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Organic micropollutants (OMPs) have been widely detected in the aqueous environment and their effect on the environment and human health has been studied. The removal of OMPs from effluents of a co...

The worldwide increasing consumption of the phosphonates 2-phosphonobutane-1,2,4-tricarboxylic acid [PBTC], 1-hydroxyethane 1,1-diphosphonic acid [HEDP], nitrilotris(methylene phosphonic acid) [NTMP], ethylenediamine tetra(methylene phosphonic acid) [EDTMP] and diethylenetriamine penta(methylene phosphonic acid) [DTPMP] over the past decades put phosphonates into focus of environmental scientists and agencies, as they are increasingly discussed in the context of various environmental problems. The hitherto difficult analysis of phosphonates contributed to the fact that very little is known about their concentrations and behavior in the environment. This work critically reviews the existing literature up to the year 2016 on the potential environmental relevance of phosphonates, their biotic and abiotic degradability, and their removal in wastewater treatment plants (WWTPs). Accordingly, despite their stability against biological degradation, phosphonates can be removed with relatively high efficiency (>80%) in WWTPs operated with chemical phosphate precipitation. In the literature, however, to our knowledge, there is no information as to whether an enhanced biological phosphorus removal alone is sufficient for such high removal rates and whether the achievable phosphonate concentrations in effluents are sufficiently low to prevent eutrophication. It is currently expected that phosphonates, although being complexing agents, do not remobilize heavy metals from sediments in a significant amount since the phosphonate concentrations required for this (>50μg/L) are considerably higher than the concentrations determined in surface waters. Various publications also point out that phosphonates are harmless to a variety of aquatic organisms. Moreover, degradation products thereof such as N-(phosphonomethyl)glycine and aminomethylphosphonic acid are regarded as being particularly critical. Despite their high stability against biological degradation, phosphonates contribute to eutrophication due to abiotic degradation (mainly photolysis). Furthermore, the literature reports on the fact that phosphonates in high concentrations interfere with phosphate precipitation in WWTPs. Thus, it is recommended to remove phosphonates, in particular from industrial wastewaters, before discharging them into water bodies or WWTPs.

Acylated derivatives of S-(2,3-dihydroxypropyl)-cysteine (S-glycerylcysteine) form the N-terminus of structural and functional proteins of bacterial origin. Synthetic lipopeptides containing tripalmitoyl-S-glycerylcysteine are derived from bacterial lipoprotein and constitute potent immunoadjuvants activating both B-lymphocytes and macrophages. There is increasingly interest in conjugates consisting of tripalmitoyl-S-glycerylcysteine linked to appropriate viral and bacterial antigens, because of their capability of inducing antigen specific antibodies and T-helper and T-killer cell specific immune responses. A new convenient synthetic pathway for the preparation of these tripalmitoyl-S-glycerylcysteinyl peptides is described. The use of N alpha-Fmoc-protected S-(2,3-dihydroxypropyl)-cysteine and its O,O'-bis acylated derivatives for the synthesis of triacyl-S-glycerylcysteinyl, O,O'-bis-acyl-S-glycerylcysteinyl and S-glycerylcysteinyl peptides of high diastereomeric purity by solid phase peptide synthesis or synthesis in solution is demonstrated.

The synthesis and characterization of lipopeptides consisting of the lipoamino acid N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteine (Pam3Cys-OH) and different peptide segments and/or spacer molecules is described. Pam3Cys-peptides, which are derived from the immunologically active N-terminus of bacterial lipoprotein, were obtained either by solution or solid phase peptide synthesis. In particular, the amphiphilic and water-soluble lipohexapeptides Pam3Cys-Ser-(Lys)4 and Pam3Cys-Ser-(Glu)4 proved to be potent macrophage and B-cell activators and non-toxic, non-pyrogenic immune adjuvants in combination with or covalently linked to antigens and haptens.

Polybrominated diphenyl ethers (PBDEs) are a group of brominated flame retardants (BFRs) used mainly as additives in different kinds of plastic material. Various PBDEs are found in all environmental compartments as well as in tissue and blood serum of animals and humans due to their persistence and tendency to bioaccumulate. Emission of PBDEs into the environment can occur during recycling of PBDE-containing plastic material or during their uncontrolled or insufficient combustion as e.g. in accidental fires or landfill fires. Under these circumstances, PBDEs can also function as precursor molecules for the formation of polybrominated dibenzodioxins (PBDDs) and dibenzofurans (PBDFs). In this study, we qualitatively investigated the reaction of two PBDE congeners, 2,2',4,4'-tetrabromo diphenyl ether (BDE 47) and 2,2',4,4',5,5'-hexabromo diphenyl ether (BDE 153), as well as hexabromobenzene (HBB), a flame retardant used in the past, when exposed to temperatures between 250 degrees C and 500 degrees C. The formed reaction products were analysed by high resolution gas chromatography-low resolution mass spectrometry (HRGC-LRMS). Among others brominated-chlorinated diphenyl ethers were formed by chlorodebromination of the PBDEs. In addition, thermolysis of BDE 47 and BDE 153 in the presence of tetrachloromethane as model substance for an organic chlorine source was studied. Thermal treatment of HBB resulted in the formation of brominated-chlorinated benzenes.

In order to enhance the sensitivity and the speed of the yeast estrogen screen (YES)-assay, which has been established in many laboratories for the determination of estrogenic activity of compounds and environmental samples, the LYES-assay, a modified version of the YES-assay including a digestion step with the enzyme lyticase, was developed. With the LYES-assay the estrogenic activities of natural (17beta-estradiol E2 and estrone), synthetic (17alpha-ethinylestradiol EE2) and pharmaceutical estrogens (diethylstilbestrol DES) as well as xenoestrogens (4-nonylphenol NP and five parabens) were determined and compared with the results obtained by other in vitro-assays namely the conventional YES-assay, the E-Screen-assay (MCF-7 breast tumor cell proliferation) and a receptor binding-assay (RB) with human estrogen receptors hER-alpha and hER-beta. In the case of E2 the LYES-assay had a significantly lower limit of quantification (LOQ) than the conventional YES-assay and even two orders of magnitude lower than the RB-assay. Compared to the E-Screen-assay the LOQ of the LYES-assay was almost one order of magnitude higher. The time required to perform the LYES-assay was as little as seven hours compared to three to five days for the conventional YES-assay. Thus, the LYES-assay is a very good alternative to existing estrogenic in vitro-assays, since it has a good sensitivity, is cheap and much faster than the other assays.

Background: Ethyl glucuronide (EtG), a direct ethanol metabolite, seems to meet the need for a sensitive and specific marker for monitoring recent alcohol consumption in different settings. Our aim was to study sensitivity, specificity, and the influence of various parameters on EtG levels in urine. Patients and Methods: Urine samples for a total of 453 patients (373 male, 80 female) were statistically analyzed. The mean age was 37.1 years (median 36, SD 12.59), body mass index was 24.7, total ethanol consumed last month was 1817.66 g (each median), and 80 patients reported cannabis use within the last 30 days. Determination of EtG was performed with a liquid chromatography-tandem mass spectrometry method with deuterium-labeled EtG as internal standard. Results: For EtG in urine, a good correlation was found with other state markers and days of sobriety. In a regression analysis, age, gender, marijuana use, kidney disease, and total grams of ethanol consumed last month were the variables that significantly influenced EtG levels in contrast to race, smoking, body mass index, cirrhosis of liver, age began drinking regularly, packs of cigarettes smoked last month, and total body water. Furthermore, in a receiver operating characteristic curve analysis to distinguish between nondrinkers and individuals sober >4 days versus individuals drinking in the recent 4 days, area under the curve was 0.834. At a cutoff of 0.145 mg/liter, sensitivity was 83.5% and specificity 68.3%. A receiver operating characteristic curve was calculated for lifetime alcohol abuse or dependence against those who had never been abusers or dependent. In this case, subjects were either never dependent or lifetime dependent, but those currently dependent were excluded. The resulting area under the curve was 0.694. At a cutoff of 0.145 mg/liter, sensitivity was 73.8% and specificity 60.3%. For those with a self-reported sobriety of less than 24 hr, the area under the curve was 0.899, sensitivity was 90.8%, and specificity was 76.5% at a cutoff of 0.435 mg/liter when we calculated nondrinkers and light drinkers against heavy drinkers and drinkers needing treatment. Cannabis-using patients showed significant differences with regard to almost all state markers when compared with nonconsuming subjects. Conclusions: Age, gender, marijuana use, kidney disease, and total grams of ethanol consumed last month should be taken into consideration when interpreting results of EtG in urine. Sensitivity and specificity seem promising. Cannabis use can be regarded as an indicator for other serious mental problems in alcohol-using subjects.

Xenin, a 25 aminoacid peptide, interacts with the neurotensin receptor subtype 1 of intestinal muscles of the guinea pig. Replacement of the C-terminal Lys -Arg peptide bond in xenin 6 by a reduced pseudo-peptide bond augmented binding affinity to isolated jejunal and colonic muscle membranes by factors of 7.7 and 21.0 respectively; the potency to contract the jejunum and to relax the colon was increased by factors of 3.2 and 1.3. The C-terminus Trp-Ile-Leu (WIL) of xenin, in contrast to the C-terminus Tyr-Ile-Leu (YIL) of neurotensin, bound competitively to the muscle membranes. WIL blocked the contractile action of xenin in the jejunum and was synergistic with the relaxing action in the colon. The Lys -Arg motif and Trp in the C-terminus of xenin are essential structures in the action of xenin on the enteral smooth muscle receptors.

The separation of oligosaccharides derivatized with various esters of aminobenzoic acid by means of reversed-phase nano-HPLC (nHPLC) with on-line ESI mass spectrometry and off-line MALDI-TOF mass spectrometry as well as MEKC is described. For this purpose methyl, ethyl and butyl aminobenzoates and heptyloxyaniline were used as derivatization agents for homologous maltodextrins and oligosaccharides from human milk. Four different C 1 8 stationary phases were tested for this purpose because the type of stationary phase was shown to have a dramatic effect on the performance of the separation. Optimal results were obtained using n-butyl aminobenzoate as label and an encapsulated ODS stationary phase. The on-line coupling of nHPLC to ESI MS allowed to separate and identify various oligosaccharides from human milk. This technique enabled the exact attribution of the molecular structure to a signal in the chromatogram. In a second approach oligosaccharides were separated by nHPLC and subsequently fractionated. The fractions were analyzed by MALDI-TOF mass spectrometry. The results obtained by this approach confirmed the ESI MS data. An analogous separation profile was obtained by using sodium dodecyl sulfate in MEKC, which proves that the retention mechanisms of both techniques are identical.

The proliferation test with human estrogen receptor-positive MCF-7 breast cancer cells (E-Screen assay) was applied for quantitative determination of total estrogenic activity in 24-h composite effluent samples from 16 municipal and two industrial sewage treatment plants (STPs) in the state of Baden-Wurttemberg, southwestern Germany. The estrogenic efficacy relative to the positive control, 17beta-estradiol, was between 26 and 74% (median, 48%) for the 16 municipal STPs. Estradiol equivalent concentrations (EEQs) were between 0.2 and 7.8 ng/L (median, 1.6 ng/L) and, thereby, were lower than those found in a pilot study, which revealed EEQs of greater than 10 ng/L in the effluents of two other STPs. The EEQs in 14 of the 16 effluent samples were very similar (0.9-3.3 ng/L), indicating a rather constant input of estrogenic substances via STPs into rivers. Additional activated charcoal filtration turned out to be very efficient in further eliminating estrogenic activity from effluents. The EEQs of the E-Screen assay and those calculated from the results of extensive chemical analysis using the estradiol equivalency factors determined for 13 natural and synthetic estrogenic substances were comparable for most of the effluent samples. 17beta-Estradiol, 17alpha-ethinylestradiol, and, to a lesser extent, estrone contributed to 90% or more of the EEQ value.

The separation of homologous maltodextrins using methyl-, ethyl- and n-butyl esters of aminobenzoic acid and n-heptyloxyaniline by means of reversed phase nano-HPLC is presented. Fused silica capillaries packed with four different stationary phases were tested, the separation of the derivatized sugars was optimized and the separation properties of these columns were tested on the basis of the van Deemter plots. UV/VIS detection was used (285 nm for the aminobenzoic esters and 275 nm for the aniline derivatives) in combination with on-line ESI triple quadrupole mass spectrometry and off-line MALDI-TOF mass spectrometry. The choice of the stationary phase had a tremendous influence on the separation. Optimal results were obtained with an encapsulated ODS stationary phase and n-butyl aminebenzoate as label for the oligosaccharides. Malto-oligosaccharides with a degree of polymerization of up to of 25 could be resolved to baseline. On-line coupling with ESI-MS yielded additional information as a result of induced fragmentation and increased sensitivity by monitoring precursor ion. Off-line mass detection with MALDI-TOF was performed on the separated derivatized saccharides collected from nano-HPLC runs. The MALDI-TOF mass spectra confirm the ESI-MS data.

The polycyclic musks tonalide ® (acetyl hexamethyltetraline = 1-(3,5,5,6,8,8-hexamethyl-6,7-dihydronaphthalen-2-yl)ethanone, AHTN), galaxolide ® (1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta( g )-2-benzopyrane, HHCB) and the degradation product HHCB-lactone were determined in water samples and brown trouts ( Salmo trutta fario ) of the river Ammer, a small catchment in the state of Baden-Wurttemberg, south-west Germany. The Ammer receives the effluent discharge of two municipal wastewater treatment plants (WWTPs) with 90,000 population equivalents. The wastewater contributes 14% of the total discharge of the river (average 1.0 m 3 /s). Water samples were collected monthly at 12 sampling points from June 2010 to May 2011. Downstream the WWTPs the median concentrations of HHCB, AHTN and HHCB-lactone were 0.26 μg/L, 0.06 μg/L and 1.0 μg/L, respectively. The effluent of the WWTPs was identified as main source of the synthetic musks in the surface water. The ratio of HHCB-lactone/HHCB showed significant seasonal variations indicating the influence of the water temperature on the degradation of HHCB in the surface water. A total of 251 trout was caught in two campaigns in October 2010 at 12 sampling points. The median concentrations of HHCB and AHTN in the trouts downstream the WWTPs significantly increased to 10.8 μg/g lipid weight (LW) and 3.7 μg/g LW, respectively.

Recently, a bacterium capable to oxidize ammonium anaerobically at a high rate was identified as novel member of the Planctomycetales (Strous, M., Fuersi, J. A., Kramer, E. H. M., Logemann, S., Muyzer, G., van de Pas-Schoonen, K. T., Webb, R. I., Kufnen, J. G., and Jetten, M. S. M.: Nature 400, 446-449, 1999). Here we investigated the microbial community structure of a trickling filter biofilm with a high anaerobic ammonium oxidation activity. Fluorescence in situ hybridization (FISH) with a set of nine probes designed for specific identification of the recently described anaerobic ammonium oxidizer demonstrated that only one probe hybridized to bacteria within the biofilm. For phylogenetic characterization of putative biofilm anaerobic ammonium oxidizers a full-cycle 16S rDNA approach was performed by using a Planctomycetales-specific forward primer for PCR amplification. Of the twenty-five 16S rDNA fragments (1364 bp in length) amplified from the biofilm, nine were affiliated to the Planctomycetales. Comparative analysis showed that these sequences were more than 98.9% similar to each other but only distantly related to the previously recognized anaerobic ammonium oxidizer (below 91% similarity) and all other organisms represented in public 16S rRNA databases (similarities of below 79%). The retrieved sequences and the previously recognized anaerobic ammonium oxidizer represent two well-separated groups of a deep-branching lineage within the Planctomycetales. Quantitative FISH analysis with a newly designed specific probe showed that the novel bacterium, provisionally classified as "Candidatus Kuenenia stuttgartiensis" constituted the dominant fraction of the biofilm bacteria. In situ probing revealed that ammonia-oxidizing bacteria of the beta-subclass of Proteobacteria were also present, albeit in significant smaller amounts, within the anoxic biofilm. Comparative sequence analysis of a stretch of the gene encoding ammonia-monooxygenase (amoA) demonstrated the occurrence of the DNA of at least three different populations of beta-subclass ammonia oxidizers within the biofilm.

Streptomyces ghanaensis (ATCC 14672) produces the phosphoglycolipid antibiotic moenomycin consisting of several components. A solid phase extraction procedure was developed which allowed a rapid isolation of both moenomycin and its biosynthetic intermediates from culture filtrates. Semi-preparative high performance liquid chromatography followed by high performance liquid chromatographv-mass spectrometry provided structural data on the different moenomycin components. In order to obtain initial information on the biosynthetic pathway, moenomycin non-producing mutants were isolated. They were shown to release intermediates with shorter lipid chains suggesting that the lipid chain synthesis probably takes place at a later stage of the moenomycin biosynthesis. Based on the biological activity and the analytical data, we assume that a modification and in particular a shorter lipid portion drastically influences the inhibitory activity of this antibiotic.

Electrospray mass spectrometry (ESI-MS), tandem massspectrometry and on-line RP-HPLC-ESI-MS were used toevaluate the composition and purity of three differentaryl ether mixtures consisting of 10 and 45 arylethers synthesized on solid support by Williamsonetherification. The libraries feature two potentialpharmacophores connected with three different spacersand serve as models for a detailed component analysis.Individual members of the library and by-products wereidentified rapidly and conveniently by product ionscans. Compound collections obtained by two differentsynthetic methods, the split/combine approach and thepremix method, showed different mass distributions inthe ESI-MS spectra. Some components were not detectedin direct ESI-MS measurements, but were found by MS/MSexperiments. Precursor ion and constant neutral lossscans allowed the identification of components withcommon structural features.

Matricine, a genuine compound of chamomile, is unstable under acid conditions. Upon steam distillation it is readily converted into chamazulene carboxylic acid and further on into chamazulene. In the present work stability tests were performed in the pH-range between 4 and 10. The decomposition of matricine in aqueous pH adjusted and in buffered solutions was monitored using an isocratic HPLC-method. In buffered solutions of pH 4 to 6 stored at room temperature for several days chamazulene carboxylic acid was found to be the first intermediate degradation product. In alkaline solutions of pH 8 to 10 however, 8-desacetyl-matricine was observed as a new degradation product. This compound was identified by UV, IR, MS and 1H-NMR. A log κ/pH-profile of matricine was established showing a stability optimum at pH 8.

Betacyanins from cell cultures of Phytolacca americana were characterized and compared with those of the stems and ripening fruits of the plant. Whereas in fruits prebetanin (betanin 6'-O-sulphate) and its isoform predominate, in the stem and cell cultures feruloylated derivatives occur as the major components. These were rigorously identified by various spectroscopic techniques (DAD-HPLC, NMR, LC-MS and electrospray MS-MS) and carbohydrate analyses as betanidin 5-O-[(5"-O-E-feruloyl)-2'-O-beta-D-apiofuranosyl] -beta-D-glucopyranoside, a new betacyanin of higher plants, and betanidin 5-O-(6'-O-E-feruloyl)-beta-D-glucopyranoside (lampranthin II), together with their isoforms.

Kanchanamycins are a new group of polyol macrolide antibiotics isolated from Streptomyces olivaceus Tü 4018. They all share a common bicyclic carbon skeleton formed by a 36-membered lactone ring and a 6-membered hemiacetal ring. A feature unusual for that class of macrolides is the terminal urea moiety observed in kanchanamycin A. The structures of the kanchanamycins were determined by electrospray MS and modern 2D NMR techniques. Due to substantial overlap of the signals intensive use of inverse detected heteronuclear correlation experiments (HSQC, HMBC, 2D-HSQC-TOCSY) was made.

In the search for a protein primer for starch synthesis, an autocatalytic self-glucosylating protein has been isolated from sweet corn. Several tryptic peptides were obtained from the [14C]glucosylated protein and were sequenced, corresponding to over 40% of the estimated total sequence (molecular mass 42 kDa). There is no homology with the amino acid sequence of the autocatalytic glycogen primer, glycogenin, nor in respect of the nature of the union between the autocatalytically added glucose and the protein, which, in the case of the corn protein, now named amylogenin, is a novel glucose-protein bond, a single β-glucose residue joined to an arginine residue.

The amino acid sequence of the novel lantibiotic epilancin K7 from Staphylococcus epidermidis K7 was determined by NMR spectroscopy. NMR spectroscopy was used because sequencing by conventional Edman degradation techniques was prohibited by internal sequence blocks owing to the presence of modified residues. Epilancin K7 consists of 31 residues, including two alpha,beta-didehydroalanine (one-letter code U) and two alpha,beta-didehydrobutyrine (O) residues, one lanthionine (A-S-A), two beta-methyllanthionines (A*-S-A), and six lysines. Epilancin K7 has a molecular mass of 3032 +/- 1.5 Da. The amino acid sequence of epilancin K7 was derived from both through-space dipolar proton-proton interactions and through-bond scalar proton-carbon interactions as detected by two-dimensional 1H-NOESY, 1H-ROESY and three-dimensional 1H-TOCSY-NOESY, and by two-dimensional 1H,13C-heteronuclear multiple-bond correlation spectroscopy, respectively. The sequence is as follows: [sequence: see text] The N-terminal residue X partly resembles an alanine but its exact nature is unclear. The organization of the sulfide-bridge-containing (beta-methyl-)lanthionines was revealed by 1H-NMR and 1H,13C-NMR spectroscopy. Epilancin K7 has a linear structure and a high positive net charge, and therefore is classified as a type-A lantibiotic. NMR analysis of a degraded though still active form of epilancin K7 showed that two N-terminal residues of epilancin K7 were missing, owing to decomposition at the alpha,beta-didehydro alanine at position 3; it was called the epilancin K7-(3-31)-peptide (peptide fragment of epilancin K7 consisting of positions 3-31). The usefulness of three-dimensional 1H-TOCSY-NOESY, and two-dimensional 1H,13C-heteronuclear multiple-bond correlation spectroscopy at natural abundance for the study of (modified) polypeptides is demonstrated.

A new member of the quinoxaline group antibiotics has been detected by HPLC-diode-array screening. The main compound produced by Streptomyces tendae strain Tü 4031 showed a high degree of similarity in the UV-visible spectral region with echinomycin and their structural similarity was confirmed by structure elucidation using electron tandem mass spectrometry and 2D nuclear magnetic resonance. The new compound, named echinoserine, is a non-cyclic form of echinomycin, but it is not a biosynthetic precursor. Echinoserine is less antibiotically active than echinomycin.

Betacyanins from the bracts of Bougainvillea glabra were isolated and characterized by a combination of spectroscopic techniques (DAD-HPLC, NMR, LC-MS, GC-MS, electrospray MS, tandem MS) as gomphrenin I (betanidin 6-O-beta-glucoside) and various derivatives of bougainvillein-v (betanidin 6-O-beta-sophoroside), i.e. mono- and diglucosylsophorosides which are acylated with 4-coumaric and caffeic acid (mono- and diesters). Besides the betacyanins, B. glabra bracts accumulated large amounts of flavonols (kaempferol and quercetin conjugates) reaching ratios of flavonol to betacyanin of 1:1.

Anthocyanins isolated and characterized from the wild carrot suspension cultures used here were 3-O-β-D-glucopyranosyl-(1→6)-[β-D-xylopyranosyl-(1→2)-]β-D

Two lipopeptide analogues of the Escherichia coli lipoprotein rendered water-soluble by polyoxyethylene were tested for mitogenicity in vitro in murine and human B lymphocytes and for adjuvant activity in vivo in mice. These highly amphiphilic lipopeptides retained the biological activity other lipopeptides usually exerted which supports the hypothesis of specific interactions of lipopeptides with membranes of reactive cells. The activation of human B lymphocytes by these lipopeptides was much less pronounced compared to that of murine cells. However, given in combination with anti-CD40 antibodies plus interleukin-4, human B lymphocytes could synergistically be stimulated to proliferate. As an adjuvant, the polyoxyethylene linked lipopeptides were almost as potent as Freund's adjuvants and other basic lipopeptides. Being water-soluble, these novel analogues are easy to apply and they are suitable for field studies as adjuvants when sonication can not usually be provided.

Different substrates were used in reaction assays for rosmarinic acid synthase, an enzyme involved in rosmarinic acid biosynthesis in cell cultures of Coleus blumei. In order to introduce the cinnamoyl moiety of the ester, 4-coumaroyl-CoA and caffeoyl-CoA were applied as substrates; for the hydroxyphenyllactic acid moiety, 4-hydroxyphenyllactate and 3,4-dihydroxyphenyllactate were used. Four different reaction products were thus produced and these were analysed by ion spray mass spectrometry and tandem mass spectrometry after purification by semi-preparative high pressure liquid chromatography. The molecular masses and the fragmentation patterns obtained by tandem mass spectrometry of the reaction products formed by the catalysis of rosmarinic acid synthase were in agreement with the structures of rosmarinic acid (caffeoyl-3′,4′-dihydroxyphenyllactate) and rosmarinic acid-like esters (4-coumaroyl-4′-hydroxyphenyllactate, 4-coumaroyl-3′,4′-dihydroxyphenyllactate, caffeoyl-4′-hydroxyphenyllactate) as expected for the substrates used in the enzyme reaction.

A novel side reaction in Fmoc-solid-phase synthesis, which occurs during removal of protecting groups and detachment from the resin, was elucidated by investigations on model peptides: During the cleavage of Pmc- or Mtr-protecting groups from arginine residues by trifluoroacetic acid in peptides with O-tert-butyl-protected aliphatic hydroxyamino acids, peptides containing O3-sulfo-serine and O3-sulfo-threonine are formed as side-products in high yields, if suitable scavengers are absent. Subsequent to their isolation and purification, the structures of these peptide sulfuric acid mono-esters could unequivocally be proven by chemical and spectroscopic (MS, NMR, IR) methods.

Anthocyanins of Daucus carota L. were investigated by pneumatically assisted electrospray (ES) mass spectrometry. In positive mode, the spectra of anthocyanins showed the natural molecular cations M+ together with ions corresponding to the aglycones. The anthocyanin compositions of extracts from suspension cultures, callus and roots of an Afghan carrot cultivar and from the central flowers of the European wild carrot were compared by using on-line high-performance liquid chromatography/ES mass spectrometry and tandem mass spectrometry. Collision-induced dissociation (CID) of M+ with argon produced the aglycone ion as the only fragment. Daughter scans revealed that all anthocyanins found in carrot contain cyanidin as aglycone. Scanning the parent ions of the cyanidin ion (m/z 287) allowed a selective and sensitive detection of up to 14 different cyanidin derivatives directly from the extracts. The presence of seven anthocyanins with M+ at m/z 949, 919, 905, 889, 863, 743 and 581 in all extracts investigated indicated a good accordance of the anthocyanin composition in tissue cultures and in intact plants.

Six anthocyanins were isolated from cell suspension cultures of an Afghan cultivar of Daucus carota by PC or HPLC. The structures of these compounds were elucidated by spectroscopic methods as cyanidin 3-O-lathyroside, cyanidin 3-O-(2''-O-beta-D-xylopyranosyl-6''-O-beta-D-glucopyranosyl-beta-D- galactopyranoside), and the latter acylated with 4-coumaric, ferulic, 4-hydroxybenzoic or sinapic acid. Unusual 1H NMR chemical shifts and 1H NOE data indicate an intramolecular copigmentation of the aglycone with these aromatic residues.

The betacyanins gomphrenin I (betanidin 6- O -β-glucoside) and its acylated forms gomphrenin II (betanidin 6- O -[6′- O -( E -4-coumaroyl)-β-glucoside]) and gomphrenin III (betanidin 6- O -[6′- O - E -feruloyl-β-glucoside]) were isolated from flowers of Gomphrena globosa and their structures elucidated by NMR spectroscopy and ion spray mass spectrometry by comparison with betanin (betanidin 5- O -β-glucoside) and its feruloyl conjugate lampranthin II (betanidin 5- O -[6′- O - E -feruloyl-β-glucoside]), which were isolated from Beta vulgaris roots and B. vulgaris cell suspension cultures, respectively. The application of this new mass spectrometric technique to this type of compound is discussed. Unusual 1 H chemical shift differences between gomphrenin I and gomphrenins II and III suggest intramolecular stacking occurring in the latter two compounds.

The use of ion-spray (i.s.) mass spectrometry for the rapid determination of molecular weight and purity is demonstrated for derivatives of cyclomalto-hexaose (αCD),-heptaose (βCD), and -octaose (γCD), and for βCD. The i.s.-mass spectra contain peaks for singly and doubly charged protonated and cationised quasi-molecular ions. Methylation of CDs yielded homogeneous compounds, whereas the synthesis of CDs containing methyl groups and an additional substituent, e.g., allyl, pentenyl, octenyl, trifluoroacetyl, or heptafluorobutanoyl, afforded mixtures of products, the compositions of which could be determined directly from the i.s.-mass spectra. The addition of lithium chloride usually facilitated the interpretation of the spectra by producing abundant (M + Li)+ ions and suppressing fragmentation. Zinc acetate favoured the formation of doubly charged molecular ions. Additional information on the structure of derivatised CDs can be obtained by i.s.-tandem mass spectrometry.

Suitable protected derivatives of trihydroxynorleucines-[(2S,4S,5S')- and (2R,4S,5S')-2-amino-4,5,6-trihydroxyhexanoic acid,] of all isomeric dihydroxynorvalines [2-amino-4,5-dihydroxypentanoic acids), of all isomeric 2-amino-6,7-dihydroxyheptanoic acids and of (2S)-2-amino-6-hydroxymethyl-7-hydroxyheptanoic acid are synthesized via the corresponding α,β-didehydro compounds, which are hydrogenated with the optically active homogeneous catalyst [Rh(COD)(DIPAMP)] + BF − 4

From a strain ofRhizopus microsporus var.rhizopodiformis a novel siderophore, named rhizoferrin, was isolated by ion-exchange column chromatography, gel filtration and preparative HPLC. Hydrolysis with 6 M HCl and subsequent gas chromatography/mass spectrometry (GUMS) of the esterified/trifluoroacetylated derivatives indicated that citric acid and diaminobutane were the only constituents. From positive fastatom-bombardment (FAB) and ion-spray tandem mass spectrometry, a molecular mass of 436 Da and the assignment of several daughter ion fragments could be obtained, which indicated the presence of two citric acid residues and one diaminobutane residue. NMR studies finally confirmedN 1,N 4-bis(1-oxo-3-hydroxy-3,4-dicarboxybutyl)-diaminobutane as the structure of rhizoferrin. The iron-binding property was demonstrated on chromeazurol S plates and its siderophore activity was confirmed by iron transport measurements in young mycelia ofR. microsporus. While rhizoferrin and also ferrioxamines B and E proved to be effective siderophores, coprogen was a poor siderophore in this fungus.

In recent years pharmaceuticals and personal care products have been detected in increasing concentrations in hospital effluents, sewage treatment plants (STP) as well as in different environmental compartments such as surface water, groundwater and soil. Little is known about the elimination of these substances during sewage treatment or about the formation of potential metabolites in the environment caused by bacterial biotransformation. To assess the biodegradability of the popular cardiovascular drug verapamil and the possible formation of potential microbial degradation products, two tests from the OECD series were used in the present study: the widely used Closed Bottle test (OECD 301 D) and the modified Zahn-Wellens test (OECD 302 B). In the Closed Bottle test, a screening test that simulates the conditions of an environmental surface water compartment, no biological degradation was observed for verapamil at concentrations of 2.33mgl(-1). In the Zahn-Wellens test, a test for inherent biodegradability which allows evaluation of aerobic degradation at high bacterial density, only a partial biological degradation was found. Analysis of test samples by high performance liquid chromatography coupled to multiple stage mass spectrometry (HPLC-MS(n)) revealed 2-(3,4-dimethoxyphenyl)-2-isopropyl-5-(methylamino)pentane nitrile, already known as D617 (Knoll nomenclature), a metabolite of mammalian metabolism, which is the major degradation product and dead-end transformation product of aerobic degradation of verapamil In recent years pharmaceuticals and personal care products have been detected in increasing concentrations in hospital effluents, sewage treatment plants (STP) as well as in different environmental compartments such as surface water, groundwater and soil. Little is known about the elimination of these substances during sewage treatment or about the formation of potential metabolites in the environment caused by bacterial biotransformation. To assess the biodegradability of the popular cardiovascular drug verapamil and the possible formation of potential microbial degradation products, two tests from the OECD series were used in the present study: the widely used Closed Bottle test (OECD 301 D) and the modified Zahn-Wellens test (OECD 302 B). In the Closed Bottle test, a screening test that simulates the conditions of an environmental surface water compartment, no biological degradation was observed for verapamil at concentrations of 2.33mgl(-1). In the Zahn-Wellens test, a test for inherent biodegradability which allows evaluation of aerobic degradation at high bacterial density, only a partial biological degradation was found. Analysis of test samples by high performance liquid chromatography coupled to multiple stage mass spectrometry (HPLC-MS(n)) revealed 2-(3,4-dimethoxyphenyl)-2-isopropyl-5-(methylamino)pentane nitrile, already known as D617 (Knoll nomenclature), a metabolite of mammalian metabolism, which is the major degradation product and dead-end transformation product of aerobic degradation of verapamil

Pep5 is a tricyclic peptide antibiotic which contains the unusual amino acids dehydrobutyrine, lanthionine and 3-methyllanthionine. It is matured from a 60-amino-acid precursor peptide (pre-Pep5) deduced from the sequence of the structural gene pepA. To study the biosynthesis of Pep5 we tried to isolate the primary translation product. We identified a peptide in crude extracts of the Pep5-producing Staphylococcus epidermidis strain using antibodies raised against a synthetic 26-residue peptide representing the leader peptide region of pre-Pep5. The putative precursor was purified by reversed-phase HPLC. The isolated peptide did not react with antibodies directed against a C-terminal fragment of mature Pep5 containing two sulfide bridges. Neither lanthionine nor 3-methyllanthionine was detected in amino acid analysis of the isolated precursor. Its amino acid sequence was identical with the sequence predicted from pepA, but Edman degradation stopped at the first threonine residue of the prolantibiotic region indicating a posttranslational modification at this position. The molecular mass of the isolated peptide was 6575.4 +/- 1.7 Da, determined by ion-spray mass spectrometry. This is in agreement with a molecule being dehydrated at the four threonine and the two serine residues in the propeptide region; such a peptide has a calculated molecular mass of 6576.7 Da. The results strongly suggest that maturation of the lantibiotic Pep5 is initiated by selective dehydration of hydroxyamino acids in the propeptide region of the primary translation product and that thioether ring formation is not closely linked to dehydration.

Tripalmitoyl-S-glycerylcysteinyl lipopeptides are B-cell and macrophage activating and may be used as low molecular weight immunogens of considerable potency and even as vaccines when conjugated with suitable epitopic structures. Selected lipopeptides carrying single Dnp haptens were found to evoke mild passive cutaneous anaphylaxis in guinea pigs sensitized against Dnp. The reactions were observed after intravenous injection whereas intradermally applied antigen was negative. The anaphylactogenicity seems unrelated to micelle or aggregate formation of the insoluble peptides which require lecithin additions as well as sonication to become solubilized. The dinitrophenylated lipopeptide tripalmitoyl-S-glyceryl-cysteinyl-seryl-lysine produced toxic reactions which were not observed with the lipopeptide devoid of Dnp. Dinitrophenylated tripalmitoyl-S-glycerylcysteiny-1,6-diaminohexane and tripalmitoyl-S-glyceryl-cysteinyl-lysine did not show these toxic reactions.

Owing to increased global UV-B radiation, a consequence of ozone destruction in the stratosphere,['] living beings need effective protection mechanisms for survival.'2, Most experiments investigating the effect of UV-€3 radiation have been carried out on cultivated plants,[41 whilst only a few have been performed on trees.I5] Here we report on the structure of sunscreen pigments formed in Scots pine seedlings (Pinus sylvestris L.) specifically as a reaction to UV-B irradiation.", 71 Analysis of methanolic extracts of needles from UV-B treated pine seedlings by reversed-phase high-performance liquid chromatography (RP-HPLC) showed that compound 1 in particular was strongly induced in cotyledons. and compound 2 in primary

A major new compound was isolated from cell suspension cultures of Arabidopsis thaliana and its structure shown to be N-(E-4-coumaroyl)-aspartate by ion spray MS and MS/MS as well as 1H NMR spectroscopy. This amide was chemically synthesized and found to be identical with the natural isolate.

Two anthocyanins from the leaves of Victoria amazonica were isolated by column chromatography including preparative HPLC and have been identified by 1 H/ 13 CNMR spectroscopy and ion spray mass spectrometry as delphinidin 3- O -(2′'- O -galloyl-β- d -galactopyranoside) and cyanidin 3- O -(2′'- O -galloyl-β- d -galactopyranoside). These pigments were also found by co-chromatography in V. cruziana .

A new mathematical model is presented which describes the denitrification process by dynamic material balance equations. In this approach the kinetic rate expressions of the single denitrification steps and the observed strong inhibition of nitrate on nitrite and nitrous oxide reduction are based exclusively on fundamental enzyme kinetics. This allows a prediction of the denitrification process in a wide range of wastewater-relevant nitrate concentrations. The model was successfully applied to the description of the kinetic behavior of a standardized denitrifying activated sludge system. Furthermore the experimentally investigated influence of hydrogen sulfide was quantified by extending the model with a non-competitive inhibition mechanism involving all steps of the denitrification process. The inhibitory effect was related to the free membrane-permeable hydrogen sulfide concentration. This means that the extent of its inhibition depends additionally on the pH-value. Even very low hydrogen sulfide concentrations lead to a strong inhibition of nitrous oxide reduction and therefore to a high release of nitrous oxide from wastewater treatment plants.

Whereas protein engineering of enzymes and structural proteins nowadays is an established research tool for studying structure-function relationships of polypeptides and for improving their properties, the engineering of posttranslationally modified peptides, such as the lantibiotics, is just coming of age. The engineering of lantibiotics is less straightforward than that of unmodified proteins, since expression systems should be developed not only for the structural genes but also for the genes encoding the biosynthetic enzymes, immunity protein and regulatory proteins. Moreover, correct posttranslational modification of specific residues could in many cases he a prerequisite for production and secretion of the active lantibiotic, which limits the number of successful mutations one can apply. This paper describes the development of expression systems for the structural lantibiotic genes for nisin A, nisin Z, gallidermin, epidermin and Pep5, and gives examples of recently produced site-directed mutants of these lantibiotics. Characterization of the mutants yielded valuable information on biosynthetic requirements for production. Moreover, regions in the lantibioties were identified that are of crucial importance for antimicrobial activity. Eventually, this knowledge will lead to the rational design of lantibiotics optimally suited for fighting specific undesirable microorganisms. The mutants are of additional value for studies directed towards the elucidation of the mode of action of lantibiotics.

Four new members of peptaibol antibiotics, designated as chrysospermins A, B, C, and D, were isolated from the mycelium of Apiocrea chrysosperma Ap101 by solvent extraction, silica gel chromatography and preparative recycling HPLC. Their structures as new nonadecapeptides were settled by detailed spectroscopic analysis and chemical degradation experiments. The chrysospermins display antibacterial and antifungal activity, and induce pigment formation by the fungus Phoma destructiva.

In the present investigation we isolated the recently discovered pentacosapeptide xenin from gastric mucosa of man, dog, pig, guinea pig, rat, and rabbit. HPLC, mass spectrometry, and amino acid sequence analysis showed xenin-25 in concentrations of 54-144 pmol/g tissue in gastric mucosa of each species. Extraction with 2% TFA followed by analytical C18 HPLC revealed 0.02-84 pmol/g xenin-25 also in hypothalamus, lung, liver, heart, kidney, adrenal gland, pancreas, testicle, skin, and duodenal, jejunal, ileal, and colonic mucosa of dog and man, respectively. Digestion of these acid extracts with pepsin liberated xenin-25 in concentrations from 2 up to 166 pmol/g tissue. Gel chromatography revealed a large molecular weight precursor of xenin-25 and evidence for an endogenous acid protease coeluting with pepsinogen capable of releasing xenin-25 from its precursor. Maximal concentrations of xenin-25 were obtained when canine gastric mucosa was incubated with 2% TFA at room temperature for 2 h. Longer incubation times led to a decline of xenin-25 concentration and to formation of xenin-16 and xenin-9, both C-terminal fragments of xenin-25. We conclude that xenin-25 is present not only in human gastric mucosa but also in the stomach of various other mammals. Xenin-25 is further present in low concentrations in many other organs where a pepsin-like protease generates xenin-25 from a large precursor and processes it to smaller fragments.

The antibacterial peptide SA-FF22, produced by the pathogen Streptococcus pyogenes strain FF22 was purified and features of its primary and secondary structure were characterised. Mass spectrometry demonstrated the pure peptide had a mass of 2794Da while, amino acid analysis revealed the presence of the unusual, thioether amino acids lanthionine and 3-methyllanthionine; thus SA-FF22 is a member of the group of antibacterial polypeptides termed lantibiotics. Furthermore, amino acid sequencing showed a unique sequence which was blocked at position 23 by a residue of the unsaturated amino acid 2,3-didehydrobutyrine. Carboxypeptidase-Y digestion could be used to demonstrate that serine occupies the C-terminal position only after complete oxidation of the thioether amino acid bridges, suggesting that the three-dimensional structure of the native peptide may prevent access of the enzyme to the C-terminus. Fragmentation of the native peptide with a variety of proteolytic enzymes failed to yield a peptide containing less than all three of the cross-linked lanthionine and methyllanthionine residues and demonstrated that all three thioether bridges overlapped. Analysis of the circular dichroism of SA-FF22 in various concentrations of 2,2,2-trifluoroethanol in water, SDS micelles and in the presence of artificial phospholipid vesicles suggested that there is significant change in its secondary structure from aqueous to lipophilic environments.

For the investigation of enzymes involved in epidermin biosynthesis it is necessary to produce sufficient amounts of preepidermin (EpiA) as a substrate and to design EpiA detection systems. Therefore, EpiA was expressed in Escherichia coli using a malE-epiA fusion. The identity of purified EpiA was confirmed by ion spray mass spectrometry and amino acid sequencing. For EpiA detection, anti-EpiA antisera were raised. Upon prolonged incubation, factor Xa not only cleaved EpiA from the fusion protein, but also less efficiently cleaved EpiA internally between R-1 and I+1. The internal factor Xa cleavage site of EpiA was masked by altering the sequence -A(-4)-E-P-R(-1)- to -A(-4)-E-P-Q(-1)- by site-directed mutagenesis.

In human neutrophils, the synthetic lipopeptide, N-palmitoyl-S-[2,3- bis(palmitoyloxy-(2RS)-propyl]-(R)-cysteinyl-(S)-seryl-(S)-lysyl-( S)-lysyl-(S) -lysyl-(S)-lysine [Pam3CysSer(Lys)4], activates NADPH-oxidase catalyzed superoxide (O2-) formation through pertussis-toxin-sensitive and pertussis-toxin-insensitive mechanisms (Seifert, R., Schultz, G., Richter-Freund, M., Metzger, J., Wiesmüller, K.-H., Jung, G., Bessler, W. G. & Hauschildt, S. (1990) Biochem. J. 267, 795-802). We studied the effects of lipopeptides on differentiation of HL-60 leukemic cells. Pam3CysSer(Lys)4 enhanced phorbol-12-myristate-13-acetate-induced O2- formation (presumably through the expression of components of NADPH oxidase) in a concentration-dependent manner with a half-maximal effect at 100 ng/ml and a maximum at 1 microgram/ml. The effect of the lipopeptide was evident after 24 h and reached a plateau after 48 h. (2S,6S)-2-Palmitoylamino-6,7- bis(palmitoyloxy)heptanoyl-(S)-seryl-(S)-lysyl-(S)-lysyl-(S) -lysyl-(S)-lysine enhanced O2- formation as well. The effects of Pam3CysSer(Lys)4 were potentiated by dibutyryl cAMP, dimethyl sulfoxide, retinoic acid, 1,25-dihydroxyvitamin D3, interferon-gamma and tumor-necrosis-factor-alpha. Pertussis toxin, but not its B-oligomer, partially inhibited enhanced O2- formation induced by Pam3CysSer(Lys)4. O2- formation induced by arachidonic acid and gamma-hexachlorocyclohexane were more sensitive to inhibition by pertussis toxin than O2- formation induced by phorbol 12-myristate 13-acetate. Enhanced O2- formation induced by dibutyryl cAMP was not affected by pertussis toxin. Unlike ATP, histamine, prostaglandin E1 and the beta-adrenergic agonist, isoproterenol, Pam3CysSer(Lys)4 did not increase cytosolic Ca2+ [( Ca2+]i) in undifferentiated HL-60 cells. Histamine but not lipopeptides stimulated high-affinity GTPase of guanine-nucleotide-binding proteins in membranes of undifferentiated HL-60 cells. In Pam3CysSer(Lys)4-differentiated HL-60 cells, the responsiveness to the [Ca2+]i-increasing agonists, N-formyl-L-methionyl-L-leucyl-L-phenylalanine, C5a and leukotriene B4, was increased, whilst the responsiveness to prostaglandin E1 and isoproterenol was decreased. Pam3CysSer(Lys)4 did not inhibit proliferation of HL-60 cells but decreased transferrin receptor expression and increased C3bi receptor expression. Pertussis toxin did not affect proliferation and expression of transferrin and C3bi receptors. Dibutyryl cAMP was considerably more effective than Pam3CysSer(Lys)4 at inducing alterations in the above parameters. Our results suggest that (a) Pam3CysSer(Lys)4 induces incomplete functional differentiation of HL-60 cells through a mechanism which does not depend on a rise in [Ca2+]i and is different from that of other differentiation-inducing substances and (b) the mechanism by which Pam3CysSer(Lys)4 induces differentiation involves pertussis-toxin-sensitive and pertussis-toxin-insensitive mechanisms.

Several arginine-rich peptides containing the C-terminus of neuropeptide Y (NPY) were prepared by solid phase peptide synthesis using Fmoc chemistry and cleaved from the resin with trifluoroacetic acid (TFA). The products were characterized by fast atom bombardment-MS, LC-thermospray-MS, ion spray-MS/MS, and Edman degradation. The side products could be identified as peptides with sulfonated arginine residues resulting from an unexpected cleavage of Mtr or Pmc protecting groups. The degree of sulfonation depended on the choice and composition of the cleavage solution. Several scavenger mixtures were used and a mixture of thioanisole/thiocresol was found to be the most efficient for suppressing sulfonation. Furthermore treatment with the enzyme arylsulfate-sulfohydrolase desulfonated the peptides yielding the correct sequence.

Synthetic lipopeptides, structurally derived from the N-terminal part of bacterial lipoprotein, constitute macrophage and B-lymphocyte activators. The molecular mechanism of macrophage activation by lipopeptides still remains unclear. The purpose of our study was to determine the route and kinetics of lipopeptide distribution in bone marrow-derived macrophages. The intracellular localization of the C-terminally biotinylated lipodipeptide Pam3Cys-Ser was investigated on ultrathin cryosections using the biotinstreptavidin-gold system. Our findings indicate that the lipopeptide penetrates the plasma membrane and can already be found within the cytoplasm, the nuclear membrane, and within the nucleus after 2 min of stimulation. The pattern of lipopeptide distribution obtained 2 min after stimulation resembles that obtained after longer incubation times (8 and 20 min). Correlating distribution patterns were observed when using the method of electron energy loss spectroscopy (EELS). These findings are a clear indication for the rapid uptake of lipopeptides into eukaryotic cells, and are of importance for further studies of the immunostimulating properties of the bacterial lipopeptides and vaccines derived therefrom.

Synthetic lipopeptide analogues of bacterial lipoprotein constitute potent polyclonal activators for monocytes/macrophages and B lymphocytes. However, the fate of the lipopeptides after their interaction with target cells is as yet unknown. In order to follow the routes and to determine the distribution of the lipopeptide within macrophages after stimulation, we investigated lipopeptide-stimulated bone marrow-derived macrophages using the novel method of electron energy loss spectroscopy (EELS). Our results show that the lipopeptide was present in different compartments of the cell. The major amount of the activator was located within the cytoplasm and the plasma membrane, and minor quantities were detected within the nuclear membrane and the nucleus. The distribution of the lipopeptides varies depending on the duration of stimulation. Our results should help to elucidate the molecular mechanisms of macrophage stimulation by lipopeptides or other cell activators.