Fakultät Naturwissenschaften

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Biologie, Ernährungs-wissenschaften und Lebensmittelwissenschaften sind die Schwerpunkte der Fakultät. Die Forschung befasst sich mit Schlüsselthemen der Life Sciences.

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Determination of potentially hazardous oxidation products in cosmetics containing lanolin or 1-(1,2,3,4,5,6,7,8-octahydro-2,3,8,8-tetramethyl-2-naphthalenyl)ethanone (OTNE)
(2019) Schrack-Belschner, Sonja Miriam Irmgard; Schwack, Wolfgang
Cosmetic products are important consumer goods in the "non-food" sector, which should not have negative effects of the human health. Critical compounds, however, can be formed by the oxidation of an unsaturated organic compound.Thereby formed oxidation products with potentially adverse properties are well known from the food sector. As the oxidation of cosmetic ingredients, however, has less been studied, the oxidation of selected cosmetic ingredients with respect to the formation of potentially critical compounds was investigated within the framework of this thesis.The oxidation of cholesterol to various cholesterol oxidation products (COPs) was investigated in a first step.COPs are known from the food sector and are suspected of causing certain diseases such as arteriosclerosis.Cosmetic products have not yet been tested for COPs, although a versatile ingredient used only in cosmetic products, lanolin, contains above-average levels of the cholesterol, which is the precursor.Total COPs contents in cosmetics containing lanolin, namely lip care products, fat creams and ointments for nursing women were in the low percent range (up to 3 %) and were thus several orders of magnitudes higher than the contents found in food.The oxidation of fragrances was studied in the second part of this work.The subject is not new as the oxidation of terpenes to contact allergens has been studied in earlier studies. The oxidation of other fragrances was hardly investigated. In order to extend our knowledge in this field, the oxidation of a synthetic fragrance frequently used in perfumes, octahydro tetramethyl naphthalenyl ethanone (OTNE) was studied. Obtained results indicated that peroxides of OTNE were formed during oxidation.It was found out that the OTNE oxidation even occurs, when perfumes are stored indoors under normal temperature and light conditions. An in-vivo test showed that OTNE oxidation can be expected on the skin after application of a perfume.
Development of a planar yeast estrogen screen as screening tool for estrogen active compounds
(2018) Schick, Dinah; Schwack, Wolfgang
Substances that disrupt or impair the hormone system (endocrine system) or that show an irreversible influence on it are referred to as endocrine disruptors or xenohormones. Concerning this, also estrogen active compounds (EAC) are endocrine disruptors, that are under suspicion of being involved in the formation of tumors or to induce disruption during development and reproduction, and are, for example, blamed for being responsible for the feminization of fish. At this, EAC can be natural (human, phytoestrogens) but also synthetic substances, which are discharged to the environment by humans (e.g. pharmaceuticals, pesticides, additives). Regarding the ubiquitous presence of EAC, suitable methods for the analysis of EAC are required. An in vitro method for the determination of EAC is the YES assay (yeast estrogen screen) that is executed in liquid solutions in microtiter plates and that works with genetically modified yeasts, which contain the human estrogen receptor (hER) and a reporter gene encoding for the enzyme beta-galactosidase. In presence of EAC, the enzyme is produced and subsequently cleaves a substrate that is used to measure the receptor activity and thus the estrogenic activity. The transfer of the YES assay to high-performance thin-layer chromatography (HPTLC) was successfully demonstrated and advanced, thus resulting in the combination of a chromatographic separation of analytes and the detection of EAC using genetically modified yeast cells directly on the HPTLC plate (HPTLC planar yeast estrogen screen, HPTLC-pYES). Usually, the substrate 4-methylumbelliferyl-beta-D-galactopyranoside is used for pYES, releasing blue fluorescing 4-methylumbelliferone (MU) after enzymatic cleavage. Various matrices, however, often contain a plenty of different components, partly showing native fluorescences (blue, red), why the detection of the blue fluorescing MU can be interfered. By applying the substrate resorufin-beta-D-galactopyranoside (RGP) and by using automated devices, the RGP-pYES as fast screening tool for EAC was developed and successfully applied to waste water samples and extracts of hops pellet samples. A screening method using HPTLC simultaneously represents a planar clean-up, why samples do not have to undergo complex steps of sample preparation or purification. The chromatographic separation in combination with the detection of estrogenic activity using genetically modified yeasts directly on the plate allowed the detection, the determination and the identification of single EAC. Using RGP, which releases orange fluorescing resorufin after enzymatic cleavage as positive signal of estrogenic activity, enabled a clear differentiation between fluorescences due to estrogenicity and the native fluorescence of sample components. Application of the RGP-pYES to spiked water samples and sewage samples showed high recovery rates and a good precision, and thus the applicability of the method as screening tool for environmental samples. By means of suitable evaluation methods, additionally the generation of dose-response curves of known and unknown EAC and thus the generation of so-called logit-log plots was possible. This enabled the determination of estradiol equivalent factors of known EAC as well as the determination of estradiol equivalent concentrations and amounts, respectively, of known and unknown EAC in liquid and solid samples. Thus, the possibility to estimate the estrogenic potential of a sample or single sample components was given. The coupling of pYES to mass spectrometry additionally allowed the identification of unknown EAC, demonstrated exemplarily by investigation of extracts of hops pellet samples, in which the only detected EAC in the hops extracts was identified as prenylnaringenin. Since the method uses a planar system, the pYES advantageously reveals all chromatographically separated sample components at one look and, as bioassay, additionally detects a possible estrogenic activity (activity at the hER) of single substances, while a differentiation between native occurring fluorescences of sample contaminants and the fluorescence as positive signal for estrogenicity of a substance is granted.
Development of strategies for the prioritization of organic trace substances in water by effect-directed analysis
(2020) Stütz, Lena; Schwack, Wolfgang
The protection of the aquatic environment and the supply of clean drinking water to people all over the world are central challenges of our time. Monitoring of the aquatic environment and the input of anthropogenic trace substances into it is therefore very important. However, since aquatic environmental samples often consist of complex substance mixtures, their characterization and evaluation is very demanding. By using generic target analysis methods, selected known anthropogenic trace substances can be detected and quantified very sensitively. For the detection of previously unknown substances, non-target analysis methods have been increasingly used in recent years. However, these methods do not provide information on the relevance of the anthropogenic trace substances occurring in water. In this context, especially all those trace substances are regarded as relevant from which a harmful effect on humans or water organisms is to be expected. For the detection of such effective substances, effect-directed analysis (EDA) can be used. In EDA, a bioassay is combined with a fractionation method and subsequent chemical analysis, the aim being to identify the bioactive substance. The separation method used in this work is high-performance thin-layer chromatography (HPTLC). After chromatography, the bioassay is performed directly on the HPTLC plate. If an effective zone appears in the bioassay, a prioritization strategy is used to clarify the identity of the substance. Due to the complex aquatic samples, a large number of different substances in a zone must still be expected despite the applied HPTLC separation, which makes it difficult to identify the effective substance. Therefore, a strategy to simplify the identification of effective substances should be developed. The aim was to reduce the complexity by multidimensional separation in such a way that chemical analysis can be used to prioritize to a few candidates in the effective fraction. In the first part of the work, a selective two-dimensional HPTLC separation was developed to reduce the number of substances in a bioactive zone. After the first separation dimension (1D) the acetylcholinesterase inhibition assay (AChE assay) was performed and afterwards only the effective zones were extracted from the HPTLC plate. The selected effective zones were separated in a second separation dimension (2D) and the bioassay was performed again. With this 2D separation, the peak capacity could be increased by a factor of 7 compared to a 1D HPTLC gradient development. If real water samples are examined for their effects, an additional structural elucidation must be carried out to clearly identify the unknown bioactive substances. In this work, the developed 2D EDA was therefore connected to a high-performance liquid chromatography (HPLC) with high-resolution mass spectrometry (HRMS) and a non-target screening (NTS) was performed. Using three water samples(drinking water, surface water and purified sewage water) spiked with six effective substances, it was shown that the developed strategy is suitable for the identification of effective substances and that these can be recovered despite repeated extraction. When applying the developed methodology to real samples, it was also possible to assign and quantify the detected effect in several waters to the substance lumichrome and to linear alkylbenzene sulfonates. Genotoxicity is a crucial endpoint for the effect assessment of water samples. However, this endpoint with metabolic activation cannot yet be performed directly on the HPTLC plate. Since many of the genotoxic substances have an indirect genotoxic effect, i.e. they only acquire their activity after metabolic activation; this endpoint was investigated in the present work with the umu assay in the microtiter plate. However, separation with HPTLC, subsequent extraction of effective zones and non-target analysis of the extracts, should also be performed for this assay. Therefore the umu assay in the microtiter plate was integrated into the existing EDA-with-HPTLC concept. In laboratory experiments, sodium hypochlorite was added to the drug metformin in order to simulate the behavior of the substance during water treatment (chlorination). The metformin sample treated with hypochlorite was examined with the umu assay and a genotoxic effect was detected. After HPTLC separation of the chlorinated sample, zones were extracted over the entire retardation range. When the extracted zones were examined with the umu assay, the genotoxic effect could be clearly assigned to one fraction. Using high-resolution mass spectrometry, the genotoxic effect could be assigned to an already known transformation product of metformin. The HPTLC separation and extraction of the zones from the plate led to a reduction of the possible effective candidate masses by a factor of 10 and thus to a clear prioritization in HRMS analysis.
Einfluss der Ernährung und von Genussmitteln auf Risikofaktoren für das Auftreten von ischämischem Herzinfarkt und Schlaganfall
(2005) Eckoldt, Joachim; Bode, Christiane
Background and aims of the study: Arteriosclerotic changes of blood vessels which contribute to coronary heart disease (CHD) and ischemic stroke are influenced by risk factors like cigarette smoking, overweight, hypertension, diabetes mellitus, missing physical exercise and nutritional factors, such as alcohol consumption. Beyond this, the concentrations of serum lipids, antioxidants, coagulation factors or other risk factors, such as C-reactive protein, and homocysteine are considered to be additional factors that indicate an enhanced or lowered risk of atherosclerosis. In this study we examined the effect of nutritional factors, in particular alcohol consumption, on various plasma components that are believed to play a role in the pathogenesis of atherosclerosis. Multiple epidemiologic studies suggest that moderate alcohol consumption reduces the mortality from cardiovascular diseases and that this effect is chiefly mediated by elevation of high-density lipoprotein (HDL). This cross-sectional study assessed the effect of moderate alcohol consumption and other life-style factors on the composition of HDL in healthy working males. An additional goal of the present study was to find out whether there is an association between alcohol consumption and the concentration of vitamins and cardio-protective substances. Methods: We included a collective of healthy men (n = 284, age 23-66 years), investigated with respect to cardiovascular risk factors. The average daily alcohol consumption, nutrient intake, smoking and other life-style factors was assessed by a computer based questionnaire. Group 1 (n = 62) comprised subjects with an average daily alcohol consumption of 0-5g, group 2 (n = 175): 5-30g, and group 3 (n = 47): 30-70g. In addition, the study design made it possible to subdivide groups 2 and 3 in a so called ?beer drinker group 2+3? (> 80 % beer), a ?wine drinker group 2+3? (> 80 % wine) and persons without preference of a certain alcoholic beverage. Results: The alcohol groups showed no significant differences in the nutritional profile (nutrients, energy intake, and metabolic rate). The markers for regular, higher alcohol consumption (g-GT and MCV) were positive correlated to the amount of alcohol consumption. There was no correlation between the great number of clinical laboratory parameters and the amount or kind of alcohol consumption. Thereby the groups are comparable in view of these laboratory parameters. Besides, there were no indications for the existence of diseases, which might influence blood lipid and vitamin concentrations. Antioxidative substances in the blood: Dietary assessment: The intake of vitamin B2, B6, B12, folic acid, retinol, ß-carotene and other carotenoids, as assessed by the computer interview was comparable in groups 1-3. The subjects in group 1 had a higher supply of the vitamins C, B1 and a-tocopherol, ?beer drinker Gr. 2+3? had a higher intake of vitamin B2, B6 and folic acid. Blood measurements: Antioxidative vitamins: The vitamin B2 status in erythrocytes (EGRAC) was lower in group 3 (vs. group 1 and 2). The plasma level of ß-carotene and ß-cryptoxanthin was lower in group 3 than in group 1. Vitamins that influence homocysteine metabolism (including homocysteine): Influence of beer and wine: The status of vitamin B6 and the concentration of free plasma pydridoxal phosphate in group 3 was significantly higher than in group 1. These results cannot explain the postulated positive influence of moderate or higher alcohol consumption through improvements of the vitamin status and the concentration of vitamins in the blood. The vitamins in beer improved the vitamin status only in case of vitamin B6, no effect was calculated in case of vitamin B2 and folic acid. Higher alcohol consumption (group 3) made the vitamin status respectively the plasma concentration of vitamin B2, ß-carotene and ß-cryptoxanthin lower compared with group 1 ? in spite of comparable supply. Coagulation factors, markers of inflammation: The coagulation factors prothrombin time and fibrinogen as well as the ?newer? risk factors C-reactive protein and homocysteine were not correlated to the amount or the kind of alcohol. Lipoproteins: The serum concentration of total cholesterol, cholesterol ester, phospholipids, apolipoprotein A-1 and A-2 was higher in group 3. Moderate and higher alcohol consumption raises the concentration of cholesterol in the high-density lipoproteins (HDL) (including subfractions) ? independent of the sort of alcoholic beverage. The concentration of cholesterol in the low- (LDL) (including subfractions), very-low (VLDL) and intermediate-density lipoproteins (IDL) in the blood was not influenced by alcohol consumption. Composition of HDL: The induced increase of HDL cholesterol was lower in the subfraction HDL3 as in the subfractions HDL2b und HDL2a. Besides we found qualitative changes of the HDL-components: the phospholipid component increased more than the other HDL-components. This phenomenon might play a beneficial role in the mechanism of atherosclerosis. Conclusions: Vitamins: The changes of antioxidative vitamins and vitamins influencing homocysteine metabolism observed in persons with moderate and increased alcohol consumption do not explain the antiatherogenic effect of alcohol. On the other hand, our study confirmed a positive association of moderate alcohol consumption with HDL plasma levels ? independent of other nutritional factors. In addition, alcohol might induce qualitative alterations of HDL composition (more pronounced increased of HDL2 relative increase of the phospholipid component). The pathophysiological significance of this phenomenon remains unclear.
Einfluss eines Glukoseentzugs auf die Strahlenempfindlichkeit von Tumorzellen und Normalzellen
(2018) Ampferl, Rena; Dittmann, Klaus
Radiotherapy is a major pillar of cancer treatment. However, the maximal dose that can be applied to a tumor is limited by side-effects of the irradiated normal tissue. Therefore, to improve treatment success, it is of significant interest to develop new treatment strategies that selectively enhance the cytotoxic effect of radiation in tumor cells while sparing healthy tissue. For this purpose, it is necessary to exploit differences between tumor cells and normal cells. Thus, tumor cells are characterized by metabolizing glucose preferentially to lactate regardless of the availability of oxygen (Warburg effect, aerobic glycolysis), while normal cells oxidize most of the glucose in the mitochondria if oxygen is present. Because the Warburg effect only produces low amounts of ATP per molecule of glucose when compared to mitochondrial glucose oxidation, tumor cells rely on high glucose supply. Hence, it was the aim of this study to investigate whether a glucose starvation during radiotherapy, which requires energy-dependent repair of DNA damage, is an appropriate strategy to selectively enhance radiosensitivity of tumor cells, but not of normal cells. It was shown that glucose starvation inhibited proliferation of the tumor cell lines A549 and FaDu, but not that of the normal fibroblasts HSF7. Moreover, deprivation of glucose induced cell death selectively in tumor cells, which occurred mainly via necrosis. Combining glucose starvation with radiotherapy led to selective radiosensitization of both tumor cell lines, which was accompanied by impaired repair of radiation-induced DNA double-strand breaks (DNA DSBs). In this context, it turned out that in tumor cells glucose is essential for the late stage of DNA DSB repair starting from 13 h after irradiation. Furthermore, an inhibition of radiation-induced histone acetylation as well as KAP1 phosphorylation could be observed in tumor cells following glucose starvation, indicating an impairment of radiation-induced chromatin relaxation. Because opening of the chromatin structure is particularly important for the repair of DNA DSBs within heterochromatin and because these DSBs are the ones that are repaired at late time points after irradiation, it can be assumed that in tumor cells glucose starvation mainly impairs the repair of heterochromatic DNA DSBs. Further investigations revealed that in tumor cells glucose starvation does not cause lack of nuclear acetyl-CoA, which is the substrate for the acetylation of histones, and therefore this could be excluded as cause of the observed inhibition of histone acetylation. However, it is known that the histone deacetylase Sirt1 is activated in response to glucose starvation. Histone deacetylation by Sirt1 could counteract radiation-induced histone acetylation, thus impairing chromatin relaxation as well as repair of DNA DSBs after irradiation. In fact, it was shown that inhibition of Sirt1 by sirtinol can partly abrogate the impaired repair of radiation-induced DNA DSBs that was observed in tumor cells under glucose-free conditions. However, the inhibitory effect of glucose starvation on DNA DSB repair in tumor cells could not only be observed under glucose-free conditions. Thus, reducing the glucose concentration to 0.5 g/l was enough to impair DSB repair following irradiation to the same degree as after total deprivation of glucose. Furthermore, it turned out that under glucose-free conditions DNA DSB repair in tumor cells was promoted by autophagy already after irradiation with 2 Gy. Finally, it was shown that, in addition to DNA DSB repair, also tumor metabolism is influenced by glucose starvation. Thus, deprivation of glucose impaired the radiation-induced switch of glucose metabolism that was characterized by increased aerobic glycolysis and decreased mitochondrial glucose oxidation, and this can also contribute to radiosensitization of the cells. In contrast to tumor cells, glucose starvation neither caused radiosensitization nor impaired the repair of radiation-induced DNA DSBs in normal fibroblasts. Moreover, in these cells, glucose starvation had no influence on histone acetylation and KAP1 phosphorylation after irradiation. These results demonstrate that glucose starvation is an appropriate in vitro strategy to selectively sensitize tumor cells to radiotherapy without influencing the radiosensitivity of normal cells.
Exo‐endo Isomerism of carboranes: Unusual geometries of C2B5X7 (X=Cl, Br) and C2B7Cl9 with exo‐skeletal BCl2 groups on carbon revealed by joint spectroscopic/computational studies
(2023) Keller, Willi; Conrad, Jürgen; Hofmann, Matthias
Reexamination of the co‐pyrolysis reactions of B2Cl4 with C2Cl4 at 350 °C and of B2Br4 with CBr4 at 300 °C in vacuo confirmed the carboranes C2B5Cl7 (1), C2B7Cl9 (2), and C2B5Br7 (3) as low‐yield products. While 1 only could be concentrated by repeated vacuum fractionation, 2 and 3 could now be isolated from the conglomerate mixtures for a full spectroscopic characterization and the compounds were verified in their geometries by detailed DFT computations. Surprisingly, the perhalogenated carboranes do not adopt the expected “all‐endo”‐geometries with cluster sizes derived by the sum of the n boron and two carbon atoms (n+2) as known from the syntheses of the parent closo‐carboranes C2BnHn+2. Instead, DFT/GIAO(ZORA)/NMR (GIAO for X=Cl, ZORA for X=Br) computations revealed that the perhalogenated carboranes favor structures with BX2 groups as exo‐skeletal ligands attached to both cage‐carbon atoms yielding the five‐vertex closo‐1,5‐(CBX2)2B3X3 (1: X=Cl; 3: X=Br) and the seven‐vertex closo‐2,4‐(CBCl2)2B5Cl5 for 2. In contrast to these perhalogenated carboranes, analogous computations on the hydrogen substituted carboranes C2BnHn+2, silaboranes Si2BnHn+2 and Si2BnXn+2 (n=5, 7) show in all cases a thermodynamic favorization of structures where all boron atoms of the formula are endo‐skeletally incorporated into the cluster frameworks.
A fast gas chromatography coupled with electron capture negative ion mass spectrometry in selected ion monitoring mode screening method for short‐chain and medium‐chain chlorinated paraffins
(2022) Schweizer, Sina; Schulz, Tobias; Vetter, Walter
Rationale Chlorinated paraffins (CPs) are a group of anthropogenic pollutants that consist of complex mixtures of polychlorinated n-alkanes of different chain lengths (~C10 to C30). Persistence, bioaccumulation, toxicity, and long-range transport of short-chain chlorinated paraffins (SCCPs, C10- to C13-CPs) have prompted their classification as persistent organic pollutants (POPs) by the Stockholm Convention in 2017. Due to the varying chain lengths and chlorination degrees, quantification of SCCPs and medium-chain chlorinated paraffins (MCCPs, C14- to C17) using gas chromatography coupled with electron capture negative ion mass spectrometry in selected ion monitoring mode (GC/ECNI-MS-SIM) is not only challenging but also very time consuming. In particular, up to eight GC runs per sample are required for the comprehensive GC/ECNI-MS-SIM quantification of SCCPs and MCCPs. These efforts are high especially if the samples do not contain CPs above the limit of detection (LOD), subsequently. Methods We developed a semi-quantitative and sensitive method for the examination of SCCPs and MCCPs in one GC run. This GC/ECNI-MS-SIM screening method was based on the recording of Cl− (m/z 35 and 37), Cl2− (m/z 70 and 72), and HCl2− (m/z 71 and 73) isotope ions and evaluation of the ratios between them. Results Correctness of the results of the screening method was verified by analysis of edible oils with and without CPs, CP standards, as well as a technical CP mixture. Polychlorinated biphenyls (PCBs) and other polyhalogenated aromatic compounds, as well as brominated flame retardants, do not form all of the fragment ions analyzed by the screening method. Conclusions After the screening, only CP-positive samples may need to be measured in detail. Measurement time will already be gained in the case of ~10% samples without CPs.
Geometrical and positional isomers of unsaturated furan fatty acids in food
(2022) Müller, Franziska; Hammerschick, Tim; Vetter, Walter
Furan fatty acids (FuFA) are important antioxidants found in low concentrations in many types of food. In addition to conventional FuFA which normally feature saturated carboxyalkyl and alkyl chains, a few previous studies indicated the FuFA co‐occurrence of low shares of unsaturated furan fatty acids (uFuFA). For their detailed analysis, the potential uFuFA were enriched by centrifugal partition chromatography (CPC) or countercurrent chromatography (CCC) followed by silver ion chromatography from a 4,7,10,13,16,19‐docosahexaenoic acid ethyl ester oil, a 5,8,11,14,17‐eicosapentaenoic acid ethyl ester oil and a latex glove extract. Subsequent gas chromatography with mass spectrometry (GC/MS) analysis enabled the detection of 16 individual uFuFA isomers with a double bond in conjugation with the central furan moiety. In either case, four instead of two uFuFA isomers previously reported in food, respectively, were detected by GC/MS. These isomers showed characteristic elution and abundance patterns in GC/MS chromatograms which indicated the presence of two pairs of cis/trans‐isomers (geometrical isomers).
High-throughput planar solid phase extraction : a new clean-up concept in multi-residue analysis of pesticides
(2014) Oellig, Claudia; Schwack, Wolfgang
Currently, the most serious problems in pesticide residue analysis by liquid chromatography (LC) or gas chromatography (GC) coupled to mass spectrometry (MS) concern the so-called “matrix effects”. The most common way to avoid these effects is the application of matrix-matched calibration standards. Nevertheless, an efficient clean-up undoubtedly is the best way to prevent matrix effects in multi-residue analysis of pesticides in food by LC–MS or GC–MS. For a totally new powerful clean-up method, called high-throughput planar solid phase extraction (HTpSPE), highly automated planar chromatographic tools were applied to remove co-extracted matrix substances entirely and to eliminate any kind of matrix related effects. For sample extraction, the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method was used to initially collect pesticides from fruits and vegetables. The received acetonitrile extracts were applied directly for the development of the novel HTpSPE clean-up. Thin-layer chromatography (TLC) was used to completely separate pesticides from matrix compounds and to focus them into a sharp zone. A two-fold development on amino-modified silica gel thin-layers with acetonitrile for the first development, and acetone for the second development in the backwards direction, was evaluated to perform the best clean-up result and collect the pesticides in a sharp, single target zone. To easily locate the pesticide zone, the Sudan II dye was added to the extracts. Following this clean-up, the target zones (pesticides) were eluted by the TLC–MS interface into vials for the LC–MS determination. HTpSPE resulted in extracts which were nearly free of co-extracted matrix and matrix effects, as shown for seven chemically representative pesticides (acetamiprid, azoxystrobin, chlorpyrifos, fenarimol, mepanipyrim, penconazole, and pirimicarb) in four different fruit and vegetable matrices (apples, cucumbers, red grapes, and tomatoes). Thanks to the very clean HTpSPE extracts, calibration can simply be performed with pure solvent standards and the quantitation by LC–MS provided excellent mean recoveries and relative standard deviations. In addition, tea samples as rather challenging matrices were chosen to apply for HTpSPE. The matrix load of tea extracts generally was too high for the available thin-layer capacity and the selectivity of the amino-modified phase was not suitable for the separation of caffeine and further matrix compounds from the target analytes (pesticides). By modifying the sample extraction, adding a pre-cleaning by dispersive solid phase extraction (dSPE) and changing the thin-layer phase to normal phase silica gel, the complete separation of pesticides and tea matrix components was possible, when again a two-fold development was applied. Caffeine and other alkaloids were completely removed. The effectiveness of HTpSPE was demonstrated by LC–MS/MS calibration curves from matrix-matched and solvent standards, which were nearly identical and by very good mean recoveries, calculated against pure solvent standards. Concerning all validation parameters, the new acetonitrile-HTpSPE procedure for tea samples was superior to the QuEChERS-dSPE method and offered highly successful results. In recent years, large-scale screening in pesticide residue analysis has gained more and more importance. Keeping this in mind, a screening strategy for HTpSPE extracts, using a high-resolution MS, was developed to analyze the cleaned extracts directly for pesticide residues without a liquid chromatographic separation. By this hyphenation, a completely new microliter-flow injection analysis–time-of-flight mass spectrometry (µL-FIA–TOFMS) screening was introduced. The novel HTpSPE–µL-FIA–TOFMS approach enabled the detection of all pesticides simultaneously in a single mass spectrum within a few minutes. The obtained mass spectra were nearly free of matrix compounds, which is especially the great benefit of the effective HTpSPE clean-up. Recovery studies by HTpSPE–µL-FIA–TOFMS against solvent standards for the matrices and pesticides under study provided excellent results, using the mass signal intensities under the entire FIA sample peak. HTpSPE clearly showed superior results concerning every tested parameter than dSPE. With the help of a self-constructed mass database searching tool, all spiked pesticides were detected and correctly identified, while only very low numbers of false-positive findings occurred. Furthermore, a non-target screening approach was successfully implemented by slightly changing the database searching process, offering a mass list of all substances, which are present in the injected extracts but not included in the mass database. Finally, the new HTpSPE–µL-FIA–TOFMS screening was successfully applied to several real samples, when the identified pesticides were quite identical compared to results of LC–MS/MS analysis of the QuEChERS-dSPE extracts.
HPTLC-bioluminescence detection: methodological improvements and the application of the method to mouthwashes
(2013) Baumgartner, Vera; Schwack, Wolfgang
For the chemical analysis of food, drugs, and environmental samples it becomes more and more important to find substances of a certain (biological) activity. For this, several biological screening assays are available. One of the most versatile is the luminescent bacteria test according to an international norm (DIN EN ISO 11348), a rapid cuvette test on cytotoxicity. The assay employs the naturally bioluminescent bacterium Vibrio fischeri, which emits blue-green light under good living conditions. Because the energy-consuming luminescence metabolism is linked directly to the bacterium?s respiratory chain, a disturbance of the bacterium?s metabolism affects the luminescence, whereas the degree of toxicity is proportional to the luminescence inhibition. Major advantage was achieved by coupling this biotest with previous separation by high-performance thin-layer chromatography (HPTLC), which allowed for a screening for individual components. The workflow consists of sample application onto an HPTLC plate, separation, drying the plate, application of the Vibrio fischeri suspension, and detection with a light-sensitive CCD camera. In the resulting image, dark zones on a brightly luminizing background indicate substances that affect the bacteria?s metabolism. No suitable image evaluation program for the effective correction and quantitative evaluation of the image after Vibrio fischeri detection was available, which was regarded as a great disadvantage. In literature, adaptations of the special corrections based on the cuvette test calculations were described, including horizontal background correction and the recalculation of the sigmoid dose-response-relationship of the bacteria?s reaction. This served as a basis for the development of a new method using existing software which did not only perform the necessary calculations but was easy and convenient enough for use in routine evaluations. Furthermore, the process of applying the aqueous bacteria suspension onto the HPTLC plates was improved. Usually, application was done by dipping with the help of a dipping device. Especially for polar substances, however, it was observed that substances can start to dissolve during this process, leading to blurring and tailing of the zones on the plate. A simple rolling device consisting of commercially available household articles was constructed. To compare rolling with dipping, octhilinone and methylparaben were chosen as test compounds. The results of rolling were far superior to dipping. However, manual rolling depended on the person who did it, and it was not possible to control pressure and velocity. To overcome this problem, a prototype of an automated rolling device was constructed and built. After the successful process optimizations, the applicability of the HPTLC-bioluminescence assay was tested on commercial mouthwashes. Mouthwashes are likely to contain antimicrobial compounds, which are not necessarily indicated on the packaging. HPTLC with biodetection was used as a rapid screening method to detect zones of interest, which were further analyzed by conventional techniques like HPLC and GC. First, the reaction of Vibrio fischeri towards more than 40 standard substances was determined. This database was used for the analysis of six commercially available mouthwashes. It revealed that not only declared preservatives are used in mouthwashes, but also other antimicrobial compounds. These were especially constituents of essential oils having antibacterial properties (anethole, carvone, menthol, thymol), but are summarized as ?aroma?, which is in compliance with legal restrictions. A most interesting question concerns the bacteria?s condition on the HPTLC plate. For the brightly luminizing background, it must be assumed that the bacteria are well alive. But no clear statement can be given for bacteria in the dark zones: they might be dead, inhibited (maybe only temporarily), or absent due to water repelling effects of the zone?s compound. A basic attempt to answer this question was made by applying a combination of classical microbiological techniques. In this dissertation it could be shown that HPTLC coupled with Vibrio fischeri detection can successfully be used in practice and is well suited to complement conventional analytical techniques. This work is meant to serve as a guideline for further research and new applications.
Improvements in the analysis of food contaminations deriving from packaging materials
(2009) Rothenbacher, Thorsten; Schwack, Wolfgang
The dissertation presents in its introduction the sources and process of food contaminations deriving from packaging materials. Subsequent legislative aspects, the analysis of food contact materials and contaminants in food are explained and examples therefore are given. The main part of the dissertation covers the following published papers: 1.T. Rothenbacher, M. Baumann and D. Fuegel. 2-Isopropylthioxanthone (2-ITX) in food and food packaging materials on the German market. Food Additives and Contaminants 2007; 24: pp. 438-444 2.T. Rothenbacher, W. Schwack. Determination of epoxidized soybean oil by gas chromatography/single quadrupole and tandem mass spectrometry stable isotope dilution assay. Rapid Communications in Mass Spectrometry 2007; 21: pp. 1937-1943 3.T. Rothenbacher, W. Schwack. Non-targeted multi-component analytical screening of plastic food contact materials using fast interpretation of deliverables via expert structure-activity relationship software Journal of AOAC INTERNATIONAL 2009; 92 (3): pp. 941-9501 4.T. Rothenbacher, W. Schwack. Rapid and nondestructive analysis of phthalic acid esters in toys of poly(vinyl chloride) by direct analysis in real time?single quadrupole mass spectrometry. Rapid Communications in Mass Spectrometry 2009; 23: pp. 2829?2835 5.T. Rothenbacher, W. Schwack. Rapid identification of additives in poly(vinyl chloride) lid gaskets by direct analysis in real time ionisation and single-quadrupole mass spectrometry. Rapid Communications in Mass Spectrometry 2010; 24: pp. 21-29
Influence of microwave irradiation and ionic liquids on multi component reactions
(2013) Mert-Balci, Fadime; Beifuss, Uwe
The present thesis focuses on the influence of microwave irradiation and ionic liquids on the outcome of two well-known three component reactions, the Groebke reaction and the Povarov reaction. The first part of the thesis deals with the influence of microwaves and ionic liquids on the Groebke reaction. The reaction of 2-aminopyridines with aldehydes and isocyanides using montmorillonite as a catalyst in toluene under microwave conditions at 160°C delivers the corresponding imidazo[1,2-a]pyridines within only seven minutes with yields ranging from 16 to 98%. The organic solvent can be replaced by ionic liquids like imidazolium and guanidinium salts. With guanidinium salts, it is possible to perform the Groebke reaction in the absence of any other catalyst and solvent under microwave conditions. The second part of this work is about the extension of the scope of typical Groebke reactions by replacing the aldehyde component with a bifunctional 2-carboxybenzaldehyde. The reaction of 2-aminopyridines with isocyanides and 2-carboxybenzaldehydes with 20 mol% methanesulfonic acid as a catalyst in toluene under microwave conditions at 160°C affords the corresponding pyrido[2?,1?:2,3]imidazo[4,5-c]isoquinolin-5(6H)-ones with yields ranging between 35 and 68%. The new method can easily be performed, is robust, and highly efficient. The third part of the thesis is focused on the intermolecular Povarov reaction. Using the reaction between aniline, benzaldehyde, and 2,3-dihydrofuran as a model reaction, the influence of ionic liquids, such as imidazolium and guanidinium salts, and microwaves on the outcome of the Povarov reaction was evaluated. It was established that the model reaction can be promoted by imidazolium salts like [bmim]BF4 under thermal as well as under microwave conditions. The reaction temperature has a strong impact on the chemical yield and the diastereoselectivity of the model reaction. At lower temperatures the formation of the endo-isomer is favored. However, the influence of microwave irradiation on yield and selectivity is not very pronounced. The Povarov reaction can also be promoted by a great number of guanidinium salts. Reactions that were performed under thermal conditions in a sealed vial demonstrated that both the chemical yield and the diastereoselectivity of the reaction are strongly influenced by a) the structure of the guanidinium ion and the nature of the anion of the guanidinium salt, and b) the concentration of the guanidinium salt. Remarkably, the Povarov can also be performed successfully in the presence of only catalytic amounts of a guanidinium salt. Finally, it was demonstrated that the guanidinium salts can be recycled and reused several times without loss of reactivity.
Investigations into heat- and light-induced terpene modifications in essential oils
(2023) Bitterling, Hannes; Vetter, Walter
Essential oils belong to secondary plant metabolites, with terpenoids and phenylpropanoids being among the main constituents in terms of quantity. Due to their lipophilic character and high volatility, they are mainly obtained by steam distillation. Citrus essential oils (agrumen oils) are an exception , since they are usually extracted from the peels by means of pressing, whereby less volatile components such as coumarins and furocoumarins are also introduced. Due to their odor and taste-giving properties, essential oils are used in the food, beverage, and cosmetics industries. In addition, due to a wide range of pharmacological properties, they are used in phytotherapy as well as in aromatherapy. However, most essential oils are highly susceptible to oxidation, polymerization, dehydrogenation, and isomerization reactions in the presence of atmospheric oxygen, light, and at high temperatures. The resulting organoleptic changes usually lead to a significant quality reduction. The formation of terpene hydroperoxides is another problem, as these are suspected of causing intolerances such as redness and itching in 1-3% of the European population upon contact with the skin. The detection of these chemical changes forms an integral part of quality control and can be prevented as far as possible by suitable production, transport, and storage strategies. Due to their volatility, essential oils are mainly analyzed by gas chromatography. However, due to their instability, the detection of hydroperoxides places considerable demands on common analytical methods. For this reason, a novel spectrophotometric method for the detection of peroxides and hydroperoxides in terpenes and essential oils was developed (paper 1). The oxidation of N-N-dimethyl-p-phenylenediamine by peroxides yielding an intensely red-colored cation (Wursters red) allowed colorimetric detection and quantitation of even smallest amounts (LOD: 0.5 ppm). The minimal sample amount of only a few milligrams, as well as simple and fast performance predestine this method for daily laboratory routine (paper 1). Among plant terpenoids, the monoterpene R-(+)-limonene is very widespread. Thus, it is not only found in citrus oils but also of in caraway oil, where its proportion amounts to almost 50%. To investigate the storage stability, R-(+)-limonene, S-(+)-carvone, different caraway oils, and the corresponding caraway seeds were stored in desiccators at 25 °C and 40 °C for eighteen months (paper 2). The samples were analyzed monthly by GC/MS and GC/FID, as well as HPLC/DAD-MS/MS. This showed that the comparison of seed, isolated essential oil, and pure substance, whichhad not been considered in storage studies so far, was of extraordinary importance. Here, both the plant matrix and the essential oil had a protective effect on individual terpenes and delayed their degradation (paper 2). Further, a clear difference between photo-oxidation and autoxidation was observed. Light-induced oxidation of terpenes primarily resulted in the formation of hydroperoxides, whereas autoxidation led to a variety of compounds such as alcohols, ketones, and epoxides. Thus, the secondary products can serve as specific markers for conclusions about the preload and quality of essential oils. In the study presented in paper 3, further photo-oxidation experiments were conducted with beta-pinene, R-(+)-limonene, and gamma-terpinene, with added furocoumarins. Furocoumarins can absorb UV-A light in the range of 320 – 380 nm and enter an energetically excited state. This energy difference between the ground state and excited state can be dissipated again by the emission of fluorescent and phosphorescent light. In this process, short-wave energy-rich UV light is converted into lower-energy visible light (bathochromic shift). For this reason, the UV light-induced degradation of the terpenes beta-pinene, R-(+)-limonene, and gamma-terpinene could be significantly reduced by adding 5% each of xanthotoxin, bergapten, bergaptol, and bergamottin. The effect of adding bergaptol was most pronounced in the photooxidation of gamma-terpinene (paper 3). Consequently, in citrus essential oils from which the natural furocoumarins had been previously removed, irradiation with UV light resulted in a strong degradation of the terpenes. This process could be markedly reduced by the re-addition of 5% of the previously removed plant-specific furocoumarins (paper 4). In summary, it can be concluded that not only the plant matrix and the essential oil as a multicomponent mixture but also potential interactions with other substances forming part of the essential oil such as furocoumarins may significantly slow down the oxidation of terpenoids.
Investigations on (photo) reactions of cosmetic UV filters towards skin proteins
(2014) Stiefel, Constanze; Schwack, Wolfgang
Although UV filters are important, widespread used cosmetic ingredients, their reaction potential towards skin proteins has hardly been studied so far. Therefore, the aim of the present thesis was to investigate the reactivity of widespread UV filter substances towards skin proteins using increasingly complex protein and skin model systems and different analytical techniques. At first, the development of a rapid high-performance thin-layer chromatographic (HPTLC) screening method on an amino phase as protein model provided an easy and rapid way to estimate the reactivity of the common UV filters benzophenone-3 (BP-3), hydroxymethoxybenzoyl sulfonic acid (HMBS), butyl methoxydibenzoylmethane (BM-DBM), 3-benzylidene camphor (3 BC), 4 methylbenzylidene camphor (4 BMC), octocrylene (OCR), ethylhexyl methoxycinnamate (EHMC), ethylhexyl salicylate (EHS), diethylhexyl butamido triazone (DEBT), ethylhexyl triazone (EHT), and octyldimethyl p-aminobenzoic acid (OD-PABA) towards amino groups under thermal and irradiation conditions. A direct comparison of the results of the screening with (photo) patch test data of the dermatological practice showed that especially those UV filters, which are known to be common triggers for (photo) allergic reactions, showed the highest tendency to bind to the amino phase. This indicates that the screening may be well suited to identify possible skin sensitizers as part of a multistage testing strategy. The observation that the reactivity of the different UV filters was influenced by both heat and UV irradiation was verified during the subsequent studies with butylamine and ethanolamine. The UV filters showed individual, time- and temperature-dependent reactivities towards the amines. Benzophenone imines, enamines, and amides were identified as typical reaction products by means of electrospray ionization mass spectrometry (ESI-MS), Fourier transform infrared (FTIR), and nuclear magnetic resonance (NMR) spectroscopy. BP-3, HMBS, the dibenzoylmethanes, OCR, and EHS showed by far the highest reactivity what was in good correlation with the previous screening, indicating a different contact-allergic potential of the UV filters. In contrast, the esters EHMC and EHT showed a significantly lower reactivity, and for the UV filters 3 BC, 4-MBC, and OD-PABA no conversion was observable at all. The formation of the reaction products had partly big influence on the respective UV filter spectra. In the case of BP-3, HMBS, and EHS, the conversions led to a strong bathochromic shift and hence to approved UVA protection. In contrary, in the case of DBM and BM-DBM and especially OCR, a breakage of the original molecule structures was observed, resulting in a significant decrease of the respective absorption strength. The same reaction tendencies could also be observed, when using Boc-protected lysine, the tetrapeptide Boc-Gly-Phe-Gly-Lys-OH (Boc-GFGK), and bovine serum albumin (BSA) as increasingly complex protein or skin models. OCR and BM-DBM confirmed to be most reactive towards the lysine side chains of the mentioned model systems, followed by DBM > BP 3 > EHS > EHMC > EHT in decreasing order. To determine the covalent binding of the UV filters to the protein BSA, beside the extraction of the unbound UV filters, the increase of the molar mass of the formed BSA-adducts was additionally exemplarily determined for EHMC and DBM by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Both methods gave comparable results. Binding to BSA did not affect the UV absorption properties of BM-DBM, EHMC, and EHT, but led to a bathochromic shift in the cases of BP-3 and EHS. For OCR, a strong hypsochromic shift and a nearly complete loss of UVA+B protection was observable. To better reflect the usual application conditions, a thin gelatine layer was chosen as further skin model. The UV filter amounts applied were adapted according to the existing ISO norm for the determination of the SPF. Afterwards, UV irradiation was performed. The binding amounts were determined both by extraction of the unbound UV filters and by isotope-ratio mass spectrometry (IRMS), where two synthetized, stable-isotope labelled UV filter analogues (EHC-d5 and DBM-d5) were used. In contrast to the esters EHMC and EHT, which showed comparatively small binding amounts, for the UV filters OCR, BP-3, EHS, and BM-DBM significant reaction tendencies towards gelatin were observed. Finally, various commercial sunscreens and personal care products with UV protection were applied on either prepared porcine skin or glass plates, followed by UV irradiation. Significant differences were observed for the amounts of UV filters extracted from glass and skin. The lower recoveries in the case of the skin indicated an additional reaction of the UV filters towards the skin samples. BP-3 showed the highest discrepancy between the recoveries from glass and skin after irradiation, followed by EHS > BM DBM > OCR > EHMC > EHT in decreasing order. The present dissertation showed that cosmetic UV filters were able to react with amino structures of different proteins under thermal and irradiation conditions. As the formation of protein adducts is seen as key event in the development of (photo) allergic reactions, the results indicate a specific skin sensitization potential of the UV filters. This is confirmed by the experience of dermatological practice. Since such reactions have partly strong influence on the respective UV filter spectra, the existing in vitro methods using PMMA or quartz glass as substrates have to be questioned, since those methods cannot capture such skin-typical reactions.
Laccase-katalysierte Dominoreaktionen von Brenzcatechinen und Hydrochinonen mit 1,3-Dicarbonylverbindungen
(2012) Hajdok, Szilvia; Beifuss, Uwe
In the present work novel domino reactions have been described which are based on the laccase-catalyzed oxidation of catechols and hydroquinones to the corresponding o- and p-quinones and their subsequent reactions with 1,3-dicarbonyl compounds. In the first part of this thesis an efficient approach to 3,4-dihydro-7,8-dihydroxy-2H-dibenzofuran-1-ones has been developed. The method includes a laccase-initiated domino reaction between cyclohexane-1,3-diones and catechols using air as an oxidant. The reactions can be carried out under mild reaction conditions without using toxic reagents. The products were obtained in yields ranging from 70 to 97% and with high purity. Byproducts were not formed. The structures of all products were unambiguously elucidated by NMR spectroscopic methods. In the second part of this work laccase-initiated domino reactions between catechols and heterocyclic 1,3-dicarbonyls have been presented. Using pyridinones, quinolinones and thiocoumarin as substrates, the corresponding benzofuropyridinones, benzofuroquinolinones and thiocoumestans were being obtained. The reactions could be easily performed to deliver the products regioselctively in yields ranging between 55 and 98%. In contrast, polycyclic dispiropyrimidinones were exclusively formed when barbituric acid derivatives were employed as substrates. The unambiguous and complete structure elucidation of all products has been achieved by NMR spectroscopic methods (HSQMBC and band-selective HMBC) as well as by X-ray crystal structure analysis. In the third part of this work laccase-catalyzed transformations between differently substituted hydroquinones and 1,3-dicarbonyls have been studied. These reactions provide a new and highly selective method for the formation of quinone bisadducts with two adjacent 1,3-dicarbonyl substituents. The only exception is the reaction of 2-chlorohydroquinone with 4-hydroxycoumarin which delivers a trisadduct. It is noteworthy that under different conditions the reaction between hydroquinones and 1,3-dicarbonyls resulted in the formation of benzofuran derivatives. The unambiguous structure elucidation of all products has been achieved by NMR spectroscopic methods including spin pattern analysis of the long-range coupled C=O carbons and 13C satellites analysis in 1H NMR spectra. The domino reactions presented in this thesis allow for the efficient and selective synthesis of numerous heterocyclic systems as well as substituted p-benzoquinones under mild reaction conditions. In most cases the products can be isolated in good to very good yields and with high purity. For the structure elucidation of the products a wide range of NMR methods was used.
Metalloporphyrine als potentiell präbiotische Moleküle und chemische Biosignaturen
(2022) Pleyer, Hannes Lukas; Strasdeit, Henry
This doctoral thesis addresses two important astrobiological aspects of metalloporphyrins. First, the possible abiotic origin of metalloporphyrins under prebiotically plausible conditions, and second, metalloporphyrins as possible molecular biosignatures in the context of the search for extraterrestrial life. This two-pronged approach allowed us to obtain a more comprehensive overview of metalloporphyrins in an astrobiological context. In presently known living organisms, metal complexes of porphyrins and porphyrinoids occur ubiquitously, whereas free base porphyrins scarcely matter. Among the best known examples are chlorophylls (magnesium complexes), which are crucially involved in photosynthesis, and the heme group, for example as a cofactor of cytochromes, which play an important role in cel¬lular respiration. In terms of evolution, porphyrinoid cofactors appear to be very old, and indeed 1.1 billion-year-old geoporphyrins have been found in sedimentary rocks. Ancestors of current porphyrinoid cofactors, simple metalloporphyrins, may have been present in the first organisms or even earlier in abiotic protometabolisms. Furthermore, the wide distribution of porphyrinoid cofactors among known organisms, as well as their participation in basic biological functions, suggests that metalloporphyrinoids may also be present in potential life forms beyond Earth. Thus, metalloporphyrinoids could be useful as chemical biosignatures in the search for life on Mars, Europa, Enceladus and beyond. The first objective of this work was to investigate whether complex formation with selected metals (Fe, Mg, Co, Ni, and Cu) is possible under prebiotically plausible conditions. Based on a prebiotic synthesis of octaalkylporphyrins at simulated primordial volcanic coasts, which has been previously described by our group, the influence of wet-dry cycles on octaethylporphyrin (OEP) and selected metal sources was to be studied. For this purpose, first a novel, automated apparatus had to be developed that allowed the simulation of conditions on the early Earth, especially at primordial volcanic coasts. In particular, this apparatus had to ensure strict exclu-sion of atmospheric oxygen and allow fluctuating changes between wet and dry phases (simu-lation of tides or rainfall). Initially, experiments were conducted to test the new apparatus. It was shown that the appa-ratus worked completely automatically and reliably over a longer period of time. In further experiments, it was tested whether oxidation-sensitive substances can be handled in the apparatus. Indeed, it was shown that it is possible to strictly exclude oxygen. Thus, the apparatus proved to be suitable for investigating the formation of metal complexes from OEP and various metal sources under the influence of alternating wet-dry cycles. Metal sources used included metal(II) chlorides, metal sulfides, basalt, and iron meteorites. The focus was on iron sources. Indeed, it was shown that iron, magnesium, cobalt, nickel and copper complexes formed with the corresponding metal sources in an unusual reaction (completely water-insoluble OEP reacted with partly also insoluble metal sources), whereby wet-dry cycles turned out to be essential. Yields ranging from 20 to 78% (relative to the porphyrin) were obtained in fresh water. In addition, the influence of artificial seawater and low pH values on complex formation was investigated. In the second part of the thesis, the stability of metalloporphyrins was investigated using the compound chlorido(octaethylporphyrinato)iron(III), [FeCl(oep)], as a model. Potentially de-structive conditions relevant to metalloporphyrins as possible chemical biosignatures were selected for the experiments performed, as well as conditions that presumably prevailed on primordial volcanic coasts. Extensive series of experiments demonstrated, among other things, that (a) the iron OEP core is stable over a pH range of 0.0 to 13.5, (b) [FeCl(oep)] is stable up to ca. 250 °C in an inert atmosphere, (c) a salt matrix protects [FeCl(oep)] from X-ray radiation but not from iron particle radiation, and (d) hypochlorite, hydrogen peroxide, chlorate, and nitric acid oxidatively decompose [FeCl(oep)] (oxidation power in descending order). On the other hand, perchlorate, which is often mentioned in connection with its occurrence in the Martian regolith, did not show any oxidation effect. Finally, the importance of metalloporphyrins as potential biosignatures is discussed in the presented work, particularly in the light of their stability and possible abiotic synthetic pathways for these compounds.
Neuartige Kupfer-katalysierte und übergangsmetallfreie Methoden zum Aufbau von Heterocyclen
(2022) Rekowski, Szymon; Beifuss, Uwe
Heterocycles are the backbones of numerous drugs and are therefore of great importance in medicinal chemistry. As a result, there is an inevitably high demand for methods to synthesize heterocycles. The requirements for new methods for the synthesis of heterocycles are nowadays very high, as they must not only be efficient and selective, but also sustainable. These prerequisites can be met by both transition metal-free and transition metal-catalyzed reactions. Thus, the transition metal-free preparation of a variety of different heterocycles can be achieved by radical, cationic and anionic cyclizations as well as by pericyclic reactions. Recently, the importance of electrochemical and photochemical methods in heterocyclic synthesis has been increasing very rapidly. In transition metal-catalyzed heterocycle synthesis, Pd- and Cu-catalysts in particular play a prominent role. For the Pd-catalyzed assembly of N-heterocycles, the intramolecular Buchwald-Hartwig amination is especially noteworthy. It is now known that many Pd-catalyzed reactions can also be carried out with Cu-catalysts. In view of the fact that Cu-catalysts are much cheaper due to the higher abundance of Cu, and that expensive ligands can usually be omitted to carry out Cu-catalyzed reactions, their enormous importance in heterocyclic synthesis is easy to understand. For example, many N-heterocycles can be prepared by intramolecular Ullmann reactions with excellent yields and high selectivities. Here, bisfunctionalized substrates with two centers of different reactivity play a major role. The aim of the present work was to develop new efficient and highly selective synthetic methods for the construction of relevant N- or O-heterocycles. In particular, Cu-catalyzed reactions with bisfunctionalized substrates were to be developed. The investigation included determining whether the corresponding reactions can also be carried out in the absence of transition metal catalysts. Benzodioxines and 2,3-dihydrobenzodioxines exhibit many interesting biological properties, but the possibilities available nowadays for their synthesis are limited. Therefore, the first part of this dissertation deals with the development of a new method for the diastereospecific construction of (Z)-2-arylidene-2,3-dihydrobenzodioxines (Z)-80 (Scheme 50) by reacting 3-aryl-substituted (Z)-1,2-dibromoarylpropenes (Z)-82 with catechols 83. While the model substrate (Z)-82a (R1 = Ph) can be prepared by reduction and subsequent bromination of a-bromocinnamaldehyde, the remaining substrates (Z)-82 were prepared in three steps from the corresponding benzaldehydes. Subsequent optimization of the model reaction under a wide variety of reaction conditions showed that the best results could be obtained under transition metal-free conditions. The highest yield of (Z)-80a was obtained when 1 equivalent of (Z)-82a (R1 = Ph, R2 = H) was reacted with two equivalents of 83a (R2 = H) in the presence of four equivalents of Cs2CO3 in DMF for 18 h at 140 °C. Remarkably, this transition metal-free domino reaction, which consists of an intermolecular O-allylation followed by an intramolecular O-vinylation, is highly diastereospecific: the use of (Z)-1,2-dibromo-3-phenyl-2-propene [(Z)-82a] exclusively delivers (Z)-2-benzylidene-2,3-dihydrobenzodioxines [(Z)-80a]. This high diastereospecifity was also observed in the reactions of all other substrates (Z)-82. The 2-arylidene-2,3-dihydrobenzodioxins (Z)-80 were obtained in yields up to 89%. This method tolerates different substituents on the aromatic moiety of (Z)-82 as well as different disubstituted catechols 83. DFT calculations conducted in collaboration with Prof. Bharatham, NIPER Nagar (Mohali), suggest that the intramolecular O-vinylation proceeds via an alkene intermediate rather than an alkyne intermediate. The diastereoselective conversion of the E-configured substrate (E)-82a (R1 = Ph) to the corresponding (E)-2-benzylidene-2,3-dihydrobenzodioxine [(E)-80a] supports this assumption. The second part of this work is devoted to the intramolecular Cu(I)-catalyzed cyclization of o-haloarylideneguanylhydrazone salts (E)-86 for the direct construction of N-1 unsubstituted 1H-indazoles 84 (Scheme 51). The synthesis of indazoles of this type is of particular interest to medicinal chemistry because they form the backbone of some important anticancer drugs. Substrates (E)-86 were prepared by condensation of o-halobenzaldehydes with aminoguanidine hydrochloride in yields up to 90%. Subsequent cyclization using 10 mol% CuI, 30 mol% DMEDA and 0.5 equivalents of Cs2CO3 afforded the 1H-indazoles 84 in yields up to 75%. The reactions were carried out at 120 °C in DMF for 5 h in a sealed glass tube. The method tolerated a full range of substituents on the aromatic moiety of the substrates. Based on DFT calculations done in collaboration with Prof. Bharatham, NIPER Nagar (Mohali), it is reasonable to assume that E/Z isomerization of substrate 86 occurs first, followed by metal complexation with subsequent C,N bond formation. The final hydrolysis of the 1H-indazole-1-carboximidamide yields the N-1 unsubstituted 1H-indazole 84.
Rapid screening of antibiotics in foods by HPTLC-FLD/EDA/MS
(2015) Chen, Yisheng; Schwack, Wolfgang
Nowadays, the usage and partly abuse of veterinary antibiotics resulted in a very pressing need to control residues in foods of animal origin. Particularly, the increasingly demanding MRL issues and the huge number of samples to be monitored raised great challenges in this field. Microbial growth inhibition assays are traditionally employed for screening purposes, while sophisticated HPLC-MS methods are alternatively used or only used for confirmation purposes. To substitute the time consuming growth inhibition assays, HPTLC as a platform hyphenated to multi detection modes was employed in this study for the development of a high throughput, sensitive and cost-efficient screening-oriented methodology for antibiotics residues. The first step was focused on tetracyclines and fluoroquinolones, which are the most problematic antibiotics in the European Union and account for the most of the used veterinary antibiotics. To prevent strong tailing effects, the separation was optimized on normal-phase silica gel plates modified with ethylenediamine tetraacetic acid (EDTA). Besides, selective and sensitive fluorescence densitometry was optimized to achieve best signal/noise ratios. Under these conditions, limits of detection (LODs) and quantitation (LOQs) were in the range 12–25 and 45–95 µg/kg, respectively. Recoveries from milk samples, spiked at 50, 100 and 150 µg/kg and extracted by a modified QuEChERS procedure, ranged from 76 to 105%. To circumvent the ion suppressions due to EDTA, HPTLC-mass spectrometry (HPTLC-MS) was optimized, allowing the selective confirmation of positive findings, also offering high sensitivity of 25 µg/kg, and meeting Commission Regulation (EU) No. 37/2010. In the second step, sulfonamides were targeted, which are the secondly most administered veterinary antibiotics in the European Union. Separation of twelve most important sulfonamides was achieved on HPTLC silica gel plates, followed by fluram derivatization and sensitive and selective quantitation by fluorescent densitometry. LODs and LOQs were determined to 15–40 and 35–70 µg/kg, respectively. Samples of bovine milk, porcine liver and kidney were extracted according to the “QuEChERS” strategy. Additionally, a confirmative detection by HPTLC-MS was optimized, offering straightforward identification of target zones. The method was validated to meet the enforced Commission Regulation (EU) No. 37/2010. Finally, a more universal screening method based on HPTLC-bioautography was developed for most of the first-line veterinary antibiotics. A comprehensive HPTLC plate test revealed that the bio-compatibility of different plate layer materials to the applied bioluminescent bacteria (A. fischeri DSM No. 7151) was surprisingly different. It was then discovered that both bright bioluminescent background and significant inhibition zones of antibiotics can only be achieved on HPTLC amino F254S plates. In this case, HPTLC was not used for the chromatographic separation of individual antibiotics extracted with acetonitrile, but in terms of planar solid phase extraction to separate bioactive matrix compounds and to focus the analytes within two distinct target zones of different polarity. Together with HPTLC-MS for identification and confirmation purposes, the developed procedure enabled the rapid, sensitive and efficient multi-class screening of antibiotic residues (16 species of 5 groups, except sulfonamides and penicillins, which only affect Gram positive bacteria). The multi-sample plate images provided the results within a few hours. Thanks to the high sensitivity and the great matrix tolerance, the established method was successfully applied to bovine milk and porcine kidney samples, each spiked at the EU MLRs.
Selektive und effiziente Laccase-katalysierte oxidative Phenolkupplungen
(2012) Constantin, Mihaela-Anca; Beifuss, Uwe
The oxidative phenolic coupling is one of the fundamental reactions of organic chemistry. In contrast to its major role in the biosynthesis of numerous natural compounds the oxidative phenolic coupling is only of little importance in organic synthesis so far. This is due to its frequent lack of regio- and stereoselectivity. Laccases are oxidases which can be employed, amongst others, for the catalysis of oxidative phenolic couplings using O2 as the oxidant. This study highlights three examples which clearly demonstrate that laccases can be used as catalysts for regio- and stereoselective oxidative couplings of phenolic compounds. The first example deals with the laccase-catalyzed oxidative dimerization of (E)-2-propenylsesamol to carpanone (a). The oxidative cyclization starts with a phenolic oxidation, which is followed by a radical coupling and an intramolecular hetero-Diels-Alder reaction. Experiments with laccases and a number of other catalysts indicate that the diastereoselectivity of the carpanone formation doesn´t depend on the nature of the catalyst but on the double bond geometry of the substrate. With (E)-2-propenylsesamol as the substrate, a 9:1-mixture of carpanone (a) and its diastereoisomer c was formed, irrespective of the catalyst used. When (Z)-2-propenylsesamol was used as the substrate, the formation of a 5:1:4-mixture of three diastereoisomers, i.e. a, c and d, was observed. When the oxidation of (E)-2-propenylsesamol with O2 as the oxidant was run in the absence of any catalyst the diastereoisomeric benzopyrans a and b were obtained in a 3:2-ratio. From a mechanistic point of view, this reaction proceeds as a Domino oxidation/intermolecular hetero-Diels-Alder reaction. The second example selected was the laccase-catalyzed oxidative coupling of sesamol, a naturally occurring phenolic antioxidant. Here, a so far unknown trimer was formed as the main product in good yield. Experiments with different catalysts indicated that the course of the oxidative coupling of sesamol depends strongly on the catalyst chosen. Finally, the laccase-catalyzed phenolic coupling of di- and trisubstituted vanillidene derivatives with O2 as the oxidant was studied. The dimerization of (E)-ferulic acid proceeded as a 8,8?-coupling with formation of a dilactone. When the disubstituted vanillidene derivatives were reacted, the diastereoselective formation of the racemic dihydrobenzo[b]furans which can be understood as the products of a 5,8?-coupling mode were formed. In contrast to the disubstituted vanillidene derivatives, the laccase-catalyzed reaction of the trisubstituted vanillidene derivatives exclusively yielded biphenyls as the result of a 5,5?-coupling.
Studien zur Synthese von Pyripyropenen und Strukturanaloga durch Cyclisierungen
(2007) Schmidt, Dietmar; Beifuss, Uwe
In 1993 Omura et al. isolated from the culture broth of Aspergillus fumigatus FO 1289 a new class of compounds that exhibit a polyoxygenated sesquiterpene and α-pyrone and therefore was called pyripyropenes. Up to now pyripyropenes represent the most potent inhibitors of the enzyme cholesterol-O-acyl transferase (ACAT) that plays a key role in human fat metabolism. For example, arteriosclerosis have its origin in a fat digestion disorder and therefore ACAT-inhibiting agents like pyripyropenes shows a new promising approach in the treatment of this cholesterol level depending diseases. In the beginning preliminary studies were performed on improving the synthesis of pyripyropene E according to literature known procedures. From the big difficulties arriving from the synthesis of the key compounds this work focussed on: a) selective γ-acylation of 2-substituted aceto acetic esters, b) effective conversion of β, δ-diketo carboxylic acids into 4-hydroxy-2H-pyran-2-ones, c) optimized cyclization of epoxyolefine substituted 6-pyridyl-4-hydroxy-2H-pyran-2-ones by variation of different parameters like Lewis acids, solvents and temperature, d) design of a new efficient method for the synthesis of 6-substituted 4-hydroxy-2H-pyran-2-ones and related heterocycles, e) application of the in a) to d) elaborated results on the efficient total synthesis of three naturally occurring compounds with a 4-hydroxy-2H-pyran-2-on skeleton. The cyclization substrate in the geranyl series, the 3-[7-(2,3-epoxy-2,3-dihydrogeranyl)]-6-(3-pyridyl)-4-hydroxy-pyran-2H-pyran-2-one, was synthesized in a nine step sequence starting from geraniol with 18 % overall yield. In the following experiments this epoxide was cyclized in liquid sulphur dioxide with twelve different Brønsted and Lewis acids. Dependent on the Lewis acid up to four products were isolated: a pyrano[4,3-b]chromen-1-one (α-pyrone), a pyrano[2,3-b]chromen-4-one (γ-pyrone), a 7-oxa-bicyclo[2.2.1]hept-2-ane and a diol generated from opening of the epoxide function. The structure of the diol was unambiguously determined by total synthesis. In the farnesyl series the cyclization substrate was assembled in total analogy to that one of the geranyl series in a nine step sequence with 20 % overall yield starting from (E,E)-farnesol. After the cyclization experiments an inseparable mixture of eight different isomers were obtained. During a series of iodocyclization experiments 3-(7-geranyl)-6-pyridyl-4-hydroxy-2H-pyran-one was reacted with iodine in acetonitrile without a base. In another experiment the corresponding enolate anion was generated by deprotonation with potassium carbonate and then was brought to reaction with iodine. Without base the iodo substituted pyrano[2,3-b]pyran-4-one derivative (γ-pyrone) was formed. The reaction of the enolate anion with iodine resulted in a mixture of two iodo substituted α-pyrones, a pyrano[4,3-b]pyran-5-one (endo product) and a furo[3,2-c]pyran-4-one (exo product). From the experience that was gained by the assembly of the cyclization substrates in the geranyl series a new synthesis of 6-substituted 4-hydroxy-2H-pyran-2-ones was developed: The main principle was the in situ release of the extraordinary sensitive 5-hydroxy-3-oxo-pent-4-enoic acids through protonation of their stable bispotassium salts with TFA at low temperature, followed by spontaneous lactonization of these acids in the reaction media TFAA leading to the corresponding pyrones in high yields. The effectiveness of this procedure was impressively demonstrated in twelve examples. The 5-phenyl-substituted bispotassium salt was used for the construction of heterocycles: one pyrazole and one isoxazole were synthesized in high yields. Based on the new pyrone synthesis the total synthesis of three natural products with a 4-hydroxy-2H-pyran-2-one framework was carried out. 1. The compound Sch-419560 isolated from Pseudomonas fluorescens was synthesized in a four step sequence starting from ethyl 2-hexylacetoacetate with 62 % overall yield. 2. The α-pyrone 3?,3?-dimethylallylconrauanalactone isolated from Garcinia conrauana Engl. (Guttiferae) was synthesized via a four step sequence starting from ethyl 2-prenylacetoacetate with 64 % overall yield. 3. Finally, the natural product aurantiacone isolated from Mimulus aurantiacus was synthesized via a seven step sequence starting from 2-hydroxybutyric acid with 62 % overall yield.

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