presión/métodos , Espectroscopía de Resonancia Magnética/métodos , Spinacia oleracea/química , Tamaño de la MuestraRESUMEN
The protein kinase ZAP-70 is involved in T-cell activation and interacts with tyrosine-phosphorylated peptide sequences known as immunoreceptor tyrosine activation motifs (ITAMs). We have studied the regulatory phosphorylation sites in the tryptic fragment containing amino acids 485-496 (ALGADDSYYTAR). The four possible peptides with phosphorylation at none, one, or both of the Y-492 and Y-493 tyrosines were specifically synthesized and analyzed by (1)H/(13)C-NMR at 600 MHz using a capillary HPLC-NMR microprobe. Unambiguous discrimination of the peptides was possible via effect of chemical shifts of phosphorylation on the aromatic tyrosine protons. With the microprobe and the detection volume of 1.5 microl, it was possible to perform structure elucidation with the very small amounts available for the various peptides. For the syringe injection, 15 microg of the analyte were used (corresponding to ca 2 mg in classical 5-mm tubes). Capillary HPLC-NMR spectra were recorded in the stopped-flow mode from less than 400 ng of each peptide, using 1D and 2D techniques ((1)H,(1)H-COSY-90, (1)H/(13)C-HSQC, and (1)H/(13)C-HMBC).
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Espectroscopía de Resonancia Magnética/métodos , Proteínas Tirosina Quinasas/análisis , Aminoácidos/química , Aminoácidos/metabolismo , Microquímica , Péptidos/análisis , Péptidos/química , Péptidos/metabolismo , Fosforilación , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/metabolismo , Sensibilidad y Especificidad , Tirosina/química , Tirosina/metabolismo , Proteína Tirosina Quinasa ZAP-70RESUMEN
Most Staphylococcus aureus strains produce the orange carotenoid staphyloxanthin. The staphyloxanthin biosynthesis genes are organized in an operon, crtOPQMN, with a sigma(B)-dependent promoter upstream of crtO and a termination region downstream of crtN. The functions of the five encoded enzymes were predicted on the basis of their sequence similarity to known enzymes and by product analysis of gene deletion mutants. The first step in staphyloxanthin biosynthesis is the head-to-head condensation of two molecules of farnesyl diphosphate to form dehydrosqualene (4,4'-diapophytoene), catalyzed by the dehydrosqualene synthase CrtM. The dehydrosqualene desaturase CrtN dehydrogenates dehydrosqualene to form the yellow, main intermediate 4,4'-diaponeurosporene. CrtP, very likely a mixed function oxidase, oxidizes the terminal methyl group of 4,4'-diaponeurosporene to form 4,4'-diaponeurosporenic acid. CrtQ, a glycosyltransferase, esterifies glucose at the C(1)'' position with the carboxyl group of 4,4'-diaponeurosporenic acid to yield glycosyl 4,4'-diaponeurosporenoate; this compound was the major product in the clone expressing crtPQMN. In the final step, the acyltransferase CrtO esterifies glucose at the C(6)'' position with the carboxyl group of 12-methyltetradecanoic acid to yield staphyloxanthin. Staphyloxanthin overexpressed in Staphylococcus carnosus (pTX-crtOPQMN) and purified was analyzed by high pressure liquid chromatography-mass spectroscopy and NMR spectroscopy. Staphyloxanthin was identified as beta-D-glucopyranosyl 1-O-(4,4'-diaponeurosporen-4-oate)-6-O-(12-methyltetradecanoate).