Browsing by Subject "NMR"

Novichok nerve agents are persistent, highly toxic chemical weapons which were added to the Chemical Weapons Convention in 2020 after their use on civilians in England. The detection and characterization of Novichok nerve agents and the degradation products formed after their exposure to decontamination products can be accomplished through complementary instrumental analyses. Chromatographic methods such as LC-MS/MS can be utilized to qualitatively detect Novichok degradation products such as hydrolysates and LC-HRMS can provide information about their structure via the elemental composition and fragmentation pathways. By contrasting these data to spectroscopic techniques such as 1H and 31P NMR, structural elucidation of decontamination products is possible as well as the determination of the kinetics of the decontamination process itself. The literature review contains a summary of all published instrumental methods with which Novichok nerve agents, degradation products, biomarkers and adducts have been analyzed and the efficacy of those methods. In the experimental research, Novichok nerve agent A-234 was decontaminated via six different decontaminants and analyzed by LC-MS/MS to identify the mass spectra of the degradation products of each, followed by LC-HRMS analysis to determine the elemental composition and fragmentation patterns of the degradation products. The A-234 rate of hydrolysis kinetics were measured by 1H and 31P NMR spectroscopy in three of the decontaminants and when possible, two dimensional analysis was used to correlate the structural data from the chromatographic analysis. Lastly, the A-234 hydrolysate was derivatized via TMSDAM methylation for GC-MS/MS analysis after testing with two silylating and three methylating agents. Decontamination of A-234 was successful within 48 hours with three decontamination agents and complete hydrolysis was observed within 5 hours with an oxidizer-containing quaternary salt based decontamination agent.

Master's thesis project includes the backbone assignment of the human activity-regulated cytoskeleton-associated protein C-lobe (hArc, Uniprot ID: Q7LC44), 7-fluoroindole-based tryptophan-labeling method, and comparing that with the 100% double-labeled and 20%(13C) fractionally labeled samples. The project focuses on the effects of 7-fluoroindole-based fluorotryptophan-labeling. hArc C-lobe has only one tryptophan, which makes the analysis easier. Typically fluorotryptophan-labeling is a costly method – fluorotryptophan itself is very expensive and attaching the fluorine to the tryptophan while expressing is expensive and complicated. Fluoroindolebased labeling circles around the problem, as indole and serine are used in procaryotic systems for tryptophan biosynthesis – meaning that fluoroindole, which is cheap, could be used as an alternative for previous methods. Fluoro-labeled tryptophan is used in protein NMR; for example, in binding studies – fluorine-probes are sensitive, and binding of ligand or protein would move these peaks, indicating binding. This project aims to get an insight into the application of this labeling method. The goal is to see if one could utilize one sample with both (1H, 15N, 13C) labeling and 7-fluorotryptophan labeling for binding and structural studies. However, fluorine is very electronegative, affecting surrounding structures and possibly sequentially nearby amino acids. This possible effect will be observed and determined by comparing the 1H15N-chemical shifts between well-established labeling methods and fluoroindolebased labeling. To determine what amino acids in the protein are affected, if they are affected, will be determined by using the backbone assignment results and the results from the sample comparisons.

Cocoa butter (CB) is the predominant continuous phase in chocolate systems and has a significant impact on the macroscopic properties of the end product. Conventional methods such as differential scanning calorimetry (DSC), pulsed nuclear magnetic resonance (pNMR), X-ray diffraction (XRD) and polarized light microscopy (PLM) have been used to study CB crystallization primarily in bulk. Potential of alternative techniques to study crystallization such as Raman spectroscopy and Fourier Transform infrared spectroscopy (FTIR) has been explored. The main objective of this thesis research was to study the feasibility of both conventional and alternative techniques to study CB crystallization in different matrices and in tempered conditions. Bulk fat (CB with 1%, 5% or without lecithin), suspensions (CB with 1% lecithin (on fat basis) and sucrose or inulin) and chocolates were sampled as such (non-tempered systems) subjected to a laboratory scale tempering procedure to produce tempered systems. Both non-tempered and tempered products were subjected to DSC, NMR, XRD, PLM, Raman spectroscopy, FTIR and diffusing wave spectroscopy (DWS), in which primary crystallization was monitored or long-term storage was assessed. A toolbox was developed comprising feasibility of complementary techniques and, moreover, the toolbox was used to study the effect of lecithin and bulking materials on the CB crystallization behavior. The tempering procedure was successfully validated for every sample, as proven by the melting profile at 6 hours through DSC. The determination of the solid fat content (SFC) from the raw free induction decay signal by NMR showed to be more useful than the scripted SFC, especially for bulk fat systems. XRD showed its feasibility to study fat polymorphism for both bulk matrices and suspensions, except when sucrose is present, due to its interference in short spacings. PLM could only be used for non-tempered bulk fat systems since in other systems sample preparation cannot be standardized to measure crystallinity. FTIR and Raman spectroscopy seemed to be useful complementary techniques and capable of differentiating polymorphic forms, as is also possible using XRD. DWS showed to be comparable with DSC with an additional improved deconvolution of crystallization peaks. This study resulted in a feasibility toolbox and was used to study the effect of lecithin concentration and bulking materials, where the addition of 1% lecithin concentration in bulk fat and usage of inulin in model suspensions improves the crystallization of the CB matrix.

Mycosporine-like Amino Acids (MAAs) are small, secondary metabolites, with the ability to absorb UV light. They are produced by cyanobacteria to act as a sunscreen. The aim of this study was to catalogue MAA genetic and chemical diversity in strains of the cyanobacterial genus Nostoc. MAAs were detected in 21 of the 68 Nostoc strains using LC/MS. Fifty four different MAAs were detected across the Nostoc strains. Glycosylated MAAs were detected in 17 of the 21 strains with hexose being the most commonly occurring sugar. Surprisingly, two structurally distinct MAAs were detected from a lichen symbiont strain, Nostoc sp. UHCC 0926. Chemical analysis detected a theoretical methylated and glycosylated variant (m/z 583, C23H39N2O15), and a suspected tri-core variant (m/z 757, C34H53N4O15) with three chromophore rings as opposed to one which is typically found. The glycosylated MAA was predicted to have a hexenimine core which was methylated and had two hexose moieties. The tri-core consisted of 2 aminohexenone cores, one on either side of a central aminohexenimine core. An 8.3 Mb draft genome sequence was obtained to identify the MAA biosynthetic gene cluster responsible for the biosynthesis of these two unusual MAAs. This resulted in the detection of two gene clusters mysA-B-C1 and mysD-C2-C3. This gene cluster organisation was compared with those of other Nostoc strains. The gene cluster organization in Nostoc sp. UHCC 0926 was unique because it was the only strain to have two gene clusters and three mysC genes despite one of the other Nostocs having the ability to produce a tri-core MAA. The strain was cultured and harvested to allow for the extraction and purification of the target MAAs. The tri-core MAA structure was confirmed by NMR. However only a putative structure for the glycosylated MAA was made. The UV absorption spectrum of the tri-core MAA had an absorption maximum at 312 nm while the glycosylated and methylated MAA had an absorption maximum at 336 nm. The investigation into the MAA production of UHCC strains expands the known chemical and genetic diversity of MAAs produced by strains of the Nostoc genus.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an evolutionarily conserved protein with pleiotropic therapeutic effects in several disease models, including Parkinson’s disease (PD), diabetes and stroke. PD is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta and many GWAS-based genes predisposing for PD are involved in oxidative stress. MANF has been shown to alleviate oxidative stress in PD models, however, the role of MANF in the antioxidant defense and mitochondrial respiration is not fully understood. By performing bulk RNA sequencing on wildtype and MANF knockout (MANF-KO) human embryonic stem cells (hESCs), we uncovered several genes involved in antioxidant defense to be up- or downregulated in MANF-KO hESC. Here we report that MANF-KO hESCs do not express the evolutionary conserved antioxidant enzyme catalase. We show that the loss of catalase makes the MANF-KO hESCs more vulnerable to hydrogen peroxide indued oxidative stress, and that MANF-KO hESCs have a reduced maximal respiration and spare respiratory capacity. Additionally, we examined if the loss of catalase in MANF-KO hESCs inhibits the differentiation of the cells to human dopaminergic neurons in vitro. We show that MANF-KO hESCs differentiate to TH+/MAP2+ cells despite a sustained deficiency of catalase, but the MANF-KO DA cultures tend to have a reduced spare respiratory capacity and higher basal glycolytic activity. To elucidate the structure-to-function relationship of MANF we utilize molecular dynamics simulations in combination with spin relaxation data from nuclear magnetic resonance spectroscopy. By examining the two-domain nature of MANF in different intracellular conditions we provide insight of the biological relevance of MANF interactions. Here we show that MANF conformational ensemble is more compact than previously reported. By simulating MANF in the presence of calcium and ATP, in neutral and low pH, we observed competitive binding of ATP and calcium to MANF. This study provides novel evidence of a regulatory role of MANF in the cellular antioxidant defense and explores the biological relevance of ATP and calcium binding to MANF.

The literature section of this thesis provides an overview of modern ion-mobility spectrometry techniques in context with recent applications in analytical chemistry. While ion-mobility spectrometry is an “old” separation technique, it has received in recent years increasing attention for its unique ability to achieve separation of isomeric molecular species. Ion-mobility spectrometry can be readily hyphenated with chromatographic and mass spectrometric techniques, introducing an additional separation dimension with the unique capability of differentiating isobaric analyte ions based on their collision cross sections. After a brief introduction into the theory of ion-mobility spectrometry, most recent applications in the field are presented with the focus being on the discrimination of small isomeric molecules. The research project section of the thesis reports the synthesis of isomerically pure standard materials of the commercially unavailable pepper alkaloids piperettine and piperettyline, and the qualitative and quantitative analysis of piperettine in selected pepper fruit samples. Strategies for the synthesis of piperettine reported in the literature are reviewed, and critically evaluated in terms of practicability and overall yields. A new, expedient, and operationally convenient synthetic approach to isomerically pure piperettine and piperettyline from inexpensive starting materials is described. In course of stability studies, both alkaloids were found to be stable in the crystalline states and as solutions in a range of organic solvents under exclusion of ambient light. However, diluted solutions of both compounds proved extremely photosensitive, with extensive double bond isomerization occurring within seconds upon sunlight exposure. An analytical method for the quantification of piperettine in pepper fruit samples was developed, involving liquid extraction, extract clean-up by solid-phase extraction, and HPLC-UV analysis. The use of a chiral stationary phase (Chiralpak IB) under optimized reversed phase condition allowed for the first time clean separation of piperettine from its naturally co-occurring isomers, and thus for its unambiguous quantification. Subsequently, this method was employed to quantify piperettine in black, green, white, and red long pepper samples. The observed piperettine content were 1.4 – 3.7 mg/g in the pepper fruit samples, representing 46 – 69% of the total sum of isomers.