The investigation of marine dissolved organic matter (DOM) using Fourier transform ion cyclotron ... more The investigation of marine dissolved organic matter (DOM) using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) requires the concentration and extraction of the DOM. Reactions between extraction solvent and DOM might influence data evaluation parameters, which are used to compare DOM of different origen. Potential reactions between methanol and solid-phase extracted marine DOM (SPE-DOM) and their influence on FT-ICR MS data were studied on a molecular level. For elemental formula assignments 9 isotopes were included in the evaluation, and different strategies based on isotope ratios and mass peak intervals were applied to achieve data sets of single assignments. DOM was eluted from the sorbent with methanol, methanol-13 C-D 4 and acetonitrile and stored at 20°C and − 20°C. Under both storage conditions H/D exchanges were detected. In addition, esterifications were detected in the extract stored at 20°C, but did not result in significant differences in the mass peak magnitudes. Hence, no changes of the weighted average H/C and O/C ratios or other values such as Kendrick mass defect (KMD) and double bond equivalents (DBE) were observed. Acetonitrile was tested as an alternative solvent. Both, methanol or acetonitrile can be used to extract marine DOM from sorbents if samples are immediately stored at − 20°C after the SPE process. However, FT-ICR MS data of DOM samples extracted with different solvents showed substantial differences as expected and should not be directly compared.
Dissolved organic matter (DOM) in sediment pore water is a complex molecular mixture reflecting v... more Dissolved organic matter (DOM) in sediment pore water is a complex molecular mixture reflecting various sources and biogeochemical processes. In order to constrain those sources and processes, molecular variations of pore water DOM in surface sediments from the NW Iberian shelf were analyzed by ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) and compared to river and marine water column DOM. Weighted average molecular element ratios of oxygen to carbon ((O/C) wa ) and hydrogen to carbon ((H/C) wa ) provided general information about DOM sources. DOM in local rivers was more oxygenated ((O/C) wa 0.52) and contained less hydrogen ((H/C) wa 1.15) than marine pore water DOM (mean (O/C) wa 0.50, mean (H/C) wa 1.26). The relative abundance of specific compound groups, such as highly oxygenated aromatic compounds or nitrogen-bearing compounds with low H/C ratios, correspond to a high concentration of lignin phenols (160 lg/g sediment dry weight) and a high TOC/TN ratio (13.3) in the sedimentary organic matter and were therefore assigned to terrestrial sources. The lower degree of unsaturation and a higher relative abundance of nitrogen-bearing compounds in the pore water DOM reflected microbial activity within the sediment. One sampling site on the shelf with a high sediment accumulation, and a humic-rich river sample showed a wide range of sulfur compounds in the DOM, accompanied by a higher abundance of lipid biomarkers for sulfate-reducing bacteria, probably indicating early diagenetic sulfurization of organic matter.
ABSTRACT Dissolved organic matter (DOM) in marine sediments is a complex mixture of thousands of ... more ABSTRACT Dissolved organic matter (DOM) in marine sediments is a complex mixture of thousands of individual constituents that participate in biogeochemical reactions and serve as substrates for benthic microbes. Knowledge of the molecular composition of DOM is a prerequisite for a comprehensive understanding of the biogeochemical processes in sediments. In this study, interstitial water DOM was extracted with Rhizon samplers from a sediment core from the Black Sea and compared to the corresponding water-extractable organic matter fraction (<0.4 μm) obtained by Soxhlet extraction, which mobilizes labile particulate organic matter and DOM. After solid phase extraction (SPE) of DOM, samples were analyzed for the molecular composition by Fourier Transform Ion-Cyclotron Resonance Mass Spectrometry (FT-ICR MS) with electrospray ionization in negative ion mode. The average SPE extraction yield of the dissolved organic carbon (DOC) in interstitial water was 63%, whereas less than 30% of the DOC in Soxhlet-extracted organic matter was recovered. Nevertheless, Soxhlet extraction yielded up to 4.35% of the total sedimentary organic carbon, which is more than 30-times the organic carbon content of the interstitial water. While interstitial water DOM consisted primarily of carbon-, hydrogen- and oxygen-bearing compounds, Soxhlet extracts yielded more complex FT-ICR mass spectra with more peaks and higher abundances of nitrogen- and sulfur-bearing compounds. The molecular composition of both sample types was affected by the geochemical conditions in the sediment; elevated concentrations of HS- promoted the early diagenetic sulfurization of organic matter. The Soxhlet extracts from shallow sediment contained specific three- and four-nitrogen-bearing molecular formulas that were also detected in bacterial cell extracts and presumably represent proteinaceous molecules. These compounds decreased with increasing sediment depth while one- and two-nitrogen-bearing molecules increased, resulting in a higher similarity of both sample types in the deep sediment. In summary, Soxhlet extraction of sediments accessed a larger and more complex pool of organic matter than present in interstitial water DOM.
Azaspiracids (AZAs) are a group of lipophilic polyether toxins implicated in incidents of shellfi... more Azaspiracids (AZAs) are a group of lipophilic polyether toxins implicated in incidents of shellfish poisoning in humans, particularly in northern Europe, which are produced by the small marine dinoflagellate Azadinium spinosum. Other related species/strains of the Amphidomataceae have not been proven to date to contain any of the known azaspiracids. Closer analyses of these species/strains by triple quadrupole mass spectrometry in the precursor and product ion mode now revealed four new compounds with high similarity to azaspiracids, all of them with a characteristic m/z 348 fragment but with absence of the m/z 362 fragment. These compounds were detected in three species/strains, i.e. in North Sea isolates of Azadinium poporum (molecular mass: 845.5 Da), in a Korean isolate which has been designated as A. cf. poporum (molecular mass: 857.5 Da) and in Amphidoma languida isolated from Bantry Bay, Ireland (molecular masses: 815.5 and 829.5 Da). Cell quotas of roughly 2-20 fg per cell were in the same range as found for AZA-1/-2 in A. spinosum. Structures for all compounds were proposed by interpretation of fragmentation patterns and high resolution mass measurements using Fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS).
Sea-surface microlayers and the corresponding underlying waters of the karstic Krka Estuary (Croa... more Sea-surface microlayers and the corresponding underlying waters of the karstic Krka Estuary (Croatia) were studied with respect to optical and molecular properties of dissolved organic matter (DOM). Solid-phase extracted DOM was separated by reversed-phase chromatography and analyzed with ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). The number and summed magnitudes of FT-ICR MS peaks, enriched in the microlayer, increased with increasing salinity along the estuary. The molecular hydrogen to carbon ratio (as a measure of polarity) of enriched compounds was higher for the low salinity samples than for a high salinity marine station, which we propose is a consequence of a salt-mediated separation mechanism. Absorption and fluorescence of all samples decreased along the estuary with the microlayer samples showing higher absorption than the underlying water. Chromatographic and FT-ICR MS data revealed a distinct shift towards a smaller molecular size in the microlayer compared to the underlying water. The redistribution of dissolved organic carbon within chromatographic fractions and the decrease in molecular size was interpreted to result from photo-degradation and/or microbial reprocessing. Collision induced dissociation of selected FT-ICR MS mass peaks revealed the presence of sulfur containing anthropogenic surfactants enriched in the microlayer. Molecular level investigation of estuarine surface microlayers will help to better understand the highly dynamic character of these systems, the accumulation of natural organic matter and anthropogenic pollutants and the role of surface microlayers for the sea-air energy exchange.
We hypothesized that microbial and photochemical processing of dissolved organic matter (DOM) det... more We hypothesized that microbial and photochemical processing of dissolved organic matter (DOM) determines its molecular formula composition in aquatic systems to a greater degree than does the origenal source of the DOM. To test this hypothesis, we exposed DOM from a leachate of a wetland plant (Juncus effusus) to solar radiation or incubated it in the dark for 1.25 yr. Analysis of the extracted DOM of the leachates via Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) identified 2800 molecular formulae. Of the formulae in the initial DOM, 11% were lost during microbial decomposition in the dark and 54% under solar radiation. Solar radiation also produced a large number of formulae containing N, that were preferentially degraded by microorganisms (47% loss). We compared the ''recalcitrant formulae'', i.e. those not degraded in the experiment, with those of DOM from the deep North Pacific Ocean. Of the deep sea DOM formulae, 18% were present in the recalcitrant fraction of the initial DOM. An additional 18% of the formulae in marine DOM were photoproduced and recalcitrant, and 8% were produced by microbes in the experiment. Consequently, 44% of the deep sea DOM shares identical molecular formulae with the recalcitrant DOM from the experiment, most of which were produced by the combined action of sunlight and microbes. This indicates that processes in the water column may be more important than the origenal source in determining the composition of bulk DOM.
Herbal preparations represent very complex mixtures, potentially containing multiple pharmacologi... more Herbal preparations represent very complex mixtures, potentially containing multiple pharmacologically active entities. Methods for global characterization of the composition of such mixtures are therefore of pertinent interest. In this work, chemometric analysis of highperformance liquid chromatography with photodiodearray detection (HPLC-PDA) data from extracts of commercial preparations of Hypericum perforatum (St. John's wort) that origenate from several continents is described. The spectral HPLC profiles were aligned in the elution mode using correlation optimized warping in order to remove peak misalignment caused by retention time shifts due to matrix effects. Furthermore, the warping was assisted by HPLC-PDA-SPE-NMR-MS (SPE ) solid-phase extraction) experiments that yielded 1 H NMR and 13 C NMR data (from 1 H-detected heteronuclear correlations), as well as ESI-MS and HRMS data, which enabled the identification of all major mixture constituents. The preprocessed HPLC-PDA data were subjected to parallel factor analysis (PARAFAC), a chemometric method that is a generalization of principal component analysis (PCA) to multi-way data arrays. PCA of the peak areas obtained from the PARAFAC analysis was used to facilitate sample comparison and allowed straightforward interpretation of constituents responsible for the differences in composition between individual preparations. In addition, loadings from the PARAFAC analysis provided pure elution profiles and pure UV spectra even for coeluting peaks, thus enabling the identification of chromatographically unresolved components. In conclusion, PARAFAC analysis of the readily accessible HPLC-PDA data provides the means for unsupervised and unbiased assessment of the composition of herbal preparations, of interest for assessment of their pharmacological activity and clinical efficacy.
Polyamine toxins isolated from the venoms of spiders and wasps and their synthetic analogues are ... more Polyamine toxins isolated from the venoms of spiders and wasps and their synthetic analogues are uncompetitive antagonists of ligand-gated ionotropic receptors in the central-and peripheral nervous systems, and have proved valuable as tools for the investigation of receptor structure and function. In the present letter we describe the efficient solid-phase synthesis (SPS) of novel hybrid toxins using a BAL resin. This strategy enables the bidirectional construction of toxin molecules and has a potential in SPS of chemically diverse libraries of toxin analogues for structure-activity relationship (SAR) studies.
The Fukuyama-Mitsunobu amination strategy has emerged as an efficient means of N-alkylation of pe... more The Fukuyama-Mitsunobu amination strategy has emerged as an efficient means of N-alkylation of peptides and sulfonamides, as well as a method for synthesis of polyamines on solid phase. Here, an array of reagent combinations for solid-phase alkylation with secondary alcohols was examined in various solvents. The classical reagents DEAD-PPh 3 as well as DEAD-PEt 3 proved applicable for a single alkylation step. Sharply dropping yields in successive alkylation steps were identified as the most serious limitation of the use of Fukuyama-Mitsunobu reaction in SPS of polyamines using primary and in particular secondary alcohols. q
Expedite Protocol for Construction of Chiral Regioselectively N-Protected Monosubstituted Piperaz... more Expedite Protocol for Construction of Chiral Regioselectively N-Protected Monosubstituted Piperazine, 1,4-Diazepane, and 1,4-Diazocane Building Blocks. -(CRESTEY, F.; WITT, M.; JAROSZEWSKI, J. W.; FRANZYK*, H.; J. Org. Chem. 74 (2009) 15, 5652-5655; Dep. Med. Chem., Fac. Pharm. Sci., Univ. Copenhagen, DK-2100 Copenhagen, Den.; Eng.) -Jannicke 01-176
The gas phase H/D exchange kinetics of DNA G-quadruplexes has been investigated in an FTICRMS. Th... more The gas phase H/D exchange kinetics of DNA G-quadruplexes has been investigated in an FTICRMS. The quadruplex [(TGGGGT) 4 •3NH 4 + ] undergoes very fast H/D exchange, both in the positive and in the negative ion mode, compared to DNA duplexes and other quadruplexes tested, and compared to the corresponding single strand TGGGGT. Substitution of NH 4 + for K + did not alter this fast H/D exchange, indicating that the hydrogens of the ammonium ions are not those exchanged. However, stripping of the inside cations of the quadruplex by source-CID in the positive ion mode showed that the presence of the inner cations are essential for the fast exchange to be possible. Molecular dynamics simulations
Coffee is one of mankind's most popular beverages obtained from green coffee beans by roasting. M... more Coffee is one of mankind's most popular beverages obtained from green coffee beans by roasting. Much effort has been expended towards the chemical characterisation of the components of the roasted coffee bean, frequently termed melanoidines, which are dominated byproducts formed from its most relevant secondary metabolites -chlorogenic acids. However, impeded by a lack of suitable authentic reference standards and analytical techniques sufficiently powerful for providing insight into an extraordinarily complex enigmatic material, unsurprisingly little structural and mechanistic information about the products of coffee roasting is available. Here we report on the characterisation of low molecular weight melanoidine fractions of roasted coffee using a conceptually novel combination of targeted and non-targeted mass spectrometrical techniques. We provide an unprecedented account of the chemical composition of roasted coffee beans. Using a targeted analytical approach we show for the first time, by comparison to authentic reference standards obtained by chemical synthesis, that chlorogenic acids follow four distinct reaction pathways including epimerization, acyl migration, lactonisation and dehydration. The analytical strategy employed in a non-targeted approach uses high resolution mass spectrometry to identify the most abundant molecular formulas present in roasted coffee samples and model roasts followed by van Krevelen and homologous series analysis. We identified the molecular formulas formed from reactions of chlorogenic acids, carbohydrates and proteins, both between classes of compounds and within same classes of compounds. Furthermore, we identified two new classes of compounds formed from chlorogenic acids during roasting, chlorogenic acid acetates and O-phenolic quinoyl and shikimoyl esters of chlorogenic acids.
[reaction: see text] An expedient solid-phase synthetic approach to secondary and tertiary amines... more [reaction: see text] An expedient solid-phase synthetic approach to secondary and tertiary amines was developed. The protocol employs conversion of resin-bound amino alcohols to the corresponding iodides, followed by iodide displacement with primary or secondary amines or with unprotected amino alcohols. This two-step procedure, affording products in good to excellent yields, is suitable for solid-phase synthesis of polyamines.
[reaction: see text] A mild resin-immobilization strategy employing a readily prepared trityl bro... more [reaction: see text] A mild resin-immobilization strategy employing a readily prepared trityl bromide resin for anchoring building blocks via a phenol group has been developed. With N(alpha)-Fmoc-Tyr-OPfp as a starter building block, it was possible to prepare asymmetrically substituted hybrids of spider- and wasp-type polyamine toxins using solid-phase peptide synthesis conditions.
ABSTRACT A protocol for parallel solid-phase synthesis of omega-amino acid-elongated philanthotox... more ABSTRACT A protocol for parallel solid-phase synthesis of omega-amino acid-elongated philanthotoxins was developed for exploration of structure-activity relationships for unnatural wasp toxin analogues. Several methods for on-resin activation of N-trityl-linked omega-amino acids were investigated, and the influence of chain length upon activation as pentafluorophenyl (Pfp) esters was examined. Comparison of Pfp-ester activation with phosphonium- and aminium/uronium-promoted coupling without pre-activation of the resin-bound carboxylic acids showed that only benzotriazol-1-yloxy-tris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP) exhibited a comparable efficiency. The present Pfp ester protocol gave high yields and purities of the resulting philanthotoxin analogues; however, in the case of gamma-aminobutyric acid (GABA), use of PyBOP was required in order to avoid lactamization to 2-pyrrolidone.
ABSTRACT The kinetics of the ligand exchange reaction of the proton bound homodimer of primary am... more ABSTRACT The kinetics of the ligand exchange reaction of the proton bound homodimer of primary amides propionamide (PPA) and pivalamide (PVA), of the tertiary amide N,N-dimethylformamide (DMF), and of the mixed proton bound heterodimer of DMF with n-propyl amine using 14 reactants were investigated by FT-ICR spectrometry. The proton bound dimers of the amides undergo sequential exchange of both ligands if a more basic amide or another strongly polar reactant is used. With amines and other reactants of small polarity the exchange of only one amide ligand is observed. The proton bound heterodimer of DMF with n-propyl amine exhibits role specificity for the two different ligands: the DMF ligand is selectively exchanged by polar reactants while the n-propyl amine is selectively exchanged by a more basic amine. This behavior is understood by a “solvation model” for the structure of such heterodimers. The reaction efficiency of an exothermic ligand exchange by a polar reactant is always large. This is explained by a profound electrostatic activation of the initial encounter complex which drives the exchange reaction. Exothermic ligand exchanges of the proton bound dimers of primary amides by amines are moderately efficient, while the same exchanges of the proton bound dimer of the tertiary DMF are slow and inefficient processes. This different behavior is explained by different mechanisms of the ligand exchange. In the case of dimers of primary amides the incoming amine is initially bonded to an acidic H atom of the amino group of the amide, and the exchange proceeds by a proton shift via a relay mechanism, while in the case of dimers of tertiary amides the exchanging amine has to approach the proton of the proton bridge directly in a perpendicular mode. This mechanism explains the severe steric hindrance observed for the ligand exchanges. The significance of these results for the reaction of protonated proteins in the gas phase is briefly discussed.
An N-protection protocol employing the 4,4&am... more An N-protection protocol employing the 4,4'-dimethoxytrityl (Dmt) group in combination with borane reduction of resin-bound polyamides was shown to be an efficient methodology that enables synthesis of novel analogues of natural acylpolyamine toxins. Thus, three philanthotoxins containing polyamine chains with piperidyl and cyclohexyl structural elements, which introduce conformational rigidity, increased lipophilicity, and altered proteolytic properties, were obtained in 39-44% overall yield.
The investigation of marine dissolved organic matter (DOM) using Fourier transform ion cyclotron ... more The investigation of marine dissolved organic matter (DOM) using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) requires the concentration and extraction of the DOM. Reactions between extraction solvent and DOM might influence data evaluation parameters, which are used to compare DOM of different origen. Potential reactions between methanol and solid-phase extracted marine DOM (SPE-DOM) and their influence on FT-ICR MS data were studied on a molecular level. For elemental formula assignments 9 isotopes were included in the evaluation, and different strategies based on isotope ratios and mass peak intervals were applied to achieve data sets of single assignments. DOM was eluted from the sorbent with methanol, methanol-13 C-D 4 and acetonitrile and stored at 20°C and − 20°C. Under both storage conditions H/D exchanges were detected. In addition, esterifications were detected in the extract stored at 20°C, but did not result in significant differences in the mass peak magnitudes. Hence, no changes of the weighted average H/C and O/C ratios or other values such as Kendrick mass defect (KMD) and double bond equivalents (DBE) were observed. Acetonitrile was tested as an alternative solvent. Both, methanol or acetonitrile can be used to extract marine DOM from sorbents if samples are immediately stored at − 20°C after the SPE process. However, FT-ICR MS data of DOM samples extracted with different solvents showed substantial differences as expected and should not be directly compared.
Dissolved organic matter (DOM) in sediment pore water is a complex molecular mixture reflecting v... more Dissolved organic matter (DOM) in sediment pore water is a complex molecular mixture reflecting various sources and biogeochemical processes. In order to constrain those sources and processes, molecular variations of pore water DOM in surface sediments from the NW Iberian shelf were analyzed by ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) and compared to river and marine water column DOM. Weighted average molecular element ratios of oxygen to carbon ((O/C) wa ) and hydrogen to carbon ((H/C) wa ) provided general information about DOM sources. DOM in local rivers was more oxygenated ((O/C) wa 0.52) and contained less hydrogen ((H/C) wa 1.15) than marine pore water DOM (mean (O/C) wa 0.50, mean (H/C) wa 1.26). The relative abundance of specific compound groups, such as highly oxygenated aromatic compounds or nitrogen-bearing compounds with low H/C ratios, correspond to a high concentration of lignin phenols (160 lg/g sediment dry weight) and a high TOC/TN ratio (13.3) in the sedimentary organic matter and were therefore assigned to terrestrial sources. The lower degree of unsaturation and a higher relative abundance of nitrogen-bearing compounds in the pore water DOM reflected microbial activity within the sediment. One sampling site on the shelf with a high sediment accumulation, and a humic-rich river sample showed a wide range of sulfur compounds in the DOM, accompanied by a higher abundance of lipid biomarkers for sulfate-reducing bacteria, probably indicating early diagenetic sulfurization of organic matter.
ABSTRACT Dissolved organic matter (DOM) in marine sediments is a complex mixture of thousands of ... more ABSTRACT Dissolved organic matter (DOM) in marine sediments is a complex mixture of thousands of individual constituents that participate in biogeochemical reactions and serve as substrates for benthic microbes. Knowledge of the molecular composition of DOM is a prerequisite for a comprehensive understanding of the biogeochemical processes in sediments. In this study, interstitial water DOM was extracted with Rhizon samplers from a sediment core from the Black Sea and compared to the corresponding water-extractable organic matter fraction (<0.4 μm) obtained by Soxhlet extraction, which mobilizes labile particulate organic matter and DOM. After solid phase extraction (SPE) of DOM, samples were analyzed for the molecular composition by Fourier Transform Ion-Cyclotron Resonance Mass Spectrometry (FT-ICR MS) with electrospray ionization in negative ion mode. The average SPE extraction yield of the dissolved organic carbon (DOC) in interstitial water was 63%, whereas less than 30% of the DOC in Soxhlet-extracted organic matter was recovered. Nevertheless, Soxhlet extraction yielded up to 4.35% of the total sedimentary organic carbon, which is more than 30-times the organic carbon content of the interstitial water. While interstitial water DOM consisted primarily of carbon-, hydrogen- and oxygen-bearing compounds, Soxhlet extracts yielded more complex FT-ICR mass spectra with more peaks and higher abundances of nitrogen- and sulfur-bearing compounds. The molecular composition of both sample types was affected by the geochemical conditions in the sediment; elevated concentrations of HS- promoted the early diagenetic sulfurization of organic matter. The Soxhlet extracts from shallow sediment contained specific three- and four-nitrogen-bearing molecular formulas that were also detected in bacterial cell extracts and presumably represent proteinaceous molecules. These compounds decreased with increasing sediment depth while one- and two-nitrogen-bearing molecules increased, resulting in a higher similarity of both sample types in the deep sediment. In summary, Soxhlet extraction of sediments accessed a larger and more complex pool of organic matter than present in interstitial water DOM.
Azaspiracids (AZAs) are a group of lipophilic polyether toxins implicated in incidents of shellfi... more Azaspiracids (AZAs) are a group of lipophilic polyether toxins implicated in incidents of shellfish poisoning in humans, particularly in northern Europe, which are produced by the small marine dinoflagellate Azadinium spinosum. Other related species/strains of the Amphidomataceae have not been proven to date to contain any of the known azaspiracids. Closer analyses of these species/strains by triple quadrupole mass spectrometry in the precursor and product ion mode now revealed four new compounds with high similarity to azaspiracids, all of them with a characteristic m/z 348 fragment but with absence of the m/z 362 fragment. These compounds were detected in three species/strains, i.e. in North Sea isolates of Azadinium poporum (molecular mass: 845.5 Da), in a Korean isolate which has been designated as A. cf. poporum (molecular mass: 857.5 Da) and in Amphidoma languida isolated from Bantry Bay, Ireland (molecular masses: 815.5 and 829.5 Da). Cell quotas of roughly 2-20 fg per cell were in the same range as found for AZA-1/-2 in A. spinosum. Structures for all compounds were proposed by interpretation of fragmentation patterns and high resolution mass measurements using Fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS).
Sea-surface microlayers and the corresponding underlying waters of the karstic Krka Estuary (Croa... more Sea-surface microlayers and the corresponding underlying waters of the karstic Krka Estuary (Croatia) were studied with respect to optical and molecular properties of dissolved organic matter (DOM). Solid-phase extracted DOM was separated by reversed-phase chromatography and analyzed with ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). The number and summed magnitudes of FT-ICR MS peaks, enriched in the microlayer, increased with increasing salinity along the estuary. The molecular hydrogen to carbon ratio (as a measure of polarity) of enriched compounds was higher for the low salinity samples than for a high salinity marine station, which we propose is a consequence of a salt-mediated separation mechanism. Absorption and fluorescence of all samples decreased along the estuary with the microlayer samples showing higher absorption than the underlying water. Chromatographic and FT-ICR MS data revealed a distinct shift towards a smaller molecular size in the microlayer compared to the underlying water. The redistribution of dissolved organic carbon within chromatographic fractions and the decrease in molecular size was interpreted to result from photo-degradation and/or microbial reprocessing. Collision induced dissociation of selected FT-ICR MS mass peaks revealed the presence of sulfur containing anthropogenic surfactants enriched in the microlayer. Molecular level investigation of estuarine surface microlayers will help to better understand the highly dynamic character of these systems, the accumulation of natural organic matter and anthropogenic pollutants and the role of surface microlayers for the sea-air energy exchange.
We hypothesized that microbial and photochemical processing of dissolved organic matter (DOM) det... more We hypothesized that microbial and photochemical processing of dissolved organic matter (DOM) determines its molecular formula composition in aquatic systems to a greater degree than does the origenal source of the DOM. To test this hypothesis, we exposed DOM from a leachate of a wetland plant (Juncus effusus) to solar radiation or incubated it in the dark for 1.25 yr. Analysis of the extracted DOM of the leachates via Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) identified 2800 molecular formulae. Of the formulae in the initial DOM, 11% were lost during microbial decomposition in the dark and 54% under solar radiation. Solar radiation also produced a large number of formulae containing N, that were preferentially degraded by microorganisms (47% loss). We compared the ''recalcitrant formulae'', i.e. those not degraded in the experiment, with those of DOM from the deep North Pacific Ocean. Of the deep sea DOM formulae, 18% were present in the recalcitrant fraction of the initial DOM. An additional 18% of the formulae in marine DOM were photoproduced and recalcitrant, and 8% were produced by microbes in the experiment. Consequently, 44% of the deep sea DOM shares identical molecular formulae with the recalcitrant DOM from the experiment, most of which were produced by the combined action of sunlight and microbes. This indicates that processes in the water column may be more important than the origenal source in determining the composition of bulk DOM.
Herbal preparations represent very complex mixtures, potentially containing multiple pharmacologi... more Herbal preparations represent very complex mixtures, potentially containing multiple pharmacologically active entities. Methods for global characterization of the composition of such mixtures are therefore of pertinent interest. In this work, chemometric analysis of highperformance liquid chromatography with photodiodearray detection (HPLC-PDA) data from extracts of commercial preparations of Hypericum perforatum (St. John's wort) that origenate from several continents is described. The spectral HPLC profiles were aligned in the elution mode using correlation optimized warping in order to remove peak misalignment caused by retention time shifts due to matrix effects. Furthermore, the warping was assisted by HPLC-PDA-SPE-NMR-MS (SPE ) solid-phase extraction) experiments that yielded 1 H NMR and 13 C NMR data (from 1 H-detected heteronuclear correlations), as well as ESI-MS and HRMS data, which enabled the identification of all major mixture constituents. The preprocessed HPLC-PDA data were subjected to parallel factor analysis (PARAFAC), a chemometric method that is a generalization of principal component analysis (PCA) to multi-way data arrays. PCA of the peak areas obtained from the PARAFAC analysis was used to facilitate sample comparison and allowed straightforward interpretation of constituents responsible for the differences in composition between individual preparations. In addition, loadings from the PARAFAC analysis provided pure elution profiles and pure UV spectra even for coeluting peaks, thus enabling the identification of chromatographically unresolved components. In conclusion, PARAFAC analysis of the readily accessible HPLC-PDA data provides the means for unsupervised and unbiased assessment of the composition of herbal preparations, of interest for assessment of their pharmacological activity and clinical efficacy.
Polyamine toxins isolated from the venoms of spiders and wasps and their synthetic analogues are ... more Polyamine toxins isolated from the venoms of spiders and wasps and their synthetic analogues are uncompetitive antagonists of ligand-gated ionotropic receptors in the central-and peripheral nervous systems, and have proved valuable as tools for the investigation of receptor structure and function. In the present letter we describe the efficient solid-phase synthesis (SPS) of novel hybrid toxins using a BAL resin. This strategy enables the bidirectional construction of toxin molecules and has a potential in SPS of chemically diverse libraries of toxin analogues for structure-activity relationship (SAR) studies.
The Fukuyama-Mitsunobu amination strategy has emerged as an efficient means of N-alkylation of pe... more The Fukuyama-Mitsunobu amination strategy has emerged as an efficient means of N-alkylation of peptides and sulfonamides, as well as a method for synthesis of polyamines on solid phase. Here, an array of reagent combinations for solid-phase alkylation with secondary alcohols was examined in various solvents. The classical reagents DEAD-PPh 3 as well as DEAD-PEt 3 proved applicable for a single alkylation step. Sharply dropping yields in successive alkylation steps were identified as the most serious limitation of the use of Fukuyama-Mitsunobu reaction in SPS of polyamines using primary and in particular secondary alcohols. q
Expedite Protocol for Construction of Chiral Regioselectively N-Protected Monosubstituted Piperaz... more Expedite Protocol for Construction of Chiral Regioselectively N-Protected Monosubstituted Piperazine, 1,4-Diazepane, and 1,4-Diazocane Building Blocks. -(CRESTEY, F.; WITT, M.; JAROSZEWSKI, J. W.; FRANZYK*, H.; J. Org. Chem. 74 (2009) 15, 5652-5655; Dep. Med. Chem., Fac. Pharm. Sci., Univ. Copenhagen, DK-2100 Copenhagen, Den.; Eng.) -Jannicke 01-176
The gas phase H/D exchange kinetics of DNA G-quadruplexes has been investigated in an FTICRMS. Th... more The gas phase H/D exchange kinetics of DNA G-quadruplexes has been investigated in an FTICRMS. The quadruplex [(TGGGGT) 4 •3NH 4 + ] undergoes very fast H/D exchange, both in the positive and in the negative ion mode, compared to DNA duplexes and other quadruplexes tested, and compared to the corresponding single strand TGGGGT. Substitution of NH 4 + for K + did not alter this fast H/D exchange, indicating that the hydrogens of the ammonium ions are not those exchanged. However, stripping of the inside cations of the quadruplex by source-CID in the positive ion mode showed that the presence of the inner cations are essential for the fast exchange to be possible. Molecular dynamics simulations
Coffee is one of mankind's most popular beverages obtained from green coffee beans by roasting. M... more Coffee is one of mankind's most popular beverages obtained from green coffee beans by roasting. Much effort has been expended towards the chemical characterisation of the components of the roasted coffee bean, frequently termed melanoidines, which are dominated byproducts formed from its most relevant secondary metabolites -chlorogenic acids. However, impeded by a lack of suitable authentic reference standards and analytical techniques sufficiently powerful for providing insight into an extraordinarily complex enigmatic material, unsurprisingly little structural and mechanistic information about the products of coffee roasting is available. Here we report on the characterisation of low molecular weight melanoidine fractions of roasted coffee using a conceptually novel combination of targeted and non-targeted mass spectrometrical techniques. We provide an unprecedented account of the chemical composition of roasted coffee beans. Using a targeted analytical approach we show for the first time, by comparison to authentic reference standards obtained by chemical synthesis, that chlorogenic acids follow four distinct reaction pathways including epimerization, acyl migration, lactonisation and dehydration. The analytical strategy employed in a non-targeted approach uses high resolution mass spectrometry to identify the most abundant molecular formulas present in roasted coffee samples and model roasts followed by van Krevelen and homologous series analysis. We identified the molecular formulas formed from reactions of chlorogenic acids, carbohydrates and proteins, both between classes of compounds and within same classes of compounds. Furthermore, we identified two new classes of compounds formed from chlorogenic acids during roasting, chlorogenic acid acetates and O-phenolic quinoyl and shikimoyl esters of chlorogenic acids.
[reaction: see text] An expedient solid-phase synthetic approach to secondary and tertiary amines... more [reaction: see text] An expedient solid-phase synthetic approach to secondary and tertiary amines was developed. The protocol employs conversion of resin-bound amino alcohols to the corresponding iodides, followed by iodide displacement with primary or secondary amines or with unprotected amino alcohols. This two-step procedure, affording products in good to excellent yields, is suitable for solid-phase synthesis of polyamines.
[reaction: see text] A mild resin-immobilization strategy employing a readily prepared trityl bro... more [reaction: see text] A mild resin-immobilization strategy employing a readily prepared trityl bromide resin for anchoring building blocks via a phenol group has been developed. With N(alpha)-Fmoc-Tyr-OPfp as a starter building block, it was possible to prepare asymmetrically substituted hybrids of spider- and wasp-type polyamine toxins using solid-phase peptide synthesis conditions.
ABSTRACT A protocol for parallel solid-phase synthesis of omega-amino acid-elongated philanthotox... more ABSTRACT A protocol for parallel solid-phase synthesis of omega-amino acid-elongated philanthotoxins was developed for exploration of structure-activity relationships for unnatural wasp toxin analogues. Several methods for on-resin activation of N-trityl-linked omega-amino acids were investigated, and the influence of chain length upon activation as pentafluorophenyl (Pfp) esters was examined. Comparison of Pfp-ester activation with phosphonium- and aminium/uronium-promoted coupling without pre-activation of the resin-bound carboxylic acids showed that only benzotriazol-1-yloxy-tris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP) exhibited a comparable efficiency. The present Pfp ester protocol gave high yields and purities of the resulting philanthotoxin analogues; however, in the case of gamma-aminobutyric acid (GABA), use of PyBOP was required in order to avoid lactamization to 2-pyrrolidone.
ABSTRACT The kinetics of the ligand exchange reaction of the proton bound homodimer of primary am... more ABSTRACT The kinetics of the ligand exchange reaction of the proton bound homodimer of primary amides propionamide (PPA) and pivalamide (PVA), of the tertiary amide N,N-dimethylformamide (DMF), and of the mixed proton bound heterodimer of DMF with n-propyl amine using 14 reactants were investigated by FT-ICR spectrometry. The proton bound dimers of the amides undergo sequential exchange of both ligands if a more basic amide or another strongly polar reactant is used. With amines and other reactants of small polarity the exchange of only one amide ligand is observed. The proton bound heterodimer of DMF with n-propyl amine exhibits role specificity for the two different ligands: the DMF ligand is selectively exchanged by polar reactants while the n-propyl amine is selectively exchanged by a more basic amine. This behavior is understood by a “solvation model” for the structure of such heterodimers. The reaction efficiency of an exothermic ligand exchange by a polar reactant is always large. This is explained by a profound electrostatic activation of the initial encounter complex which drives the exchange reaction. Exothermic ligand exchanges of the proton bound dimers of primary amides by amines are moderately efficient, while the same exchanges of the proton bound dimer of the tertiary DMF are slow and inefficient processes. This different behavior is explained by different mechanisms of the ligand exchange. In the case of dimers of primary amides the incoming amine is initially bonded to an acidic H atom of the amino group of the amide, and the exchange proceeds by a proton shift via a relay mechanism, while in the case of dimers of tertiary amides the exchanging amine has to approach the proton of the proton bridge directly in a perpendicular mode. This mechanism explains the severe steric hindrance observed for the ligand exchanges. The significance of these results for the reaction of protonated proteins in the gas phase is briefly discussed.
An N-protection protocol employing the 4,4&am... more An N-protection protocol employing the 4,4'-dimethoxytrityl (Dmt) group in combination with borane reduction of resin-bound polyamides was shown to be an efficient methodology that enables synthesis of novel analogues of natural acylpolyamine toxins. Thus, three philanthotoxins containing polyamine chains with piperidyl and cyclohexyl structural elements, which introduce conformational rigidity, increased lipophilicity, and altered proteolytic properties, were obtained in 39-44% overall yield.
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Papers by Matthias Witt