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Preparation and analytical investigation of amiton and amiton-like compounds closely related to the chemical weapons convention. “relevance of organo(thio)phosphates in the hazard defense sector”
Preparation and analytical investigation of amiton and amiton-like compounds closely related to the chemical weapons convention. “relevance of organo(thio)phosphates in the hazard defense sector”
The recent developments in the Middle East especially, the terroristic acts going on in Syria and Iraq put a new complexion on this work which focusses on the preparation and analytical investigation of Amiton and several homologs thereof and make it even more important. Amiton is an organophosphate nerve agent, formerly known as a pesticide, and falls under the Chemical Weapons Convention (CWC) whereas the other compounds prepared in the course of this thesis are not covered by the CWC. However, the aim of this thesis was not to identify new chemical warfare agents but to contribute to a better understanding of the existing ones. Basically, the work on hand can be divided into five main parts: Part 1: Preparation of Organophosphates and their Precursors The synthesis of the Amiton-like compounds was achieved in good yields by the coupling of an appropriate amino alcohol in its enolate form to the respective phosphorylchloride. In the case that the necessary educts were not commercially available the synthesis of the precursors was performed. Finally, nineteen Amiton-like compounds were prepared of which twelve having not been reported in the literature before. Additionally, most of the compounds could be transferred to the corresponding quaternary ammonium salts of which some also yielded single crystals. The latter ones were subjected to X-ray single crystal structure analysis and the structures are described for the first time in this thesis. A separate study addresses the isomerization of Amitons which are capable of the so called thiono-thiol isomerization. Thus far, no decisive explanation of the mechanism in the literature can be identified so that a new experimental series to describe the facts for Amiton was necessary. It was found that the decay of the thiono educt obeys a first order reaction kinetic which is dependent on the solvent. Part 2: Characterization by Analytical Instruments To fully characterize the prepared Amitons and their precursors the most recent and available analytical instruments were used. Especially, these are IR and Raman spectroscopy, HPLC- and GC- mass spectrometry as well as NMR spectroscopy. Furthermore, the obtained spectra could be uploaded into existing databases of the respective analytical instrument and methods for their recovery were developed. In particular, during the measurement of GC-MS spectra it was observed that some of the substances undergo thiono-thiol isomerization while being analyzed and thus are difficult to identify. Still until today this phenomenon is not fully described in literature but could be addressed in a separate part of this work. Moreover, the mass spectra differ from each other only in a very few peaks due to the very close relationship of the molecules analyzed in this study. Thus, in-depth investigations by two-dimensional mass spectrometry had to be conducted for unambiguous identification. The thus obtained results are reported in a separate part of this thesis. Part 3: Toxicological Studies Knowing the toxicological parameters of the prepared compounds is of central importance for safe handling of those compounds. Moreover, an assessment of the toxicity is also crucial in terms of a possible terroristic threat. Organophosphates have the fateful property to inhibit cholinesterases which are essential for the signal transduction in vertebrates. Most often this process is irreversible and finally leading to breakdown of the organism. For the determination of the inhibitory constants for acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activity a classical Ellman-essay was employed. It was found that all compounds but Amiton showed a weak inhibitory potential towards AChE. Additionally, these molecules were found to have a very similar effect towards BuChE. Furthermore, the found results strongly indicate that or prove that the respective inhibitory effect can be tuned very precisely by proper choosing and combining of the substituents of the nitrogen atom as well as the phosphorus atom. Part 4: Special Investigations Exceeding the so far described investigations detailed studies on special topics were run. In a separate series of experiments the vapor pressure data of Amitons prepared in this study were determined by employing the so called transpiration method approach. This involves a controlled gas stream which transports material of the sample under exact control of temperature and measurement time which is proportional to the vapor pressure of the sample. This method was applied for volatile substances so far and was successfully extended in close cooperation with Martin Härtel, LMU, to toxic and thermo-labile compounds like some of the synthesized ones. Knowledge of the vapor pressure and the enthalpy of vaporization allow the estimation of the detectability of a substance from the air. These values are of great importance, because, especially the hand-held and field portable instruments of the Bundeswehr are mainly based on the principle of gas phase detection. The initially obtained mass spectra are looking very similar to each other and finally were only distinguishable by their respective Kovats indices. The evaluation of the mass spectra itself results only in very few cases in a clear identification of the compound. For this reason the prepared substances were subjected to a two-dimensional mass spectrometric experiment, to allow an unambiguous assignment. As a major result of these investigations full mass spectrometric fragmentation pathways could be described for the Amiton molecules for the first time. As a rule these show two main fragmentation pathways. The first one is based on the fragmentation of the nitrogen containing side chain, which is at the same time dominating the mass spectrum. The second pathway is based on the fragmentation of the phosphorus moiety and shows fewer fragments. But, the latter one is at the same time important for the solution of the chalcogen configuration of the central phosphorus atom and thus a complete resolution of the structure by means of mass spectrometry. Another question was the detection of the substances from different environmental matrices. Usually sampling procedures and samples, like soil or water samples require mostly an intensive sample preparation, e.g. liquid-liquid extraction or solid-phase extraction. Furthermore, in the context of chemical warfare analysis no methods for the work up of so called complex matrices from the environment are described until today. In the conduct of this work a single automated method for solid-phase microextraction (SPME) was developed, which is capable of the extraction and direct processing of phosphororganic compounds from media: air, water and vegetation (foliage and grass) by direct immersion of a PDMS/DVB fiber. Part 5: Detection and Analysis with Military Detection Equipment and Procedures A major part of this work was dedicated to the evaluation of the Bundeswehr detection equipment and procedures with respect to the detection and analysis of the compounds prepared in the frame of this thesis. Almost all instruments could be tested. The obtained results very clearly demonstrate that the detection of the compounds is possible but that a distinct identification is tricky, particularly as some results are, but, comprehensible, contrary to others. Additionally, two techniques that have not been used so far could be initially tested. First, this was an instrument based on flame photometry and to all challenged compounds correctly responded. Second, a promising benchtop NMR instrument could be used to analyze the compounds. The thus obtained results force the conclusion, that NMR spectroscopy is an absolutely meaningful and necessary supplementation to the so far available instrumentation and offers a fast, affordable and absolutely reliable method to limit the possible compound class of the unknown analyte.
Organophosphate, Amiton, synthesis, vapor pressure, direct immersion, complex matrix, SPME, toxicology, spectrum, isomerization, rearrangement, enthalpy of vaporization, vapor pressure, kinetic study, enzyme inhibition, AChE, BuChE
Althoff, Marc André
2018
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Althoff, Marc André (2018): Preparation and analytical investigation of amiton and amiton-like compounds closely related to the chemical weapons convention: “relevance of organo(thio)phosphates in the hazard defense sector”. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

The recent developments in the Middle East especially, the terroristic acts going on in Syria and Iraq put a new complexion on this work which focusses on the preparation and analytical investigation of Amiton and several homologs thereof and make it even more important. Amiton is an organophosphate nerve agent, formerly known as a pesticide, and falls under the Chemical Weapons Convention (CWC) whereas the other compounds prepared in the course of this thesis are not covered by the CWC. However, the aim of this thesis was not to identify new chemical warfare agents but to contribute to a better understanding of the existing ones. Basically, the work on hand can be divided into five main parts: Part 1: Preparation of Organophosphates and their Precursors The synthesis of the Amiton-like compounds was achieved in good yields by the coupling of an appropriate amino alcohol in its enolate form to the respective phosphorylchloride. In the case that the necessary educts were not commercially available the synthesis of the precursors was performed. Finally, nineteen Amiton-like compounds were prepared of which twelve having not been reported in the literature before. Additionally, most of the compounds could be transferred to the corresponding quaternary ammonium salts of which some also yielded single crystals. The latter ones were subjected to X-ray single crystal structure analysis and the structures are described for the first time in this thesis. A separate study addresses the isomerization of Amitons which are capable of the so called thiono-thiol isomerization. Thus far, no decisive explanation of the mechanism in the literature can be identified so that a new experimental series to describe the facts for Amiton was necessary. It was found that the decay of the thiono educt obeys a first order reaction kinetic which is dependent on the solvent. Part 2: Characterization by Analytical Instruments To fully characterize the prepared Amitons and their precursors the most recent and available analytical instruments were used. Especially, these are IR and Raman spectroscopy, HPLC- and GC- mass spectrometry as well as NMR spectroscopy. Furthermore, the obtained spectra could be uploaded into existing databases of the respective analytical instrument and methods for their recovery were developed. In particular, during the measurement of GC-MS spectra it was observed that some of the substances undergo thiono-thiol isomerization while being analyzed and thus are difficult to identify. Still until today this phenomenon is not fully described in literature but could be addressed in a separate part of this work. Moreover, the mass spectra differ from each other only in a very few peaks due to the very close relationship of the molecules analyzed in this study. Thus, in-depth investigations by two-dimensional mass spectrometry had to be conducted for unambiguous identification. The thus obtained results are reported in a separate part of this thesis. Part 3: Toxicological Studies Knowing the toxicological parameters of the prepared compounds is of central importance for safe handling of those compounds. Moreover, an assessment of the toxicity is also crucial in terms of a possible terroristic threat. Organophosphates have the fateful property to inhibit cholinesterases which are essential for the signal transduction in vertebrates. Most often this process is irreversible and finally leading to breakdown of the organism. For the determination of the inhibitory constants for acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activity a classical Ellman-essay was employed. It was found that all compounds but Amiton showed a weak inhibitory potential towards AChE. Additionally, these molecules were found to have a very similar effect towards BuChE. Furthermore, the found results strongly indicate that or prove that the respective inhibitory effect can be tuned very precisely by proper choosing and combining of the substituents of the nitrogen atom as well as the phosphorus atom. Part 4: Special Investigations Exceeding the so far described investigations detailed studies on special topics were run. In a separate series of experiments the vapor pressure data of Amitons prepared in this study were determined by employing the so called transpiration method approach. This involves a controlled gas stream which transports material of the sample under exact control of temperature and measurement time which is proportional to the vapor pressure of the sample. This method was applied for volatile substances so far and was successfully extended in close cooperation with Martin Härtel, LMU, to toxic and thermo-labile compounds like some of the synthesized ones. Knowledge of the vapor pressure and the enthalpy of vaporization allow the estimation of the detectability of a substance from the air. These values are of great importance, because, especially the hand-held and field portable instruments of the Bundeswehr are mainly based on the principle of gas phase detection. The initially obtained mass spectra are looking very similar to each other and finally were only distinguishable by their respective Kovats indices. The evaluation of the mass spectra itself results only in very few cases in a clear identification of the compound. For this reason the prepared substances were subjected to a two-dimensional mass spectrometric experiment, to allow an unambiguous assignment. As a major result of these investigations full mass spectrometric fragmentation pathways could be described for the Amiton molecules for the first time. As a rule these show two main fragmentation pathways. The first one is based on the fragmentation of the nitrogen containing side chain, which is at the same time dominating the mass spectrum. The second pathway is based on the fragmentation of the phosphorus moiety and shows fewer fragments. But, the latter one is at the same time important for the solution of the chalcogen configuration of the central phosphorus atom and thus a complete resolution of the structure by means of mass spectrometry. Another question was the detection of the substances from different environmental matrices. Usually sampling procedures and samples, like soil or water samples require mostly an intensive sample preparation, e.g. liquid-liquid extraction or solid-phase extraction. Furthermore, in the context of chemical warfare analysis no methods for the work up of so called complex matrices from the environment are described until today. In the conduct of this work a single automated method for solid-phase microextraction (SPME) was developed, which is capable of the extraction and direct processing of phosphororganic compounds from media: air, water and vegetation (foliage and grass) by direct immersion of a PDMS/DVB fiber. Part 5: Detection and Analysis with Military Detection Equipment and Procedures A major part of this work was dedicated to the evaluation of the Bundeswehr detection equipment and procedures with respect to the detection and analysis of the compounds prepared in the frame of this thesis. Almost all instruments could be tested. The obtained results very clearly demonstrate that the detection of the compounds is possible but that a distinct identification is tricky, particularly as some results are, but, comprehensible, contrary to others. Additionally, two techniques that have not been used so far could be initially tested. First, this was an instrument based on flame photometry and to all challenged compounds correctly responded. Second, a promising benchtop NMR instrument could be used to analyze the compounds. The thus obtained results force the conclusion, that NMR spectroscopy is an absolutely meaningful and necessary supplementation to the so far available instrumentation and offers a fast, affordable and absolutely reliable method to limit the possible compound class of the unknown analyte.