Heterocycles [h]fused onto 4-oxoquinoline-3-carboxylic acid, part IV. Convenient synthesis of substituted hexahydro [1,4]thiazepino[2,3-h]quinoline-9-carboxylic acid and its tetrahydroquino[7,8-b]benzothiazepine homolog.

Substituted [1,4]thiazepino[2,3-h]quinolinecarboxylic acid 3 is prepared by PPA-catalyzed thermal lactamization of the respective 8-amino-7-[(2-carboxyethyl)thio]-1,4-dihydroquinoline-3-carboxylic acid 9. The latter synthon is obtained by reduction of the 8-nitro-1,4-dihydroquinoline precursor 8 which, in turn, is made accessible via interaction of 3-mercaptopropionic acid with 7-chloro-1-cyclopropyl-6-fluoro-8-nitro-1,4-dihydroquinoline-3-carboxylic acid 7 in the presence of triethylamine. A benzo-homolog of 3, namely tetrahydroquino[7,8-b]benzothiazepine-3-carboxylic acid 6, is analogously prepared via the reaction of 2-mercaptobenzoic acid with 7, followed by reduction of the resulting 7-[(2-carboxyphenyl)thio]-8-nitro product 10 into the corresponding 8-amino derivative 11, and subsequent lactamization. The structures assigned to 3, 6 and 8-11 are based on microanalytical and spectral (IR, MS, NMR) data.

A novel approach for ganglioside structural analysis based on electrospray multiple-stage mass spectrometry.

A powerful method for detailed structural analysis based on electrospray ionization high-capacity ion-trap multiple-stage mass spectrometry (MS) is for the first time introduced in glycolipidomics. The method was optimized for accurate structural elucidation of human brain gangliosides and specifically applied to normal adult human hippocampus-associated structures. The multiple-stage MS experiments reported here allowed for a complete structural characterization of the oligosaccharide moiety of a GM1 ganglioside species. This was achieved by elucidating the sequence and identification of the GM1a structural isomer from the sialic acid attachment site at the neutral oligosaccharide chain. Moreover, the determination of the d18:1/18:0 sphingoid base/fatty acid composition of the ceramide moiety could be confirmed by this method. The novel protocol developed here proves high potential for rapid, reliable, and reproducible investigation of complex lipid-linked carbohydrates such as polysialylated gangliosides or species carrying some other groups that easily cleave off.

Facile synthesis of some novel pyrido[3',2':4,5]thieno[2,3-b][1,4]thiazine-8-carboxylic acids.

Model tetrahydropyrido[3',2':4,5]thieno[2,3-b][1,4]thiazines 9a-c were synthesized via reductive lactamization, using sodium dithionite, of the respective 2-[(carboxyalkyl)thio]-3-nitro-4,7-dihydrothieno[2,3-b]pyridine-5-carboxylic acids 7a-c. The latter derivatives were made via interaction of 2-chloro-7-cyclopropyl-3-nitro-4,7-dihydrothieno[2,3-b]pyridine-5-carboxylic acid (6) with each of alpha-mercaptoacetic, alpha-mercaptopropionic, and alpha-mercaptosuccinic acids and triethylamine in aqueous acetone at room temperature. The structures of 7a-7c and 9a-9c are supported by microanalytical and spectral (IR, MS, NMR) data. Compounds 9a and 9c showed potent inhibitory activity against the IGROV1 (Ovarian Cancer) cell line.

Even-electron ions: a systematic study of the neutral species lost in the dissociation of quasi-molecular ions.

The collision-induced dissociations of the even-electron [M + H](+) and/or [M - H](-) ions of 121 model compounds (mainly small aromatic compounds with one to three functional groups) ionized by electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) have been studied using an ion trap instrument, and the results are compared with the literature data. While some functional groups (such as COOH, COOCH(3), SO(3)H in the negative ion mode, or NO(2) in both the positive and negative ion modes) generally promote the loss of neutrals that are characteristic as well as specific, other functional groups (such as COOH in the positive ion mode) give rise to the loss of neutrals that are characteristic, but not specific. Finally, functional groups such as OH and NH(2) in aromatic compounds do not lead to the loss of a neutral that reflects the presence of these substituents. In general, the dissociation of [M + H](+) and [M - H](-) ions generated from aliphatic compounds or compounds containing an aliphatic moiety obeys the even-electron rule (loss of a molecule), but deviations from this rule (loss of a radical) are sometimes observed for aromatic compounds, in particular for nitroaromatic compounds. Thermochemical data and ab initio calculations at the CBS-QB3 level of theory provide an explanation for these exceptions. When comparing the dissociation behaviour of the even-electron [M + H](+) and/or [M - H](-) ions (generated by ESI or APCI) with that of the corresponding odd-electron [M](+) ions (generated by electron ionization, EI), three cases may be distinguished: (1) the dissociation of the two ionic species differs completely; (2) the dissociation involves the loss of a common neutral, yielding product ions differing in mass by one Da, or (3) the dissociations lead to a common product ion.

Oligosaccharide analysis by capillary-scale high-pH anion-exchange chromatography with on-line ion-trap mass spectrometry.

A capillary-scale high-pH anion-exchange chromatography (HPAEC) system for the analysis of carbohydrates was developed, in combination with two parallel on-line detection methods of sub-picomolar sensitivity: (1) pulsed amperometric detection (PAD); (2) capillary-scale desalting followed by electrospray ion-trap (IT) mass spectrometry (MS). The capillary chromatographic system combined the superb selectivity of HPAEC that allows routine separation of isomeric oligosaccharides with the information on monosaccharide sequence and linkage positions obtained by MS/MS fragmentation using the IT-MS. The applicability of the system in biomedical research was demonstrated by its use for the analysis of a urine sample of a GM1-gangliosidosis patient. Isomeric glycans in the sample could be resolved by HPAEC and assigned on the basis of the monosaccharide linkage information revealed by on-line IT-MS/MS.

Operational variables in high-performance liquid chromatography-electrospray ionization mass spectrometry of peptides and proteins using poly(styrene-divinylbenzene) monoliths.

Capillary reversed-phase high-performance liquid chromatography (RP-HPLC) utilizing monolithic poly(styrene-divinylbenzene) columns was optimized for the coupling to electrospray ionization mass spectrometry (ESI-MS) by the application of various temperatures and mobile phase additives during peptide and protein analysis. Peak widths at half height improved significantly upon increasing the temperature and ranged from 2.0 to 5.4 s for peptide and protein separations at 70 degrees. Selectivity of peptide elution was significantly modulated by temperature, whereas the effect on proteins was only minor. A comparison of 0.10% formic acid (FA), 0.050% trifluoroacetic acid (TFA), and 0.050% heptafluorobutyric acid (HFBA) as mobile phase additives revealed that highest chromatographic efficiency but poorest mass spectrometric detectabilities were achieved with HFBA. Clusters of HFBA, water, and acetonitrile were observed in the mass spectra at m/z values >500. Although the signal-to-noise ratios for the individual peptides diverged considerably both in the selected ion chromatograms and extracted mass spectra, the average mass spectrometric detectabilities varied only by a factor of less than 1.7 measured with the different additives. Limits of detection for peptides with 500 nl sample volumes injected onto a 60 mm x 0.20 mm monolithic column were in the 0.2-13 fmol range. In the analysis of hydrophobic membrane proteins, HFBA enabled highest separation selectivity at the cost of lower mass spectral quality. The use of 0.050% TFA as mobile phase additive turned out to be the best compromise between chromatographic and mass spectrometric performance in the analysis of peptides and proteins by RP-HPLC-ESI-MS using monolithic separation columns.

[Methyl-D3]2hypericin as internal standard for quantification in human plasma.

The multistep synthesis and negative ion-ESI fragmentation pattern of [methyl-D(3)](2)hypericin (1-D(6)) is described. The application of 1-d(6) as internal standard for the quantification of hypericin (1) in the ng mL(-1) range in human plasma by isotope-dilution LC-MS is demonstrated. The hypericin-containing plasma samples are spiked with 1-D(6), deproteinized and extracted with ethyl acetate. The extracts are injected into a HPLC-ESI-ion-trap system and the mass-separated negative ions from 1 and 1-D(6) are analysed. From their intensities linear standard curves over the concentration range from 1 to 10 ng mL(-1) are obtained. Accuracy, precision and recovery are discussed.