Reactions of singlet oxygen and singlet oxygen mimics with dicyclopropylethylenes: Comparison of reactivity and mechanisms

An extensive study of the reaction pathways of 1,1-dicyclopropyl ethylene, cis- and trans- 1,2-dicyclopropylethylenes has been undertaken with different electrophiles 4-methyl-1,2,4-triazoline-3,5-dione (MTAD), tetracyanoethylene (TCNE), and singlet oxygen $\rm(\sp1O\sb2).$ Comparison of reactivity and reaction mechanisms among the electrophiles is investigated. Singlet oxygen exhibits significantly lower reactivity compared to the other electrophiles. MTAD and TCNE react with dicyclopropylethylenes to produce predominantly $\sp{\prime\prime}2+2\sp{\prime\prime}$ adducts and a small amount of the "ene" adducts. The $\sp{\prime\prime}2+2\sp{\prime\prime}$ is the major product presumably because of the high activation energy leading to the highly strained "ene" products. Solvent trapping studies provide strong evidence of a "stepwise" mechanism, involving a zwitterionic or aziridinium imide as an intermediate from the study of the reactions products of dicyclopropylethylenes and MTAD. ^

Variables affecting the precision and accuracy of the Intoxilyzer 5000

The Intoxilyzer 5000 was tested for calibration curve linearity for ethanol vapor concentration between 0.020 and 0.400g/210L with excellent linearity. Calibration error using reference solutions outside of the allowed concentration range, response to the same ethanol reference solution at different temperatures between 34 and 38$\sp\circ$C, and its response to eleven chemicals, 10 mixtures of two at the time, and one mixture of four chemicals potentially found in human breath have been evaluated. Potential interferents were chosen on the basis of their infrared signatures and the concentration range of solutions corresponding to the non-lethal blood concentration range of various volatile organic compounds reported in the literature. The result of this study indicates that the instrument calibrates with solutions outside the allowed range up to $\pm$10% of target value. Headspace FID dual column GC analysis was used to confirm the concentrations of the solutions. Increasing the temperature of the reference solution from 34 to 38$\sp\circ$C resulted in linear increases in instrument recorded ethanol readings with an average increase of 6.25%/$\sp\circ$C. Of the eleven chemicals studied during this experiment, six, isopropanol, toluene, methyl ethyl ketone, trichloroethylene, acetaldehyde, and methanol could reasonably interfere with the test at non-lethal reported blood concentration ranges, the mixtures of those six chemicals showed linear additive results with a combined effect of as much as a 0.080g/210L reading (Florida's legal limit) without any ethanol present. ^

Synthesis of azulenylsilane nitrones as diagnostic tools for superoxide detection

The superoxide radical is considered to play important roles in physiological processes as well as in the genesis of diverse cytotoxic conditions such as cancer, various cardiovascular disorders and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD) and Alzheimer’s disease (AD). The detection and quantification of superoxide within cells is of critical importance to understand biological roles of superoxide and to develop preventive strategies against free radical-mediated diseases. Cyclic nitrone spin traps such as DMPO, EMPO, DEPMPO, BMPO and their derivatives have been widely used in conjunction with ESR spectroscopy to detect cellular superoxide with some success. However, the formation of unstable superoxide adducts from the reaction of cyclic nitrones with superoxide is a stumbling block in detecting superoxide by using electron spin resonance (ESR). A chemiluminescent probe, lucigenin, and fluorogenic probes, hydroethidium and MitoSox, are the other frequently used methods in detecting superoxide. However, luceginen undergoes redox-cycling producing superoxide by itself, and hydroethidium and MitoSox react with other oxidants apart from superoxide forming red fluorescent products contributing to artefacts in these assays. Hence, both methods were deemed to be inappropriate for superoxide detection. In this study, an effective approach, a selective mechanism-based colorimetric detection of superoxide anion has been developed by using silylated azulenyl nitrones spin traps. Since a nitrone moiety and an adjacent silyl group react readily with radicals and oxygen anions respectively, such nitrones can trap superoxide efficiently because superoxide is both a radical and an oxygen anion. Moreover, the synthesized nitrone is designed to be triggered solely by superoxide and not by other commonly observed oxygen radicals such as hydroxyl radical, alkoxyl radicals and peroxyl radical. In vitro studies have shown that these synthesized silylated azylenyl nitrones and the mitochondrial-targeted guanylhydrazone analog can trap superoxide efficiently yielding UV-vis identifiable and even potentially fluorescence-detectable orange products. Therefore, the chromotropic detection of superoxide using these nitrones can be a promising method in contrast to other available methods.

Covalent protein adduction by drugs of abuse

Recreational abuse of the drugs cocaine, methamphetamine, and morphine continues to be prevalent in the United States of America and around the world. While numerous methods of detection exist for each drug, they are generally limited by the lifetime of the parent drug and its metabolites in the body. However, the covalent modification of endogenous proteins by these drugs of abuse may act as biomarkers of exposure and allow for extension of detection windows for these drugs beyond the lifetime of parent molecules or metabolites in the free fraction. Additionally, existence of covalently bound molecules arising from drug ingestion can offer insight into downstream toxicities associated with each of these drugs. This research investigated the metabolism of cocaine, methamphetamine, and morphine in common in vitro assay systems, specifically focusing on the generation of reactive intermediates and metabolites that have the potential to form covalent protein adducts. Results demonstrated the formation of covalent adduction products between biological cysteine thiols and reactive moieties on cocaine and morphine metabolites. Rigorous mass spectrometric analysis in conjunction with in vitro metabolic activation, pharmacogenetic reaction phenotyping, and computational modeling were utilized to characterize structures and mechanisms of formation for each resultant thiol adduction product. For cocaine, data collected demonstrated the formation of adduction products from a reactive arene epoxide intermediate, designating a novel metabolic pathway for cocaine. In the case of morphine, data expanded on known adduct-forming pathways using sensitive and selective analysis techniques, following the known reactive metabolite, morphinone, and a proposed novel metabolite, morphine quinone methide. Data collected in this study describe novel metabolic events for multiple important drugs of abuse, culminating in detection methods and mechanistic descriptors useful to both medical and forensic investigators when examining the toxicology associated with cocaine, methamphetamine, and morphine.

Covalent protein adduction of nitrogen mustards and related compounds

Chemical warfare agents continue to pose a global threat despite the efforts of the international community to prohibit their use in warfare. For this reason, improvement in the detection of these compounds remains of forensic interest. Protein adducts formed by the covalent modification of an electrophilic xenobiotic and a nucleophilic amino acid may provide a biomarker of exposure that is stable and specific to compounds of interest (such as chemical warfare agents), and have the capability to extend the window of detection further than the parent compound or circulating metabolites. This research investigated the formation of protein adducts of the nitrogen mustard chemical warfare agents mechlorethamine (HN-2) and tris(2-chloroethyl)amine (HN-3) to lysine and histidine residues found on the blood proteins hemoglobin and human serum albumin. Identified adducts were assessed for reproducibility and stability both in model peptide and whole protein assays. Specificity of these identified adducts was assessed using in vitro assays to metabolize common therapeutic drugs containing nitrogen mustard moieties. Results of the model peptide assays demonstrated that HN-2 and HN-3 were able to form stable adducts with lysine and histidine residues under physiological conditions. Results for whole protein assays identified three histidine adducts on hemoglobin, and three adducts (two lysine residues and one histidine residue) on human serum albumin that were previously unknown. These protein adducts were determined to be reproducible and stable at physiological conditions over a three-week analysis period. Results from the in vitro metabolic assays revealed that adducts formed by HN-2 and HN-3 are specific to these agents, as metabolized therapeutic drugs (chlorambucil, cyclophosphamide, and melphalan) did not form the same adducts on lysine or histidine residues as the previously identified adducts formed by HN-2 and HN-3. Results obtained from the model peptide and full protein work were enhanced by comparing experimental data to theoretical calculations for adduct formation, providing further confirmatory data. This project was successful in identifying and characterizing biomarkers of exposure to HN-2 and HN-3 that are specific and stable and which have the potential to be used for the forensic determination of exposure to these dangerous agents.

Reactions of singlet oxygen and singlet oxygen mimics with dicyclopropylethylenes: comparison of reactivity and mechanisms

An extensive study of the reaction pathways of 1,1- dicyclopropyl ethylene, cis- and trans- 1,2-dicyclopropylethylenes has been undertaken with different electrophiles 4-methyl-1,2,4- triazoline-3,5-dione (MTAD), tetracyanoethylene (TCNE), and singlet oxygen (102). Comparison of reactivity and reaction mechanisms among the electrophiles is investigated. Singlet oxygen exhibits significantly lower reactivity compared to the other electrophiles. MTAD and TCNE react with dicyclopropylethylenes to produce predominantly "2+2" adducts and a small amount of the "ene" adducts. The "2+2" is the major product presumably because of the high activation energy leading to the highly strained "ene" products. Solvent trapping studies provide strong evidence of a "stepwise" mechanism, involving a zwitterionic or aziridinium imide as an intermediate from the study of the reactions products of dicyclopropylethylenes and MTAD.

Covalent Protein Adduction by Drugs of Abuse

Recreational abuse of the drugs cocaine, methamphetamine, and morphine continues to be prevalent in the United States of America and around the world. While numerous methods of detection exist for each drug, they are generally limited by the lifetime of the parent drug and its metabolites in the body. However, the covalent modification of endogenous proteins by these drugs of abuse may act as biomarkers of exposure and allow for extension of detection windows for these drugs beyond the lifetime of parent molecules or metabolites in the free fraction. Additionally, existence of covalently bound molecules arising from drug ingestion can offer insight into downstream toxicities associated with each of these drugs. This research investigated the metabolism of cocaine, methamphetamine, and morphine in common in vitro assay systems, specifically focusing on the generation of reactive intermediates and metabolites that have the potential to form covalent protein adducts. Results demonstrated the formation of covalent adduction products between biological cysteine thiols and reactive moieties on cocaine and morphine metabolites. Rigorous mass spectrometric analysis in conjunction with in vitro metabolic activation, pharmacogenetic reaction phenotyping, and computational modeling were utilized to characterize structures and mechanisms of formation for each resultant thiol adduction product. For cocaine, data collected demonstrated the formation of adduction products from a reactive arene epoxide intermediate, designating a novel metabolic pathway for cocaine. In the case of morphine, data expanded on known adduct-forming pathways using sensitive and selective analysis techniques, following the known reactive metabolite, morphinone, and a proposed novel metabolite, morphine quinone methide. Data collected in this study describe novel metabolic events for multiple important drugs of abuse, culminating in detection methods and mechanistic descriptors useful to both medical and forensic investigators when examining the toxicology associated with cocaine, methamphetamine, and morphine.