This work describes the application of spiroligomers as serine hydrolases mimetics. Through collaboration with Kendall Houk's group, for the first time in the Schafmeister lab, we demonstrate that "theozymes" can be successfully used as models to design highly functionalized spiroligomer constructs for organocatalysis. We demonstrate a structure-function relationship between the structure of a series of bi-functional and tri-functional spiroligomer based transesterification catalysts and their catalytic activity. First, we designed and synthesized a series of stereochemically and regiochemically diverse bi-functional spiroligozymes to identify the best arrangement of a pyridine as a general base catalyst and an alcohol nucleophile to accelerate attack on vinyl trifluoroacetate as an electrophile. The best bifunctional spiroligozyme reacts with vinyl trifluoroacetate to form an acyl-spiroligozyme conjugate 2.7x103-fold faster than the background reaction with benzyl alcohol. We then incorporated an additional urea functional group to activate the acyl-spiroligozyme intermediate through hydrogen bonds and enable acyl transfer to methanol. The best trifunctional spiroligozyme carries out multiple turnovers and acts as a transesterification catalyst with k1/kuncat of 2.2x103 and k2/kuncat of 1.3x102. Quantum mechanical calculations identified four transition states in the catalytic cycle and provided a detailed view of every stage of the transesterification reaction. With the aim of accelerating the k2, we sought to design better oxyanion holes that hold multiple hydrogen bonding groups in close proximity of the catalytic groups. A macrocyclic motif would be a good candidate to force the oxyanion hole arm to arrange hydrogen-bonding groups in a precise three-dimensional constellation for transition state stabilization. In Chapter 4, we introduce an in silico designed macrocyclic spiroligomer, which overlays well with catalytic active site of an inhibitor bound-esterase. Finally, we detail our effort to develop new methodologies for rapidly synthesizing spiroligomers on solid-support. This would allow us to efficiently permute their structures for diverse applications such as organocatalysts, host molecules, and biologically related applications such as inhibiting protein-protein interactions. / Chemistry

This thesis research presents the synthesis and first application of bis-amino acid-based spiroligomers towards the development of organocatalysis, from small molecules to moderate size spiroligomers, and to macromolecules. By synthesizing a toolbox of cyclic monomers called "bis-amino acids", the Schafmeister group has developed an approach to construct both small and macromolecules named "Spiroligomers". These molecules arrange catalytic functional groups in a shape-persistent and programmable backbone. Unlike proteins and small peptides, spiroligomers do not fold; rather, their polycyclic backbone structures are controlled by the sequence and stereochemistry of the component monomers. Firstly, we demonstrated a structure/catalytic activity relationship together with computational modeling that suggests that a specific hydrophobic interaction between the modified pro4 catalyst and the aldehyde substrate is responsible for an observed rate enhancement in the aldol reaction. For the moderate size molecules, several spiroligomer libraries were prepared through solid phase or solution phase synthesis and screened for either the alcohol kinetic resolution reaction or the aldol reaction. The poor activity and selectivity suggest that the scaffolds involved cannot create the necessary chiral environment for asymmetric catalysis. Finally, a synthetic method of macromolecules using cross metathesis coupling was developed and a series of tetra-functionalized macrocyclic spiroligomers were synthesized. Three of these macromolecules were examined as asymmetric catalysts in the aldol reaction and gave moderate activity and selectivity. The NMR analysis of these macromolecules indicates their dynamic nature. As the first application of bis-amino acid based macromolecules in organocatalysis area, although these catalysts only generated moderate activity and selectivity, they provided evidence that changing the configuration of one stereocenter of the fourteen available within these macromolecules can alter the selectivity. This synthetic methodology also provides an effective way to create more complicated pocket like spiroligomer macromolecules for the future applications in catalysis and molecular recognition. / Chemistry

Indiana University-Purdue University Indianapolis (IUPUI) / Canines can detect different diseases simply by smelling different biological sample types, including urine, breath and sweat. This has led researchers to try and discovery unique volatile organic compound (VOC) biomarkers. The power of VOC biomarkers lies in the fact that one day they may be able to be utilized for noninvasive, rapid and accurate diagnostics at a point of care using miniaturized biosensors. However, the identity of the specific VOC biomarkers must be demonstrated before designing and fabricating sensing systems. Through an extensive series of experiments, VOCs in urine are profiled by solid phase microextraction (SPME) coupled to gas chromatography-mass spectrometry (GC-MS) to identify biomarkers for breast cancer using murine models. The results from these experiments indicated that unique classes of urinary VOCs, primarily terpene/terpenoids and carbonyls, are potential biomarkers of breast cancer. Through implementing chemometric approaches, unique panels of VOCs were identified for breast cancer detection, identifying tumor location, determining the efficacy of dopaminergic antitumor treatments, and tracking cancer progression. Other diseases, including COVID-19 and hypoglycemia (low blood sugar) were also probed to identify volatile biomarkers present in breath samples. VOC biomarker identification is an important step toward developing portable gas sensors, but another hurdle that exists is that current sensors lack selectivity toward specific VOCs of interest. Furthermore, testing sensors for sensitivity and selectivity is an extensive process as VOCs must be tested individually because the sensors do not have modes of chromatographic separation or compound identification. Another set of experiments is presented to demonstrate that SPME fibers can be coated with materials, used to extract standard solutions of VOCs, and analyzed by GC-MS to determine the performance of various gas sensing layers. In the first of these experiments, polyetherimide (PEI) was coated onto a SPME fiber and compared to commercial polyacrylate (PAA) fibers. The second experiment tuned the extraction efficiency of polyvinylidene fluoride (PVDF) - carbon black (CB) composites and showed that they had higher sensitivity for urinary VOC extraction relative to a polydimethylsiloxane (PDMS) SPME fiber. These results demonstrate SPME GC-MS can rapidly characterize and tune the VOC adsorption capabilities of gas sensing layers.

Measuring lipid oxidation is useful as a means of monitoring oxidative stress, such as that induced by clinical conditions or environmental exposure. Characteristic volatile compounds, often with low threshold odors, are secondary products of lipid oxidation reactions. Metallic flavor in food and beverages has been linked with oxidation of lipids in the oral cavity. Breath, an emerging medium for analysis of internal condition, is one means of measuring the metal-induced lipid oxidation responsible for this flavor. This project analyzes the breath of human subjects, as well as lipid oxidation of in vitro samples to identify compounds responsible for producing metallic flavor, which result from the oxidation of lipids in the oral cavity. Because these analytes are found at extremely low (picomolar to nanomolar) concentrations, preconcentration of samples prior to gas chromatography-mass spectrometry analysis is crucial. This study utilizes both solid phase microextraction (SPME) and micromachined silicon micropreconcentrators to concentrate compounds in breath to optimize analysis. / Master of Science

The next generation of toxic chemicals and hazardous wastes from sophisticated chemical industries will demand the environmental agencies to employ biological methods over the conventional physical and chemical remediation methods. Over the past decade, natural metallo-enzymes have been identified to degrade some of the major chemical contaminants through electron transfer pathways. However, these natural enzymes are less stable in organic solvents and they are not effective for the degradation of toxic compounds such as polychlorinated biphenyls or dioxins. This thesis explores the use of protein design approaches to produce chemically and molecularly modified enzymes, which are highly stable, possess little substrate specificity, and have higher activity than the natural enzymes. The experiments presented in this thesis make use of solid phase synthesis and site-directed mutagenesis for the synthesis and production of these enzymes and popular chromatographic techniques for their purification. The partial characterization of these proteins revealed the essential structural features of these proteins, and their catalytic activity was demonstrated by the use of peroxidase assays. / Master of Science

Sweet cream liquid buttermilk and skimmed milk ingredients were heat processed and/or exposed to fluorescent light to determine changes in potential flavor compounds. Solid phase microextraction-gas chromatography/flame ionization detection was used to analyze the concentrations of the volatile compounds (2-butanone, 2-pentanone, acetaldehyde, diacetyl, hexanal, methyl sulfide) found in the two components. Pasteurized unoxidized skimmed milk had measurable levels of 2-butanone, acetaldehyde, and diacetyl. Pasteurization of skimmed milk increased concentration of 2-pentanone and methyl sulfide to measurable levels. However only 2-butanone and acetaldehyde were detectable in oxidized skimmed milk. All liquid buttermilk ingredient treatments had measurable concentrations of 2-butanone, 2-pentanone, and acetaldehyde. Pasteurization of unoxidized liquid buttermilk increased the concentration of diacetyl and hexanal to measurable levels whereas oxidized buttermilk, both pasteurized and unpasteurized, had measurable levels of hexanal and methyl sulfide. Nonfat (.3%) dairy beverages were formulated using the same components to determine if the volatiles in liquid buttermilk might enhance the flavor of nonfat milk. Triangle tests and hedonic tests were performed on the nonfat formulations to determine their overall difference and overall acceptance. There was no significant difference between formulations. / Master of Science

Yes / The duocarmycins are potent antitumour agents with potential in the development of antibody drug conjugates (ADCs) as well as being clinical candidates in their own right. In this paper, we describe the synthesis of a duocarmycin monomer (DSA) that is suitably protected for utilisation in solid phase synthesis. The synthesis was performed on a large scale and the resulting racemic protected Fmoc-DSA subunit was separated by supercritical fluid chromatography (SFC) into the single enantiomers. Application to solid phase synthesis methodology gave a series of monomeric and extended duocarmycin analogues with amino acid substituents. The DNA sequence selectivity was similar to previous reports for both the monomeric and extended compounds. The substitution at the C-terminus of the duocarmycin caused a decrease in antiproliferative activity for all of the compounds studied. An extended compound containing an alanine at the C-terminus was converted to the primary amide or to an extended structure containing a terminal tertiary amine but this had no beneficial effects on biological activity. / MJS was funded by Novartis and UEA. We thank the EPSRC Mass Spectrometry Service, Swansea. We thank Richard Robinson and Julia Hatto at Novartis for help in the large scale synthesis.

No description available.

Separation technologies have occupied a central role in the current practices of analytical methods used for drug analysis today. As the emphasis in contemporary drug analysis shifts towards ultra-trace concentrations, the contribution from unwanted matrix interferences takes on greater significance. In order to single out a trace substance with confidence from a rapidly expanding list of drug compounds (and their metabolites) in real complex specimens, analytical technologies must evolve to keep up with such trends. Today, the task of unambiguous identification in forensic toxicology still relies heavily upon chromatographic methods based on mass spectrometric detection, in particular GC-MS in electron ionisation (EI) mode. Although the combined informing power of (EI) GC-MS has served faithfully in a myriad of drug application studies to date, we may ask if (EI) GC-MS will remain competitive in meeting the impending needs of ultra-trace drug analysis in the fut ure? To what extent of reliability can sample clean-up strategies be used in ultra-trace analysis without risking the loss of important analytes of interest? The increasing use of tandem mass spectrometry with one-dimensional (1D) chromatographic techniques (e.g. GC-MS/MS) at its simplest, considers that single-column chromatographic analysis with mass spectrometry alone is not sufficient in providing unambiguous confirmation of the identity of any given peak, particularly when there are peak-overlap. Where the mass spectra of the individual overlapping peaks are highly similar, confounding interpretation of their identities may arise. By introducing an additional resolution element in the chromatographic domain of a 1D chromatographic system, the informing power of the analytical system can also be effectively raised by the boost in resolving power from two chromatographic elements. Thus this thesis sets out to address the analytical challenges of modern drug analysis through the application of high resolut ion comprehensive two-dimensional gas chromatography (GC„eGC) to a series of representative drug studies of relevance to forensic sciences.

D'entre els defectes organolèptics associats al vi, en destaca l'anomenat "gust de suro" habitualment vinculat a la presència de cloroanisoles, els quals són productes de l'activitat microbiana formats a partir dels corresponents clorofenols. La present tesi doctoral recull, en primer lloc, metodologies analítiques adreçades principalment a la determinació dels compostos clorofenòlics (2,4,6-triclorofenol, 2,3,4,6-tetraclorofenol i pentaclorofenol) en el control de qualitat dels taps suro, emprant dissolucions hidroalcohòliques com a medi de maceració o d'extracció i utilitzant les tècniques d'extracció en fase sòlida (SPE) i microextracció en fase sòlida (SPME) acoblades a la cromatografia de gasos (GC).En segon lloc, per tal de dur a terme l'anàlisi de cloroanisoles juntament amb els seus precursors en matrius de suro s'ha avaluat un mètode basat en l'extracció amb dissolvent orgànic, el qual ha estat aplicat per a l'estudi de diferents sistemes d'eliminació d'aquests anàlits en la matriu citada. En darrer lloc, s'han proposat metodologies per l'anàlisi de mostres de vi, en les quals d'una banda s'han determinat els compostos clorofenòlics utilitzant la SPME i de l'altra el 2,4,6-tricloroanisole i el 2,4,6-tribromoanisole mitjançant l'acoblament de la SPE i la injecció de grans volums (LVI) en el sistema cromatogràfic. / The organoleptic defect known as cork taint is associated with a musty or mouldy aroma in wine. Chloroanisoles, which are produced through a process of detoxification by fungal methylation of chlorophenolic compounds, are considered to be the main contributory substances. In the present work, analytical methods for the determination of chlorophenols (2,4,6-trichlorophenol, 2,3,4,6-tetrachloropheol and pentachlorophenol) in cork matrices using the solid-phase extraction (SPE) and the solid-phase microextraction (SPME) as preconcentration techniques were evaluated. Therefore, hydroalcoholic solutions have been proposed as extractant solvent in order to test the quality of cork stoppers.Furthermore, a methodology based on extraction with an organic solvent for the simultaneous determination of chloroanisoles and chlorophenols in cork matrices was proposed. This methodology was applied with the aim of checking the efficiency of several washing treatments to remove the target analytes in naturally contaminated cork samples.Finally, different procedures have been developed for the analysis of wine samples. On one hand, chlorophenols were quantified by employing a SPME method. On the other hand, a reliable SPE method coupled to large volume injection was proposed for the determination of both 2,4,6-trichloroanisole and 2,4,6-tribromoanisole.