Palladium catalyzed reductions play an important role in many organic synthetic processes. When combined with flow chemistry, they offer an efficient approach for modifying reduction-sensitive functional groups. The preparation and modification of pyrrolidine-based paramagnetic building blocks require more vigorous conditions than their unsaturated analogues, and yields are generally low. The objective of this study is to demonstrate that flow-chemical reduction provides a viable method for obtaining nitroxide stable free radicals with saturated rings from their unsaturated counterparts. Because reduction of the nitroxide group is unavoidable during palladium catalyzed hydrogenation, a second aim is to investigate the behavior of the nitroxide group under varying pressure and temperature conditions in flow systems. While saturation of the carbon–carbon double bond in pyrroline aldehydes or esters and reduction of the nitroxide to the corresponding hydroxylamine occur readily under mild conditions, conversion of the nitroxide to the amine in significant quantities requires elevated temperatures. This suggests that pressure can be used to control secondary functional group reductions while avoiding overreduction of the nitroxide. We anticipate that this work will contribute to the selective synthesis of pyrrolidine- and piperidine-type nitroxide stable free radicals using a green methodology.

Imidazo[1,2-a]pyridines possessing carboxamido and ester functionalities in 2- and 6-positions were synthesised in palladium-catalysed amino- and alkoxy/aryloxycarbonylation using a great variety of amines and alcohols/ phenols as N- and O-nucleophiles, respectively. The corresponding iodoheteroaromatics, used as substrates, were synthesised from the substituted 2-aminopyridines, terminal alkynes and iodine in copper-catalysed oxidative ring-closure-iodination reaction sequence. Mono- and dinuclear copper-2-aminopyridine complexes, used as pre-formed catalysts, were characterised by X-ray crystallography. The amides and esters were obtained in moderate to high yields mainly depending on the nucleophile and not on the structure of the 2-iodo[1,2-a] pyridine substrates.

The toxic nature of bacterial endotoxins is affected by the structural details of lipid A, including the variety and position of acyl chains and phosphate group(s) on its diglucosamine backbone. Negative-ion mode tandem mass spectrometry is a primary method for the structure elucidation of lipid A, used independently or in combination with separation techniques. However, it is challenging to accurately characterize constitutional isomers of lipid A extracts by direct mass spectrometry, as the elemental composition and molecular mass of these molecules are identical. Thus, their simultaneous fragmentation leads to a composite, so-called chimera mass spectrum. The present study focuses on the phosphopositional isomers of the classical monophosphorylated, hexaacylated Escherichia coli-type lipid A. Collision-induced dissociation (CID) was performed in an HPLC-ESI-QTOF system. Energy-resolved mass spectrometry (ERMS) was applied to uncover the distinct fragmentation profiles of the phosphorylation isomers. A fragmentation strategy applying multi-levels of collision energy has been proposed and applied to reveal sample complexity, whether it contains only a 4′-phosphorylated species or a mixture of 1- and 4′-phosphorylated variants. This comparative fragmentation study of isomeric lipid A species demonstrates the high potential of ERMS-derived information for the successful discrimination of co-ionized phosphorylation isomers of hexaacylated lipid A.

Urbanization with reduced microbial exposure is associated with an increased burden of asthma and atopic symptoms. Conversely, environmental exposure to endotoxins in childhood can protect against the development of allergies. Our study aimed to investigate whether the renaturation of the indoor environment with aerosolized radiation-detoxified lipopolysaccharide (RD-LPS) has a preventative effect against the development of ragweed-induced Th2-type airway inflammation. To explore this, cages of six-week-old BALB/c mice were treated daily with aerosolized native LPS (N-LPS) or RD-LPS. After a 10-week treatment period, mice were sensitized and challenged with ragweed pollen extract, and inflammatory cell infiltration into the airways was observed. As dendritic cells (DCs) play a crucial role in the polarization of T-cell responses, in our in vitro experiments, the effects of N-LPS and RD-LPS were compared on human monocyte-derived DCs (moDCs). Mice in RD-LPS-rich milieu developed significantly less allergic airway inflammation than mice in N-LPS-rich or common environments. The results of our in vitro experiments demonstrate that RD-LPS-exposed moDCs have a higher Th1-polarizing capacity than moDCs exposed to N-LPS. Consequently, we suppose that the aerosolized, non-toxic RD-LPS applied in early life for the renaturation of urban indoors may be suitable for the prevention of Th2-mediated allergies in childhood.

The carotenoid composition of petals, florets, and full inflorescences of Calendula officinalis grown in sun and shade was investigated by the HPLC-DAD-MS method. The total carotenoid content of flowers grown in the shade was higher than those grown in the sun (1.154 and 0.872 mg/g in petals), while no differences were found in the proportion of individual carotenoids. In all samples, 29 components were identified, from which the main carotenoids, besides lutein, were 5,8-epoxy-carotenoids with 8S and 8 ' R configurations. The main 5,8-epoxy-carotenoid was chrysanthemaxanthin with the 8S configuration and not flavoxanthin with the 8R configuration, as published earlier. In addition, (all-E,8 ' R)- and (9Z,8 ' R)-luteoxanthin were detected in larger amounts. The 5,8-epoxy-carotenoids (flavoxanthin, chrysanthemaxanthin, and luteoxanthin epimers) were also prepared via an acid-catalyzed reaction of the parent carotenoid 5,6-epoxides. The structures of the epimers were elucidated by NMR measurements.

Lutein and its cis-isomers occur in a lot of plants, including a variety of flowers. In this study, lutein isomers were produced via iodine-catalyzed isomerization, and four cis-isomers (9Z-, 9′Z-, 13Z-, and 13Z′) were isolated by means of column chromatography and semipreparative HPLC. The structures of the 9′Z- and 13′Z-isomers were elucidated via NMR measurements. These compounds were used as standards for the HPLC-DAD-MS determination of the carotenoid composition of the flowers of 20 plant species, in which lutein and its geometrical isomers are the main components. The flowers showed great variation in their cis- and trans-lutein content, and also in the presence or absence of other carotenoids, such as violaxanthin, neoxanthin, β-cryptoxanthin, and β-carotene. Some of the investigated flowers were found to be rich sources of lutein without zeaxanthin.

Pseudomonas aeruginosa is a priority target pathogen for antibiotic research and development because of its high resistance to a wide range of antibiotics. Acquisition of resistance is sometimes associated with modifications of the lipid A component present in the outer membrane of most Gram-negative bacteria. For a deeper understanding of subtle chemical variations of lipid A in relation to biological properties, the structural elucidation of lipid A species within a bacterial strain is of crucial importance. However, the simultaneous presence of monophosphorylated positional isomers in naturally heterogeneous lipid A samples cannot easily be recognized with direct MS measurements or with conventional LC-MS strategies. Herein, we present the application of a non-aqueous capillary electrophoresis (NACE) method coupled to positive and negative ion electrospray tandem mass spectrometry with collision-induced dissociation (CID) activation technique for the in-depth analysis of the lipid A isolate of P. aeruginosa PAO1. The main advantage of our NACE strategy is its separation power regarding the site of phosphorylation of the lipid A structures, which makes it a good orthogonal technique to chromatographic characterizations of the bacterial lipid A composition, where the separation is mainly based on acylation differences of the species. Overall, the results of this electrophoretic approach revealed hitherto unreported isomeric monophosphorylated PAO1 lipid A constituents, including both phosphate and acyl chain positional isomers. The parallel fragmentation in the complementary positive and negative ion modes enabled the unequivocal assignment of the phosphorylation site and position of acyl chains in lipid A compounds of three acylation families ranging from tetra- to hexa-acylation. Moreover, C1-monophosphorylated lipid A species have been identified in P. aeruginosa for the first time.

The sterically highly un-favoured position-7 of a steroidal skeleton was functionalised in a reaction sequence of conventional synthetic reactions and palladium-catalysed aminocarbonylation. The synthesis was based on the use of chenodeoxycholanic acid as starting material, which was transformed to 24-carboxamide, followed by oxidation of 7-hydroxy group. The resulting 7-oxo functionality was converted to the corresponding iodoalkene by the Barton's method. The 7-iodo-6-ene functionality underwent high-yielding aminocarbonylation in the presence of palladium-phosphine in situ catalysts and various amines as N-nucleophiles. The new 7-carboxamido compounds were obtained in good isolated yields, via highly chemoselective reactions, under relatively mild conditions.

The carotenoid composition of the flower of Telekia speciosa was investigated for the first time by HPLC-DAD-MS. In addition to the main carotenoid lutein and its geometrical isomers, 5,6-epoxy-carotenoids, namely violaxanthin, lutein 5,6-epoxide and antheraxanthin, were detected in larger amounts. In addition, β-carotene 5,6-epoxide and β-carotene 5,6,5′,6′-diepoxide were found, which occurs very rarely in plants. For unambigous identification, β-carotene 5,6-epoxide and β-carotene 5,6,5′,6′-diepoxide were prepared semisynthetically, and they were characterized by 1H and 13C NMR and HPLC-CD methods.

This paper presents the genome sequence of a Shigella sonnei mutant strain (S. sonnei 4351) and the effect of mutation in lipopolysaccharide biosynthesis on bacterial fitness. Lipopolysaccharides are the major component of the outer leaflet of the Gram-negative outer membrane. We report here a frameshift mutation of the gene gmhD in the genome of S. sonnei 4351. The mutation results in a lack of epimerization of the core heptose while we also found increased thermosensitivity, abnormal cell division, and increased susceptibility to erythromycin and cefalexin compared to the S. sonnei 4303. Comparative genomic analysis supplemented with structural data helps us to understand the effect of specific mutations on the virulence of the bacteria and may provide an opportunity to study the effect of short lipopolysaccharides.

Lipid A, the inflammatory portion of lipopolysaccharides (LPS, endotoxins), is the main component of the outer membrane of Gram-negative bacteria. Its bioactivity in humans and animals is strictly related to its chemical structure. In the present work, the fragmentation patterns of the singly charged monosodium [M + Na]+ and disodium [M - H + 2Na]+ adducts, as well as the protonated form of monophosphorylated lipid A species were investigated in detail using positive-ion electrospray ionization-based tandem (MS/MS) and multistage mass spectrometry (MSn) with low-energy collision-induced dissociation (CID). Several synthetic and native lipid A samples were included in the study. We found that the fragmentation pattern of disodiated lipid A is quite similar to that of the well-characterized deprotonated lipid A molecule (typically detected in the negative-ion mode), while the fragmentation pattern of monosodiated lipid A contains fragment ions similar to those of both protonated and deprotonated lipid A molecules. In summary, we propose a new mass spectrometry approach based on the fragmentation regularities of only positively charged precursor ions to dissect the location of the phosphate group and fatty acid moieties on monophosphorylated lipid A. Moreover, this study provides a better understanding of the so-called "chimera mass spectra", which are commonly detected during the fragmentation of native lipid A samples containing both C-1 and C-4' phosphate positional isomers but rarely identified in negative-ion mode.

Jipi-japa (Carludovica palmata) and zamia (Zamia dressier() are endemic species and are found mostly in the forests of Central America including Panama. In theory both the jipi-japa fruit and the stem and seeds of zamia are edible, although were only consumed occasionally mostly before modern times. The complete and detailed carotenoid analysis of the fruits of jipi-japa and the brown leaves of zamia was achieved using HPLC-DAD-MS technique, and co-chromatography with authentic samples. Plant extracts were subjected to HPLC separation on both C30 and C18 columns to make possible a better identification. Altogether 22 components were detected and 15 identified from the total extract of jipi-japa, and 32 components were detected and 17 identified from the extract of brown zamia leaves. From the identified carotenoids quite a number has special structures, which have recently been described in some of our previous papers on Central American plants (Murillo et al., 2011a, 2018; Gulyds-Fekete et al., 2013). It was found that almost 50 % of the total carotenoid content can be attributed to capsorubin in both plants. This carotenoid is present usually as a minor (2-3 %) camtenoid in other sources such as red pepper. Other camtenoids with lc end group are present in significant amounts, as well. Total carotenoid content was determined as the sum of yellow and red carotenoids (see Supplement). The main carotenoid, capsorubin, was isolated by open column chromatography on calcium carbonate and aluminium oxide and characterized by UV-vis, H-1 and C-13 NMR methods and compared to literature data. Capsorubin is a potent antioxidant even among carotenoids and have important biological effects. These two plants could be good sources for capsorubin either for isolation in gram amounts or for direct consumption.

Lipid A represents a heterogeneous group of bacterial outer membrane phosphoglycolipids, which play a major role in the pathogenesis of Gram-negative sepsis. The number and position of phosphoryl and acyl groups in lipid A molecules are key structural determinants in their bioactivities. In this study, a NACE-ESI-MS/MS method was developed for the simultaneous analysis of lipid A isomers possessing a different degree of phosphorylation and acylation. Various C4'- and C1-monophosphorylated lipid A isobars, as well as acylation isomers, were baseline separated within 43 min in a separation medium of methanol/dichloromethane/triethylamine/acetic acid 60:40:1.08:0.36 (v/v/v/v). Both normal and reverse CE polarities could be applied for proper detection of the analytes owing to the combination of a suction effect caused by the nebulizer gas at the outlet end of the capillary and external pressure applied on the inlet vial. The separated lipid A species could be identified unequivocally by their characteristic fragmentation patterns through CID performed in both negative- and positive-ionization modes. The uniqueness of the NACE-ESI-MS/MS method lies in its simplicity and reliability for proving the phosphorylation isomerism (C1 or C4') and acylation pattern of native lipid A species or those designed for therapeutic applications.

Lipid A is the anchor of endotoxins on the surface of Gram-negative bacteria. In the human body, it is a prominent stimulator of the immune system, but it may also cause dangerous medical conditions, such as endotoxic shock and sepsis. To reveal the specific structural parts and molecular heterogeneity of lipid A isolates, such as the site of phosphorylation and type of fatty acyl chains, a pressure-assisted non-aqueous capillary electrophoresis – tandem mass spectrometry method was developed. Baseline separation of both phosphorylation and acylation isomers was achieved. Identification was carried out from the tandem mass spectra recorded in the positive ionization mode. B-type ions are formed by diagnostic neutral losses. B2 type ions are confirming the site of phosphorylation, while B1 type ions and other fragments are enabling the characterization of acylation isomers. This novel method should be regarded as an orthogonal technique to formerly developed LC‐MS/MS methods in the screening of bacterial samples or lipid A based therapeutics. Kivonat Az endotoxinok a lipid A reszukkel agyazodnak be a Gram-negativ bakteriumok sejtmembranjaba. Az emberi szervezetben a lipid A hatekonyan stimulalja az immunrendszert, de akar sulyos egeszsegi allapotokat is előidezhet, mint az endotoxikus sokk, vagy a szepszis. A szerkezetenek reszleteinek megismeresere es a lipid A izolatum alkotoinak valtozatossagat – ugymint a foszforilacios helyek es a kapcsolodo zsirsavlancok tipusat – feltarando kifejlesztettunk egy nyomassal segitett nemvizes kapillaris elektroforezis–tandem tomegspektrometrias modszert. A foszforilacios- es az acilacios izomereknel is sikeres alapvonali elvalasztast ertunk el. A szerkezeti informaciot a pozitiv ion modban felvett tandem tomegspektrumok szolgaltattak. Diagnosztikus semleges vesztesekkel B-tipusu ionok keletkeznek. A B2-tipusu ionok azonositjak a foszforilacios poziciot, mig a B1 ionok az acilacios izomerek jellemzeset teszik lehetőve. Jelen modszer a korabban kifejlesztett LC-MS/MS modszerek kiegeszitője lehet a bakterialis eredetű mintak vizsgalataban es a lipid A klinikai alkalmazasakor.

Fatty acids everywhere, ionic liquids and proteins, endotoxin structure and function. Kivonat Zsirsavak mindenhol, ionfolyadekok es feherjek, endotoxin szerkezet es funkcio.

Objective: Clinical manifestations of Gram-negative bacteria mediated diseases can be influenced by how the host senses their major microbe-associated molecular pattern, the cell wall lipopolysaccharide (LPS). Keystone periodontal pathogens can produce a heterogeneous population of LPS molecules, with strikingly different host-microbiome interactions and immune outcomes. Design: Structure-function correlations of salivary LPS extracts in patients with periodontitis before and after periodontal treatment and healthy volunteers were analysed by comparing its lipid A and carbohydrate chain chemical structure and evaluating its endotoxin activity and inflammatory potential. Results: Salivary LPS extracts from periodontitis patients were characterised by high m/z lipid A mass-spectrometry peaks, corresponding to over-acylated and phosphorylated lipid A ions and by a combination of rough and smooth LPS carbohydrate moieties. In contrast, gingival health was defined by the predominance of low m/z lipid A peaks, consistent with under-acylated and hypo-phosphorylated lipid A molecular signatures, with long and intermediate carbohydrate chains as determined by silver staining. Total, diseased salivary LPS extracts were stronger inducers of the recombinant factor C assay and triggered significantly higher levels of TNF-alpha, IL-8 and IP-10 production in THP-1 cells, compared to almost immunosilent healthy samples. Interestingly, salivary LPS architecture, endotoxin activity, and inflammatory potential were well conserved after periodontal therapy and showed similarities to diseased samples. Conclusions: This study sheds new light on molecular pathogenic mechanisms of oral dysbiotic communities and indicates that the regulation of LPS chemical structure is an important mechanism that drives oral bacteria-host immune system interactions into either a symbiotic or pathogenic relationship.

Much interest is at present focused on bacterial endotoxins, also known as lipopolysaccharides (LPS), as they are responsible for the development of clinical symptoms of Gram-negative sepsis which is the leading cause of death in intensive care units. Endotoxicity is associated with the special phosphoglycolipid part of LPS, termed lipid A. Main challenges in the structural elucidation of lipid A arise from its amphiphilic character and inherent heterogeneity. A mass spectrometry-based de novo method combined with reversed-phase liquid chromatography for the detailed structural characterization of complex lipid A mixtures (obtained by mild acid hydrolysis of LPS) from different bacterial sources has been developed. Tandem mass spectrometric measurements were performed with an electrospray-ionisation quadrupole time-of-flight (ESI-Q-TOF) mass spectrometer in both negative-and positive-ionization modes in order to explore fragmentation pathways. It was found that characteristic product ions in the positive-ion mode could be used for the unambiguous assignment of the phosphorylation site, whereas the use of both ionization modes provided consistent and/or complementary information about the fatty acyl distribution between the two glucosamine moieties of lipid A. Since the immunostimulatory (advantageous) vs. proinflammatory (endotoxic) effect of the lipid A is closely related to the fine chemical structure, our relatively simple structural elucidation strategy could offer great potential in the bioanalysis of native lipid A samples and lipid A-based vaccines.

In this study, we report the detailed analysis of the fragmentation patterns of positively charged lipid A species based on their tandem mass spectra obtained under low-energy collision-induced dissociation conditions of an electrospray quadrupole time-of-flight mass spectrometer. The tandem mass spectrometry experiments were performed after the separation of the compounds with a reversed-phase high performance liquid chromatography method. We found that both, phosphorylated and nonphosphorylated lipid A molecules can be readily ionized in the positive-ion mode by adduct formation with triethylamine added to the eluent. The tandem mass spectra of the lipid A triethylammonium adduct ions showed several product ions corresponding to inter-ring glycosidic cleavages of the sugar residues, as well as consecutive and competitive eliminations of fatty acids, phosphoric acid, and water following the neutral loss of triethylamine. Characteristic product ions provided direct information on the phosphorylation site(s), also when phosphorylation isomers (ie, containing either a C1 or a C4' phosphate group) were simultaneously present in the sample. Continuous series of high-abundance B-type and low-abundance Y-type inter-ring fragment ions were indicative of the fatty acyl distribution between the nonreducing and reducing ends of the lipid A backbone. The previously reported lipid A structures of Proteus morganii O34 and Escherichia coli O111 bacteria were used as standards. Although, the fragmentation pathways of the differently phosphorylated lipid A species significantly differed in the negative-ion mode, they were very similar in the positive-ion mode. The complementary use of positive-ion and negative-ion mode tandem mass spectrometry was found to be essential for the full structural characterization of the C1-monophosphorylated lipid A species.

A fast and simple method with CZE coupled to ESI/QTOF‐MS was optimized and validated for quantitative determination of organic acids (lactic acid, succinic acid, malic acid, tartaric acid, shikimic acid, and citric acid) in red wines. The BGE was ammonium acetate and the separation of the analytes was performed in a polybrene‐coated capillary in the presence of EOF. The sample preparation included dilution and filtration of the wine. The method showed satisfactory performance characteristics: good linearity for each organic acid, with correlation coefficients ranging from r 2 = 0.9902 (shikimic acid) to r 2 = 0.9990 (tartaric acid). The limit of quantification was between 0.0034 mM (for shikimic acid) and 0.107 mM (for citric acid), and the recovery data fell between 95.8% (malic acid) and 102.7% (lactic acid); the total run time was less than 4 min. The RSD values for the interday repeatability and intraday reproducibility were between 3.44 and 9.50%, and between 1.75 and 8.29%, respectively. Seventeen Macedonian red Vranec wines were studied demonstrating a wide variation in the organic acids’ concentration, which should be most probably due to the variation of the climate conditions in the vine areas.