Instrumentation and development of a mass spectrometry system for the study of gas-phase biomolecular ion reactions

Gas-phase reactions of biomolecular ions are highly relevant to the understanding of structures and functions of the biomolecules. Mass spectrometry is a powerful tool in investigating gas-phase ion chemistry. Various mass spectrometers have been developed to explore ion/molecule reactions, ion/ion reactions, ion/photon reactions, ion/radical reactions etc., both at atmospheric pressure and in vacuum. In-vacuum reactions have an advantage of involving pre-selecting the ions for the reactions using a mass analyzer. Over the decades, a variety of mass analyzers have been employed in the research of ion chemistry. Hybrid configurations, such as quadrupole ion trap with a time-of-flight and or a quadrupole ion trap tandem with an Orbitrap, have been utilized to improve the performances for both the reaction (in trapping mode) and the mass analysis (accurate mass measurements). Complicated instrument structures, including ion optics, multiple mass analyzers and differential pumping for high vacuum, are typically required for the mass spectrometers for gas phase ion chemistry study. An alternative approach is to simplify the instrumentation by using pulsed discontinuous atmospheric pressure interfaces for introducing ionic or neutral reactants and a single ion trap as both the reactor and the mass analyzer. Such a simple mass spectrometry system was set up and demonstrated using two discontinuous atmospheric pressure interfaces in the study for this thesis. It was capable of carrying out ion/molecule and ion/ion reactions at an elevated pressure without the needs of ion optics or differential pumping system. Together with a pyrolysis radical source, in-vacuum ion/radical reactions were performed and their associated chemistry was studied. Radicals are important intermediates related to biochemical processes and biological functions. There are very limited techniques to monitor the reactive intermediates in-situ during a multi-step reaction in aqueous phase. On the other hand, these intermediates can be cooled down and preserved into a single-step procedure in gas-phase reactions since they only occur via collisions. Therefore, the fundamental study of gas-phase radical ion chemistry will provide evidences of the reactivity, energetics, and structural information of biological radicals, which has the potential to solve puzzles of aging, disease biomarker identification, and enzymatic activities. Using the system described above, a new reaction between protonated alkyl amines and pyrolysis formed cyclopropenylidene carbene was discovered, as the first experimental evidence of the reactivity of cyclopropenylidene. Given the important role of cyclopropenylidene in the combustion chemistry, organic synthesis, and interstellar chemistry, it is highly desirable to establish a fundamental understanding of their physical and chemical properties. The amine/cyclopropenylidene reactions were systematically studied using both theoretical calculation and experimental evidences. A proton-bound dimer reaction mechanism was proposed, with the amine and the carbene sharing a proton to form a complex as the first step, which was closely related to the high gas-phase basicity of cyclopropenylidene. Subsequent unimolecular dissociation of the complex yielded three possible reaction pathways, including proton-transfer to the carbene, covalent product formation, and direct separation. These reactions were studied with a variety of alkyl amines of different gas-phase basicities. For the covalent complex formation, partial protonation on cyclopropenylidene within the dimer facilitates subsequent nucleophilic attack to the carbene carbon by the amine nitrogen and leads to a C-N bond formation. The highest yield of covalent complex was achieved with the gas-phase basicity of the amine slightly lower but comparable to cyclopropenylidene. The results demonstrated a new reaction pathway of cyclopropenylidene besides the formation of cyclopropenium, which has long been considered as a dead end in interstellar carbon chemistry. Further reactivity study of cyclopropenylidene towards biomolecular ions was also carried out for nucleobases, nucleosides, amino acids, peptides, proteins, and lipids. The reaction to form proton-bound dimer for protonated biomolecular ions remained as the dominant reaction pathway. Interestingly, other possible reaction pathways, such as modifications of thiyl group or disulfide bonds, double bond addition, and single bond insertion, were inhibited in gas-phase ion/carbene reactions. Such results inferred that the reactivity of neutral species was not directly applicable to ion reactions, with the proton involved in the gas-phase biomolecular ion reactions

Some perspectives on the design and discovery of new multi-component reactions

This thesis is divided into three parts. Part one presents an overview of multi-component reactions, contrasting isocyanide based and non-isocyanide based multi-component reactions, and gives examples of the most important examples of these types of reactions. In addition, a brief discussion of 1,3-dipolar cycloadditions is given to serve as a framework for the discussion in part two of the results obtained. Part two is divided into three sections and discusses two conceptually different approaches to the development of new multi-component reactions. The first discusses the use of combinatorial methods for the generation and screening of reaction libraries and the limitations encountered in this approach. The second section deals with the use of isocyanides in a 1,4-cycloaddition followed by a 1,3-dipolar cycloaddition affording isoxazolines. A series of isocyanides were successfully employed both in an intra- and intermolecular fashion. Furthermore, the results gained from attempts using electron rich dipolarophiles as trapping agents in the latter 1,3-dipolar cycloaddition, suggest an alternative mechanism proceeding g through an intermediate nitronate, rather than the nitrile oxide as previously assumed. The initial low yields were improved upon by the use of lithium perchlorate as a promoter of the cycloaddition reaction. The third section details the attempts made at utilising silylated nucleophiles to generate silyinitronates from nitroalkenes and their subsequent use in inter- and intramolecular 1,3-dipolar cycloadditions. Part three describes the experimental procedures employed and results obtained

Supramolecular sensing at the solid-liquid interface with phosphonate cavitands

The base of supramolecular chemistry rests on molecular recognition, that is the selective recognition of substrate molecules (guest) by synthetic receptors (host). The present thesis deals with the selective recognition properties of tetraphosphonate cavitands towards N-methylpyridinium and N-alkylammonium salts. In the first part of the thesis an extensive study of the thermodynamics of the complexation properties of tetraphosphonate cavitands towards N-methylpyridinium salts in solution via Isothermal Titration Calorimetry (ITC) is reported. The information obtained by the ITC in the recognition process of the receptor towards N-methylpyridinium salts were then exploited for the design of a new type of non-covalently linked-cavitand-stopped rotaxane. In the second part of the work, the complexation properties of cavitands were assessed towards N-alkylammonium salts at the solid-liquid interface via microcantilevers reaching an unprecedented real-time label-free selectivity. ITC was used as an independent tool for confirmation and rationalization of the results obtained with microcantilevers-based sensor. This approach has been then benchmarked by differentiating biological important molecules like sarcosine and glycine in water, reaching unique performances. The results obtained for the N-alkylammonium salts series opened the route to use microcantilevers for the online monitoring and the label-free sensing of biologically active ammonium-based molecules like drugs. The first experiments performed to test the recognition properties of tetraphosphonate cavitands towards drugs at the solid-liquid interface are described.La base della chimica supramolecolare risiede nel riconoscimento molecolare, cioè nel selettivo riconoscimento delle molecola di substrato (guest) da parte del recettore sintetico (host). La presente tesi si occupa delle selettive proprietà di riconoscimento dei cavitandi tetrafosfonati verso i sali di N-metilpiridinio e di N-alchilammonio. Nella prima parte della tesi è riportato uno studio estensivo relativo alla termodinamica delle proprietà complessanti dei cavitandi tetrafosfonati verso i sali di N-metilpiridinio tramite Titolazione Calorimetrica Isoterma (ITC). Le informazioni ottenute tramite ITC riguardanti il processo di riconoscimento del recettore con i sali di N-metilpiridinio sono state successivamente sfruttate per il design di una nuova tipologia di rotaxano, utilizzando i cavitandi come stopper non covalentemente legati all'asse. Nella seconda parte del lavoro sono state studiate le proprietà complessanti dei cavitandi nei confronti dei sali di N-alchilammonio all'interfaccia solido-liquido via microcantilever, raggiungendo una selettività label-free in tempo reale senza precedenti. La calorimetria, in questo caso, è stata usata come strumento indipendente per la conferma e la razionalizzazione dei risultati ottenuti tramite microcantilever. Questo approccio è stato poi testato differenziando molecole biologiche chiave come sarcosina e glicina in acqua, raggiungendo performance uniche. I risultati ottenuti per la serie di sali di N-alchilammonio ha aperto la strada per l'utilizzo dei microcantilever nel monitoraggio on-line e nel sensing label-free di molecole contenenti il gruppo amminico biologicamente attive come le droghe. Nell'ultima parte del lavoro vengono descritti gli esperimenti preliminari eseguiti per testare le proprietà di riconoscimento dei cavitandi tetrafosfonati verso le droghe all'interfaccia solido-liquido

Polymer Mimetics for Soil Modeling and Detection of Biomarkers

The population of the world is increasing day by day and is expected to reach 9.8 billion by the year 2050. The ever-increasing demand for agricultural products is putting an unprecedented strain on the world\u27s soils as the human population continues to expand. Soil degradation caused by over-farming and the agrochemicals (fertilizers, pesticides, etc.) used in agriculture is a growing problem, although its causes remain murky. In addition, little is understood about the molecular-level interactions of substances that are subsequently introduced to soils, such as agricultural chemicals (ACs). Therefore, it is expected that these constraints may be circumvented by the synthesis of natural mimics of soil, known as Engineered Soil Surrogates (ESSs), to associate their bulk attributes with structure compositions. A series of polymeric ESSs were synthesized and the sorption behavior of Norflurazon as the ACs was observed and compared to the sorption behavior of a natural soil, Pahokee peat. On the other hand, the world is currently dealing with the drug overdose epidemic mainly due to the illicit use of synthetic opioids, primarily fentanyl. More than 70000 people died due to fentanyl overdose in 2021, which is often mixed with other drugs such as heroin, cocaine, etc. with or without the knowledge of the end-users. Therefore, the detection of fentanyl by law enforcement agencies and end-users is of utmost importance. Molecularly Imprinted Polymer (MIP) based sensors can be a solution to this problem. A MIP was made by using methacrylic acid (MAA), and ethylene glycol dimethacrylate (EGDMA) as the functional monomer and cross-linking monomer respectively, and benzyl fentanyl as the target template. Binding sites that are complementary to the analyte in size and shape are revealed after the template has been removed. Selectivity studies comprising various drugs such as heroin, cocaine, and methamphetamine showed that the synthesized MIP can selectively detect benzyl fentanyl. Finally, a molecularly imprinted hydrogel employing diffraction grating techniques was developed to detect microRNA. The hydrogels imprinted with the miR-21 DNA target were sensitive to the target sequence\u27s reintroduction and selective among comparable nucleotide sequences

Electroanalytical sensors using lipophilic cyclodextrins

Lipophilic dialkylated-a-, β- and γ-cyclodextrin derivatives were used as selective ionophores for a series of clinically relevant ammonium ions, and as enantioselective ionophores for both a- and β-aryl ammonium ions. Sensitive and selective potentiometric detection of the local anaesthetics lidocaine and bupivacaine was achieved by using 2,3,6 trioctyl-β-cyclodextrin as the ionophore, leading to micromolar detection limits. Interference studies showed that the simulated clinical electrolyte background caused minimal interference whereas organic interferents of similar size and charge caused some perturbation of the electrode response at a concentration of 10 mmol dm(^-3). An electrode comprising a plasticized biocompatible membrane matrix, TECOFLEX, with 2,6 didodecyl-β-cyclodextrin was incorporated in a flow injection analysis system and the response to lidocaine studied in the presence of human serum. Human serum caused no adverse effects to the electrochemical response of the electrode. These electrodes are, therefore, very suitable for on-line detection of local anaesthetics. Potentiometric detection of tricyclic antidepressants using didodecyl-a-, β- and γ- cyclodextrins as the ionophore, gave micromolar detection limits. Interference from simulated clinical electiolyte background and selected organic interferents gave similar results to those discussed above. In order to lower the detection limit to sub-nanomolar levels modified amperometric electrodes were assembled by depositing a membrane comprising plasticised TECOFLEX, 2,3,6 triethyl-β-cyclodextrin and TKB on the working electrode of a screen printed electrode. Lipophilic 2,6 didodecyl-a- and β-cyclodextrins exhibited enantiomeric discrimination in the binding of propranolol, ephedrine, amphetamine and methamphetamine. These results were confirmed using potentiometric and NMR techniques

Increasing drug retention in lung tissue through conjugation with polyethylene-glycol

The pulmonary delivery of drugs is an attractive route of administration because of the large surface area and high permeability of the airway epithelium. The large majority of inhaled drugs are used to manage asthma and Chronic Obstructive Pulmonary Disorder (COPD), such as inhaled corticosteroids and β2-adrenergic receptor agonists. Local delivery of small molecules often results in sub-optimal pharmacokinetics characterised by short absorption times (tmax) and high systemic concentrations (Cmax). Numerous drug delivery strategies have been attempted to increase lung retention time, including drug encapsulation in microspheres, the use of polymeric excipients, or the formation of low solubility drugs. So far, drug conjugation strategies have been limited to decreasing the prodrug solubility. The non-permanent conjugation of small molecules to a large hydrophilic polymer has not been studied for pulmonary delivery. The rationale behind such a strategy is that small molecules are mainly absorbed through the epithelium by passive diffusion, the absorption rates being positively correlated to the drug lipophilicity and molecular weight. This project has therefore been looking at the production, characterisation, in vitro and ex vivo evaluation of polyethylene glycol (PEG)-ester conjugates for the sustained delivery of drugs to the lung. This thesis presents the successful oxidation and subsequent esterification of PEG of various molecular weights with prednisolone (a corticosteroid) and salbutamol (a β2-adrenergic receptor agonist). This study illustrated the feasibility of a polymeric drug conjugate strategy for sustained release of drugs to the lung. The conjugates exhibited good in vitro stability which was translated into improved pharmacokinetics and longer residence time ex vivo in the isolated and perfused rat lung. Further studies must be conducted to fully assess the role of esterases in the pulmonary hydrolysis of the conjugates and in vivo experiments would be necessary to verify the safety of the conjugates and efficacy of the drug

Proceedings of the Thirteenth International Conference on Time-Resolved Vibrational Spectroscopy

The thirteenth meeting in a long-standing series of “Time-Resolved Vibrational Spectroscopy” (TRVS) conferences was held May 19th to 25th at the Kardinal Döpfner Haus in Freising, Germany, organized by the two Munich Universities - Ludwig-Maximilians-Universität and Technische Universität München. This international conference continues the illustrious tradition of the original in 1982, which took place in Lake Placid, NY. The series of meetings was initiated by leading, world-renowned experts in the field of ultrafast laser spectroscopy, and is still guided by its founder, Prof. George Atkinson (University of Arizona and Science and Technology Advisor to the Secretary of State). In its current format, the conference contributes to traditional areas of time resolved vibrational spectroscopies including infrared, Raman and related laser methods. It combines them with the most recent developments to gain new information for research and novel technical applications. The scientific program addressed basic science, applied research and advancing novel commercial applications. The thirteenth conference on Time Resolved Vibrational Spectroscopy promoted science in the areas of physics, chemistry and biology with a strong focus on biochemistry and material science. Vibrational spectra are molecule- and bond-specific. Thus, time-resolved vibrational studies provide detailed structural and kinetic information about primary dynamical processes on the picometer length scale. From this perspective, the goal of achieving a complete understanding of complex chemical and physical processes on the molecular level is well pursued by the recent progress in experimental and theoretical vibrational studies. These proceedings collect research papers presented at the TRVS XIII in Freising, German

Pseudomonas aeruginosa PA3859: From Structure to Function

The aim of this work has been the structural and functional study of a putative carboxylesterase purified from P. aeruginosa, namely PA3859. The protein has been purified from the wild type and a preliminary biochemical charcterization was carried out. The PA3859 gene was then cloned and the recombinant protein was expressed in E. coli (Chapter 2). The recombinant PA3859 was successfully crystallized and its 3D crystal structure was determined (Chapter 3 and 4). Starting from the enzyme 3D structure, an approach involving in silico, in vitro and in vivo assays lead to the reliable determination of the PA3859 physiological function (Chapter 5, 6, 7)

Design Strategies of Fluorescent Biosensors Based on Biological Macromolecular Receptors

Fluorescent biosensors to detect the bona fide events of biologically important molecules in living cells are increasingly demanded in the field of molecular cell biology. Recent advances in the development of fluorescent biosensors have made an outstanding contribution to elucidating not only the roles of individual biomolecules, but also the dynamic intracellular relationships between these molecules. However, rational design strategies of fluorescent biosensors are not as mature as they look. An insatiable request for the establishment of a more universal and versatile strategy continues to provide an attractive alternative, so-called modular strategy, which permits facile preparation of biosensors with tailored characteristics by a simple combination of a receptor and a signal transducer. This review describes an overview of the progress in design strategies of fluorescent biosensors, such as auto-fluorescent protein-based biosensors, protein-based biosensors covalently modified with synthetic fluorophores, and signaling aptamers, and highlights the insight into how a given receptor is converted to a fluorescent biosensor. Furthermore, we will demonstrate a significance of the modular strategy for the sensor design

Post combustion CO2 capture with substituted ethanolamines and piperazines : Ab initio and DFT studies

The emission of greenhouse gases in the atmosphere, particularly carbon dioxide, caused by fossil fuel combustion, has been claimed to be responsible for global warming. Capturing of carbon dioxide from exhaust gases by using aqueous amine solutions is an important process for the reduction of the emission of these gases from power plants. Monoethanolamine (MEA) is used as an industrial solvent for carbon dioxide capture from exhaust gases. However, the use of MEA for CO2 capture from power plants is expensive. Therefore, finding the cheaper solvent for CO2 capture is needed to reduce the CO2 emissions into the atmosphere and thereby saving the biodiversity in the world.</p