Heme d1 is a modified tetrapyrrole playing an important role in denitrification by acting as the catalytically essential cofactor in the cytochrome cd1 nitrite reductase of many denitrifying ...bacteria. In the course of heme d1 biosynthesis, the two propionate side chains on pyrrole rings A and B of the intermediate 12,18‐didecarboxysiroheme are removed from the tetrapyrrole macrocycle. In the final heme d1 molecule, the propionate groups are replaced by two keto functions. Although it was speculated that the Radical S‐adenosyl‐l‐methionine (SAM) enzyme NirJ might be responsible for the removal of the propionate groups and introduction of the keto functions, this has not been shown experimentally, so far. Here, we demonstrate that NirJ is a Radical SAM enzyme carrying two iron–sulfur clusters. While the N‐terminal 4Fe‐4S cluster is essential for the initial SAM cleavage reaction, it is not required for substrate binding. NirJ tightly binds its substrate 12,18‐didecarboxysiroheme and, thus, can be purified in complex with the substrate. By using the purified NirJ/substrate complex in an in vitro enzyme activity assay, we show that NirJ indeed catalyzes the removal of the two propionate side chains under simultaneous SAM cleavage. However, under the reaction conditions employed, no keto group formation is observed indicating that an additional cofactor or enzyme is needed for this reaction.
The heme d1 biosynthesis enzyme NirJ belongs to the Radical SAM protein family and binds two iron–sulfur clusters. One of the clusters is required for the typical SAM cleavage reaction. Overall NirJ catalyzes the removal of two propionate side chains from the substrate 12,18‐didecarboxysiroheme.
Different phenylenediamines were used to explore anodic oxidation in solution during electrospray ionization (ESI) mass spectrometry analysis. In our experiments, a series of unknown ionic species ...was detected in the phenylenediamine solutions. Our results propose that reactions of phenylenediamines with species formed by anodic oxidation of typical ESI solvents during the electrospray ionization process such as formaldehyde are producing these peaks. Identification of these compounds inter alia suggests formal alkylation, a reaction not reported so far as a result of electrolytic oxidation in the prospective organic solvents.
•The first reported LC-UHR-QqTOF method for identification of the simeprevir's reactive metabolites.•LC-MS/MS method for determination of simeprevir’s metabolic stability.•Nineteen simeprevir ...metabolites were identified by the proposed LC-UHR-QqTOF method.•Two different chromatographic methods have been established.
Liquid chromatography coupled to a triple quadrupole and, alternatively, to an ultrahigh-resolution quadrupole time-of-flight (UHR-QqTOF) mass spectrometers was used to collect qualitative and quantitative information from incubations of the anti-hepatitis C drug simeprevir with human and rat liver microsomes, respectively, supplemented with NADPH and glutathione. For this, different chromatographic methods using two different chromatographic columns, Kinetex® 2.6 µm C18 (50 × 3 mm) and Atlantis T3 (100 Å, 3 µm, 4.6 mm × 150 mm), have been employed. For determination and structural characterization of the reactive metabolites, we used information obtained from high-resolution mass spectrometry, namely accurate mass data to calculate the elemental composition, accurate MS/MS fragmentation patterns for confirmation of structural proposals, and the high mass spectral resolution to eliminate false-positive peaks. In this study, the use of high-resolution mass spectrometry (HR-MS) enabled the identification of 19 simeprevir metabolites generated by O- respectively N-demethylation, oxidation, dehydrogenation, hydrolysis, and formation of glutathione conjugates. The in silico study provides insights into the sites of simeprevir most amenable to reactions involving cytochrome P450. The developed methods have been successfully applied to analyze simeprevir and its metabolites simultaneously; based on this data, potential metabolic pathways of simeprevir are discussed. In general, the obtained results demonstrate that simeprevir is susceptible to form reactive simeprevir-glutathione adducts and cyclopropansulfonamide, which may explain the implication of simeprevir in idiosyncratic adverse drug reactions (IADRs) or hepatotoxicity.
Raffinose wurde aus den Samen der Blauen Süßlupine durch Extraktion mit 50% Ethanol gewonnen und über eine Kationen‐Austauschersäule sowie schließlich durch analytische HPLC mit Hilfe eines ...RI‐Detektors gereinigt. Alle analytischen Spektren sind vollständig entweder im Hauptteil oder in der supporting information wiedergegeben. Die NMR‐ sowie die Massenspektren werden eingehend interpretiert und mit theoretischen Berechnungen der 13C chemischen Verschiebungen verglichen. Das Projekt stellt eine Fortsetzung des Buches “Classics in Spectroscopy” von S. Berger und D. Sicker (Wiley‐VCH 2009) dar.
Raffinose has been obtained by extraction with 50% ethanol from the seeds of the Sweet Blue Lupine and purified first via a Cation exchange column and finally by analytical HPLC using a RI‐detector. All analytical spectra were recorded and are reproduced either in the main part or in the supporting information. The NMR‐ and mass‐spectra have been interpreted and compared with theoretical calculations of the 13C chemical shifts. The project is a follow up of the recent book “Classics in Spectroscopy” by S. Berger und D. Sicker (Wiley‐VCH 2009).
Meist hat der Volksmund ja recht: “Jedes Böhnchen – ein Tönchen...”. Aber warum nur setzen uns Hülsenfrüchte derart unter Druck? Das liegt am reichlich enthaltenen Trisaccharid Raffinose. Für uns ist es unverdaulich, weil wir nicht über das Enzym α‐Galactosidase verfügen. So wird Raffinose zu einem gefundenen Fressen für Darmbakterien, die sie vergären können, wobei aber Gase wie Methan entstehen. Raffinose steht über das Enzym Raffinase in enger Beziehung zu Saccharose, der weltweit in größter Menge durch Kristallisation gereinigten “Chemikalie”: 175 Mio. Tonnen/Jahr. Es ist der “Raffinade‐Zucker”, den wir verzehren. Wir beschreiben Raffinose in der Welt der Saccharide, die Isolierung aus Lupinensamen und alle analytischen Spektren.
Abstract
Raffinose wurde aus den Samen der Blauen Süßlupine durch Extraktion mit 50% Ethanol gewonnen und über eine Kationen‐Austauschersäule sowie schließlich durch analytische HPLC mit Hilfe eines ...RI‐Detektors gereinigt. Alle analytischen Spektren sind vollständig entweder im Hauptteil oder in der supporting information wiedergegeben. Die NMR‐ sowie die Massenspektren werden eingehend interpretiert und mit theoretischen Berechnungen der
13
C chemischen Verschiebungen verglichen. Das Projekt stellt eine Fortsetzung des Buches “Classics in Spectroscopy” von S. Berger und D. Sicker (Wiley‐VCH 2009) dar.
Raffinose has been obtained by extraction with 50% ethanol from the seeds of the Sweet Blue Lupine and purified first via a Cation exchange column and finally by analytical HPLC using a RI‐detector. All analytical spectra were recorded and are reproduced either in the main part or in the supporting information. The NMR‐ and mass‐spectra have been interpreted and compared with theoretical calculations of the
13
C chemical shifts. The project is a follow up of the recent book “Classics in Spectroscopy” by S. Berger und D. Sicker (Wiley‐VCH 2009).
1α-Amino-1,6,9-trideoxy forskolin was synthesized starting from drimenal and an isoprenoid C
5 unit. A tricyclic labdane with the entire forskolin skeleton was available in only four steps. Barton's ...nitrite photolysis was applied to functionalize C-1.
Graphic
The interaction of a moenomycin derivative with the enzyme penicillin binding protein 1b (PBP 1b) has been studied by means of STD NMR. The results obtained initiated the synthesis of a number of ...moenomycin derivatives modified in unit A including a moenomycin-ampicillin conjugate and determination of their antibiotic activities. A protocol is described that allows studying the interaction of moenomycin analogues with PBP 1b by fluorescence correlation spectroscopy.
A new approach for the synthesis of moenomycin trisaccharide analogues is reported in which three monosaccharide building blocks are used and allows the introduction of different substituents at the ...6‐position of unit E (Scheme 2). Furthermore, a new procedure for the introduction and manipulation of unit F is described (Scheme 3).
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