Adsorption von Wertstoffen aus Schwarzlauge und deren weitere Verwendung in der elektrochemischen Synthese

Die vorliegende Arbeit umfasst die Entwicklung von Ionenaustauscherharzen zur Gewinnung natürlicher Phenole aus Schwarzlauge sowie die elektrochemische Synthese aromatischer Nitrile. Die in Schwarzlauge gelösten Phenole Guajakol, Acetovanillon, Vanillin und 4,4‘-Dihydroxy-3,3‘-dimethoxylstilben können durch Ionenaustausch gewonnen werden. Dabei ist ein Ionenaustauscherharz erforderlich, welches im Gegensatz zu bestehenden Harzen zwischen organischen und anorganischen Ionen differenziert. Dahingehend wurden Ionenaustauscherharze entwickelt, welche durch entsprechende funktionelle Gruppen van der Waals- und pi-Wechselwirkungen adressieren. Die Ionenaustauscherharze wurden durch Funktionalisierung von Merrifield-Harz mit N- und P-Nukleophilen hergestellt. Die Phenolate wurden aus Schwarzlauge an die funktionalisierten Harze adsorbiert. Nach Freisetzung mit Säure erfolgte die Quantifizierung mittels Gaschromatographie gegen einen internen Standard, wobei die hergestellten Ionenaustauscherharze verglichen mit bestehenden Harzen ein vermindertes Adsorptionsvermögen aufwiesen. Für die elektrochemische Nitrilsynthese wurden zwei Strategien verfolgt. Die Reaktion von in situ-erzeugten Iminen führte lediglich zu geringen Mengen Nitril, wobei die eingesetzte Ni(O)OH-Elektrode in Gegenwart von Ammoniak instabil war. Die elektrochemische Reaktion von Oximen an Graphit und Blei hingegen lieferte in einer Domino-Oxidation-Reduktion-Sequenz Nitrile in Ausbeuten von bis zu 81%. Zudem waren Bleibronzekathoden kompatibel mit bromierten Substraten. Ferner führte die Zugabe eines Protonendonors zur Unterdrückung der kathodischen Reaktion und somit zu Nitril-N-oxiden, welche Bausteine in der Synthese von Antibiotika wie z.B. Dicloxacillin sind.This thesis comprises the development of ion exchange resins for the adsorption of phenolic components from black liquor as well as the electrochemical synthesis of nitriles from aldehydes. The phenols guaiacol, acetovanillone, vanillin and 4,4’-dihydroxy-3,3’-dimethoxystilbene can be adsorbed from black liquor by ion exchange. In contrast to common commercial resins, an ion exchange resin is necessary which enables differentiation between organic and inorganic ions. For this purpose, ion exchange resins addressing van der Waals and pi interactions were developed. The resins were synthesized by the functionalization of Merrifield resin with N and P nucleophiles, respectively. The functionalized resins were used for the adsorption of the phenolates from black liquor. By using acid, the phenols were set free, and quantification was carried out by gas chromatography with an internal standard. The synthesized resins exhibited a lower adsorption capacity compared to common commercial resins. In regard to the electrochemical nitrile synthesis, two paths were followed. The reaction of an in situ-generated imine led to the corresponding nitrile only in low yield, whereas the applied Ni(O)OH electrode appeared to be instable towards ammonia. In contrast, the electrochemical reaction of oximes at graphite and lead proceeded in a domino oxidation reduction sequence leading to nitriles in yields up to 81%. Additionally, leaded bronze cathodes were compatible with brominated substrates. Finally, proton donating additives enabled suppression of the cathodic reaction leading to nitrile N-oxides which are building blocks in the synthesis of antibiotics such as dicloxacillin.V, 225 Seite

ETD on small intact proteins in an ultra high resolution quadrupole TOF mass spectrometer

Comunicaciones a congreso

Electron capture and transfer dissociation: Peptide structure analysis at different ion internal energy levels

We decoupled electron-transfer dissociation (ETD) and collision-induced dissociation of charge-reduced species (CRCID) events to probe the lifetimes of intermediate radical species in ETD-based ion trap tandem mass spectrometry of peptides. Short-lived intermediates formed upon electron transfer require less energy for product ion formation and appear in regular ETD mass spectra, whereas long-lived intermediates require additional vibrational energy and yield product ions as a function of CRCID amplitude. The observed dependencies complement the results obtained by double-resonance electron-capture dissociation (ECD) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and ECD in a cryogenic ICR trap. Compared with ECD FT-ICR MS, ion trap MS offers lower precursor ion internal energy conditions, leading to more abundant charge-reduced radical intermediates and larger variation of product ion abundance as a function of vibrational post-activation amplitude. In many cases decoupled CRCID after ETD exhibits abundant radical c-type and even-electron z-type ions, in striking contrast to predominantly even-electron c-type and radical z-type ions in ECD FT-ICR MS and especially activated ion-ECD, thus providing a new insight into the fundamentals of ECD/ET

Electron Transfer Dissociation Mass Spectrometry of Hemoglobin on Clinical Samples

A mass spectrometry-based assay combining the specificity of selected reaction monitoring and the protein ion activation capabilities of electron transfer dissociation was developed and employed for the rapid identification of hemoglobin variants from whole blood without previous proteolytic cleavage. The analysis was performed in a robust ion trap mass spectrometer operating at nominal mass accuracy and resolution. Subtle differences in globin sequences, resulting with mass shifts of about one Da, can be unambiguously identified. These results suggest that mass spectrometry analysis of entire proteins using electron transfer dissociation can be employed on clinical samples in a workflow compatible with diagnostic application

A new method for accurate assessment of DNA quality after bisulfite treatment

The covalent addition of methylgroups to cytosine has become the most intensively researched epigenetic DNA marker. The vast majority of technologies used for DNA methylation analysis rely on a chemical reaction, the so-called ‘bisulfite treatment’, which introduces methylation-dependent sequence changes through selective chemical conversion of non-methylated cytosine to uracil. After treatment, all non-methylated cytosine bases are converted to uracil but all methylated cytosine bases remain cytosine. These methylation dependent C-to-T changes can subsequently be studied using conventional DNA analysis technologies. The bisulfite conversion protocol is susceptible to processing errors, and small deviation from the protocol can result in failure of the treatment. Several attempts have been made to simplify the procedure and increase its robustness. Although significant achievements in this area have been made, bisulfite treatment remains the main source of process variability in the analysis of DNA methylation. This variability in particular impairs assays, which strive for the quantitative assessment of DNA methylation. Here we present basic mathematical considerations, which should be taken into account when analyzing DNA methylation. We also introduce a PCR-based assay, which allows ab initio assessment of the DNA quality after bisulfite treatment and can help to prevent inaccurate quantitative measurement resulting from poor bisulfite treatment

Electron Capture and Transfer Dissociation: Peptide Structure Analysis at Different Ion Internal Energy Levels

We decoupled electron-transfer dissociation (ETD) and collision-induced dissociation of charge-reduced species (CRCID) events to probe the lifetimes of intermediate radical species in ETD-based ion trap tandem mass spectrometry of peptides. Short-lived intermediates formed upon electron transfer require less energy for product ion formation and appear in regular ETD mass spectra, whereas long-lived intermediates require additional vibrational energy and yield product ions as a function of CRCID amplitude. The observed dependencies complement the results obtained by double-resonance electron-capture dissociation (ECD) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and ECD in a cryogenic ICR trap. Compared with ECD FT-ICR MS, ion trap MS offers lower precursor ion internal energy conditions, leading to more abundant charge-reduced radical intermediates and larger variation of product ion abundance as a function of vibrational post-activation amplitude. In many cases decoupled CRCID after ETD exhibits abundant radical c-type and even-electron z-type ions, in striking contrast to predominantly even-electron c-type and radical z-type ions in ECD FT-ICR MS and especially activated ion-ECD, thus providing a new insight into the fundamentals of ECD/ETD. (J Am Soc Mass Spectrom 2009, 20, 567-575) (C) 2009 Published by Elsevier Inc. on behalf of American Society for Mass Spectrometr

Top-down analysis of 30-80 kDa proteins by electron transfer dissociation time-of-flight mass spectrometry

Electron transfer dissociation (ETD)-based top-down mass spectrometry (MS) is the method of choice for in-depth structure characterization of large peptides, small- and medium-sized proteins, and non-covalent protein complexes. Here, we describe the performance of this approach for structural analysis of intact proteins as large as the 80kDa serotransferrin. Current time-of-flight (TOF) MS technologies ensure adequate resolution and mass accuracy to simultaneously analyze intact 30-80kDa protein ions and the complex mixture of their ETD product ions. Here, we show that ETD TOF MS is efficient and may provide extensive sequence information for unfolded and highly charged (around 1 charge/kDa) proteins of ∼30kDa and structural motifs embedded in larger proteins. Sequence regions protected by disulfide bonds within intact non-reduced proteins oftentimes remain uncharacterized due to the low efficiency of their fragmentation by ETD. For serotransferrin, reduction of S-S bonds leads to significantly varied ETD fragmentation pattern with higher sequence coverage of N- and C-terminal regions, providing a complementary structural information to top-down analysis of its oxidized form. Figure ETD TOF MS provides extensive sequence information for unfolded and highly charged proteins of ~30 kDa and above. In addition to charge number and distribution along the protein, disulfide bonds direct ETD fragmentation. For intact non-reduced 80 kDa serotransferrin, sequence regions protected by disulfide bonds oftentimes remain uncharacterized. Reduction of disulfide bonds of serotransferrin increases ETD sequence coverage of its N- and C-terminal regions, providing a complementary structural information to the top-down analysis of its oxidized for

Top-down analysis of 30–80 kDa proteins by electron transfer dissociation time-of-flight mass spectrometry

Electron transfer dissociation (ETD)-based top-down mass spectrometry (MS) is the method of choice for in-depth structure characterization of large peptides, small- and medium-sized proteins, and non-covalent protein complexes. Here, we describe the performance of this approach for structural analysis of intact proteins as large as the 80 kDa serotransferrin. Current time-of-flight (TOF) MS technologies ensure adequate resolution and mass accuracy to simultaneously analyze intact 30-80 kDa protein ions and the complex mixture of their ETD product ions. Here, we show that ETD TOF MS is efficient and may provide extensive sequence information for unfolded and highly charged (around 1 charge/kDa) proteins of similar to 30 kDa and structural motifs embedded in larger proteins. Sequence regions protected by disulfide bonds within intact non-reduced proteins oftentimes remain uncharacterized due to the low efficiency of their fragmentation by ETD. For serotransferrin, reduction of S-S bonds leads to significantly varied ETD fragmentation pattern with higher sequence coverage of N- and C-terminal regions, providing a complementary structural information to top-down analysis of its oxidized form

Identification of RNA molecules by specific enzyme digestion and mass spectrometry: software for and implementation of RNA mass mapping

The idea of identifying or characterizing an RNA molecule based on a mass spectrum of specifically generated RNA fragments has been used in various forms for well over a decade. We have developed software—named RRM for ‘RNA mass mapping’—which can search whole prokaryotic genomes or RNA FASTA sequence databases to identify the origin of a given RNA based on a mass spectrum of RNA fragments. As input, the program uses the masses of specific RNase cleavage of the RNA under investigation. RNase T1 digestion is used here as a demonstration of the usability of the method for RNA identification. The concept for identification is that the masses of the digestion products constitute a specific fingerprint, which characterize the given RNA. The search algorithm is based on the same principles as those used in peptide mass fingerprinting, but has here been extended to work for both RNA sequence databases and for genome searches. A simple and powerful probability model for ranking RNA matches is proposed. We demonstrate viability of the entire setup by identifying the DNA template of a series of RNAs of biological and of in vitro transcriptional origin in complete microbial genomes and by identifying authentic 16S ribosomal RNAs in a ‘small ribosomal subunit RNA’ database. Thus, we present a new tool for a rapid identification of unknown RNAs using only a few picomoles of starting material

Dysregulation of DGCR6 and DGCR6L: psychopathological outcomes in chromosome 22q11.2 deletion syndrome

Chromosome 22q11.2 deletion syndrome (22q11DS) is the most common microdeletion syndrome in humans. It is typified by highly variable symptoms, which might be explained by epigenetic regulation of genes in the interval. Using computational algorithms, our laboratory previously predicted that DiGeorge critical region 6 (DGCR6), which lies within the deletion interval, is imprinted in humans. Expression and epigenetic regulation of this gene have not, however, been examined in 22q11DS subjects. The purpose of this study was to determine if the expression levels of DGCR6 and its duplicate copy DGCR6L in 22q11DS subjects are associated with the parent-of-origin of the deletion and childhood psychopathologies. Our investigation showed no evidence of parent-of-origin-related differences in expression of both DGCR6 and DGCR6L. However, we found that the variability in DGCR6 expression was significantly greater in 22q11DS children than in age and gender-matched control individuals. Children with 22q11DS who had anxiety disorders had significantly lower DGCR6 expression, especially in subjects with the deletion on the maternal chromosome, despite the lack of imprinting. Our findings indicate that epigenetic mechanisms other than imprinting contribute to the dysregulation of these genes and the associated childhood psychopathologies observed in individuals with 22q11DS. Further studies are now needed to test the usefulness of DGCR6 and DGCR6L expression and alterations in the epigenome at these loci in predicting childhood anxiety and associated adult-onset pathologies in 22q11DS subjects