Nitrogen-containing heterocycles are of vital importance for the pharmaceutical and agrochemical industries. The Beauchemin group has been studying rare, amphoteric nitrogen-substituted isocyanates over the past years, and showed that their [3+2] alkene cycloaddition and cascade reactions provide access to a variety of NNCO containing heterocyclic compounds. This triggered interest into the reactivity of the parent N-isothiocyanates, which are also rare, and led to the discovery of aminothiocarbonylation reactions. The products formed are azomethine imines which contain a cyclic -aminothiocarbonyl motif, thus providing a cycloaddition route to these useful dipoles from simple starting materials. Such aminothiocarbonylation reactions were developed with both alkenes and imines as substrates.Apart from cyclic azomethine imine formations, efforts have also been made toward forming the acyclic azomethine imines as intermediates. These intermediates undergo [3+2] cycloaddition to form thiocarbamoyl pyrazolidine derivatives, and a preliminary substrate scope for this new intermolecular reactivity is presented. Other preliminary results include an unexpected Chugaev type reactivity. Collectively, these results show that N-isothiocyanates hold significant potential for the development of new reactivity.

Glutathione transferases (GSTs) are good candidates for investigations of enzyme evolution, due to their broad substrate specificities and structural homology. The primary role of GSTs is to act as phase II detoxifying enzymes protecting the cell from toxic compounds of both endo- and exogenous origins. The detoxification is conducted via conjugation with glutathione (GSH), which facilitates their removal from the body. The work presented in this thesis has supported a theory for enzyme evolution when the multiple pathway to novel functions can been seen to involve a “generalist” state from which “specialist” states with a new activities can evolve. The generalist has broader specificity and lower activity than the specialist. The term quasi-species is used for a group or cluster of enzyme variants with similar functional properties, and this entity has been suggested as the fittest group for further evolution. This is based on studies of the evolution of new GST variants in two generation. Three diverging clusters or quasi-species, with diverging substrate selectivity, were identified from a GST M1/M2 library, by using directed evolution (family DNA shuffling), multiple substrate screening and multivariate statistics as tools. One of the clusters was M1-like and the other was M2-like, both functionally and structurally. The third quasi-species diverged orthogonally from the parent-like distributions. Its functional character can be referred to as a “generalist” as it had lower activities with most of the substrates assayed except for epoxy-3-(4-nitrophenoxy)-propane (EPNP) and p-nitrophenyl acetate (pNPA). Another round of family DNA shuffling was made with selected variants from the “generalist” quasi-species. From the second generation three quasi-species emerged with diverging functions and sequences. The major cluster contained enzyme variants that represented a direct propagation of the generalists. Diverging from the generalists was a cluster with high specificity with isothiocyanates (ITCs). Increased ITC specificity and decreased epoxide specificity was observed among the novel variants (specialists). The change in functional properties was attributed to a Tyr116His substitution in the active site. These results demonstrate the usefulness of multivariate analysis in the quest for novel enzyme quasi-species in a multi-substrate space, and how minimal changes in the active site can generate distinctive functional properties. An application of our method could be identification of enzyme quasi-species that have lost their sensitivity with alternative inhibitors.

Thesis (Ph. D.)--University of Wisconsin--Madison, 2007. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 2007. / Includes bibliographical references. Also available on the Internet.

No description available.

Depuis longtemps les réactifs isothiocyanates (ITCs) sont largement utilisés dans le domaine de la bioconjugaison. Ces électrophiles forts réagissent avec les cystéines et les lysines des protéines pour former une liaison stable. Cette réactivité click permet de réaliser des marquages sélectifs ainsi que des fonctionnalisations de protéines. Cependant, les ITCs ne sont pas faciles à synthétiser et à isoler et leur stabilité ne permet pas une conservation optimale.Le but de ce projet est de développer le système enzymatique myrosinase-glucosinolate (MG) comme outil de conjugaison capable de former un ITC in situ. Le tandem MG est un mécanisme de défense des plantes de l’ordre des Brassicales bien connu. Dans ce système biochimique, la myrosinase opère comme thioglucosidase, en hydrolysant les glucosinolates (GLs), pour générer des ITCs. L’avantage de ce tandem enzymatique est de produire les ITCs à partir de précurseurs solubles dans l’eau, non toxiques, sous conditions douces.Afin d’explorer cet outil enzymatique, deux types de GLs non naturels ont été conçus. En raison de l’importance de l’interaction lectine-mannose dans les mécanismes d’adhésion bactérienne, nous avons conçu une petite librairie de GLs intégrant un mannoside. De cette façon il est possible d’étudier des lectines bactérienne (FimH). Le second type de glucosinolate est caractérisé par une deuxième fonction chimique, permettant de réaliser des réactions orthogonales.Le système MG a été évalué dans plusieurs approches de bioconjugaison telles que le marquage sélectif d’une lectine, la synthèse de néoglycoprotéines et la fonctionnalisation de nanoparticules. / Since many decades, Isothiocyanate (ITCs) reagents are widely used in bioconjugation approaches to create a stable bond onto the lysin and cysteine residues of proteins and peptides. Thanks to this click reaction it is possible to achieve a selective ligation or a high functionalization of proteins. On the others hand, isothiocyanates are not easy to prepare, to isolate, to store and most of the time insoluble in water.The aim of this work is to explore the myrosinase-glucosinolate (MG) enzymatic tandem as a ligation system, in order to release ITCs in-situ. The MG tandem is a well-known mechanism of defense in plants of the order Brassicales. In this biochemical system, myrosinase acts as a thioglucosidase, hydrolyzing glucosinolates (GLs) to liberate transient species that spontaneously form ITCs. The advantage of this enzymatic tandem is to generate ITCs from a stable non-toxic GLs precursor, soluble in water, using mild conditions.In order to develop this enzymatic tool, two kind of unnatural GLs were synthetized. Due to the important role of the mannoside-lectins interaction in the bacterial adhesion mechanism, at first, we designed a small library of GL bearing mannosides, in order to target and study the bacterial lectin FimH. The second kind of GL possess a chemical function allowing to study orthogonal reactions. After, the ability of the myrosinase to hydrolyze those unnatural GLs was investigated, as well their chemical reactivity.Then, the performances of the MG system was studied in different approaches of bioconjugation, such as: the synthesis of neoglycoproteins (NGPs), the site selective labeling of a lectin and the functionalization of gold nanoparticles. The feasibility of these strategies confirmed that the MG system can be used as an enzymatic tool in some bioconjugation approaches.

Glutathione transferases (GSTs, EC.2.5.1.18) are multifunctional enzymes that are universally distributed in all cellular life forms. They play important roles in metabolism and detoxication of endogenously produced toxic compounds and xenobiotics. GSTs have gained considerable interest over the years for biomedical and biotechnological applications due to their involvement in the conjugation of glutathione (GSH) to a vast array of chemical species. Additionally, the emergence of non-detoxifying functions of GSTs has further increased their biological significance. The present work encompasses four scientific studies aimed at investigating human as well as fruit fly Drosophila melanogaster GSTs. Paper I presents the immobilization of GSTs on nanoporous alumina membranes. Kinetic analyses with 1-chloro-2,4-dinitrobenzene followed by specificity screening with alternative substrates showed a good correlation between the data obtained from immobilized enzymes and the enzymes in solution. Furthermore, immobilization showed no adverse effects on the stability of the enzymes. Paper II presents inhibition studies of human hematopoietic prostaglandin D2 synthase (HPGDS), a promising therapeutic target for anti-allergic and anti-inflammatory drugs. Our screening results with an FDA-approved drug library revealed a number of effective inhibitors of HPGDS with IC50 values in the low micromolar range. Paper III concerns the toxicity of organic isothiocyanates (ITCs) that showed high catalytic activities with GSTE7 in vitro. The in vivo results showed that phenethyl isothiocyanate (PEITC) and allyl isothiocyanate in millimolar dietary concentrations conferred toxicity to the adult fruit flies leading to death or shortened life-span. The transgenic female flies overexpressing GSTE7 showed increased tolerance against PEITC toxicity compared to the wild-type. However, the effect was opposite in male flies overexpressing GSTE7 after one week exposure. Notably, the transgene enhanced the oviposition activity of flies with and without ITCs exposure. Paper IV highlights Drosophila GSTs as efficient catalysts of the environmental pollutant and explosive 2,4,6-trinitrotoluene and the related 2,4-dinitrotoluene degradation. This result suggests the potential of GST transgenes in plants for biotransformation and phytoremediation of these persistent environmental pollutants.

E. coli O157:H7 has been found to survive in dry sausages and cause disease. Isothiocyanates have been studied for their capacity to eliminate pathogens from foods and are attractive from the consumer perspective because of their natural origin. There is a need to better understand how isothiocyanates kill microorganisms and their behaviour in food matrices. It was found that glutathione and cysteine naturally present in meat can react with AIT, forming a conjugate with no or low bactericidal activity against an E. coli O157:H7. In addition, AIT presented higher anti-E. coli activity at lower pH values; therefore, it should be more efficient in acid foods. AIT was also found to inhibit the activity of thioredoxin reductase and acetate kinase; hence, enzymatic inhibition may represent a way in which AIT kills E. coli O157:H7. Mustard powder is used as a spice (active myrosinase) and/or binder (inactive myrosinase) in meat products. Both of these powders killed E. coli O157:H7 in dry fermented sausage. This was not expected since the powder lacking myrosinase is not able to produce isothiocyanates. Starter cultures and E. coli were found to consume significant amounts of glucosinolates. Pediococcus pentosaceus UM 121P and Staphylococcus carnosus UM 123M (higher myrosinase-like activity) were compared against P. pentosaceus UM 116P + S. carnosus UM 109M for their ability in reducing E. coli viability in dry sausage. Sausage batches containing powders of hot mustard, cold mustard, autoclaved mustard and no powder were prepared. Both pairs of starters yielded similar results. Reduction >5 log CFU/g of E. coli O157:H7 occurred after 31 d for hot powder and 38 d for cold powder; there was no reduction in the control. E. coli O157:H7 itself has greater effect on glucosinolate degradation than either pair of starters, which may be more important in determining its survival. Autoclaved powder caused >5 log CFU/g reduction after 18 d. This may be the result of synergistic/additive interaction among E. coli O157:H7 myrosinase-like activity, the presence of newly formed/released antimicrobials in the autoclaved powder and the multiple hurdles present in the dry sausage. Autoclaved mustard powder has potential as a novel food ingredient for the meat industry.

E. coli O157:H7 has been found to survive in dry sausages and cause disease. Isothiocyanates have been studied for their capacity to eliminate pathogens from foods and are attractive from the consumer perspective because of their natural origin. There is a need to better understand how isothiocyanates kill microorganisms and their behaviour in food matrices. It was found that glutathione and cysteine naturally present in meat can react with AIT, forming a conjugate with no or low bactericidal activity against an E. coli O157:H7. In addition, AIT presented higher anti-E. coli activity at lower pH values; therefore, it should be more efficient in acid foods. AIT was also found to inhibit the activity of thioredoxin reductase and acetate kinase; hence, enzymatic inhibition may represent a way in which AIT kills E. coli O157:H7. Mustard powder is used as a spice (active myrosinase) and/or binder (inactive myrosinase) in meat products. Both of these powders killed E. coli O157:H7 in dry fermented sausage. This was not expected since the powder lacking myrosinase is not able to produce isothiocyanates. Starter cultures and E. coli were found to consume significant amounts of glucosinolates. Pediococcus pentosaceus UM 121P and Staphylococcus carnosus UM 123M (higher myrosinase-like activity) were compared against P. pentosaceus UM 116P + S. carnosus UM 109M for their ability in reducing E. coli viability in dry sausage. Sausage batches containing powders of hot mustard, cold mustard, autoclaved mustard and no powder were prepared. Both pairs of starters yielded similar results. Reduction >5 log CFU/g of E. coli O157:H7 occurred after 31 d for hot powder and 38 d for cold powder; there was no reduction in the control. E. coli O157:H7 itself has greater effect on glucosinolate degradation than either pair of starters, which may be more important in determining its survival. Autoclaved powder caused >5 log CFU/g reduction after 18 d. This may be the result of synergistic/additive interaction among E. coli O157:H7 myrosinase-like activity, the presence of newly formed/released antimicrobials in the autoclaved powder and the multiple hurdles present in the dry sausage. Autoclaved mustard powder has potential as a novel food ingredient for the meat industry.

Nitrogen-containing heterocyclic compounds are very important to the pharmaceutical and agrochemical industries, among others. Over the past few years, the Beauchemin group has been exploring reactivity of N-substituted isocyanates and as part of this has developed a metal-free alkene aminocarbonylation process relying on imino-isocyanates to form azomethine imines. The azomethine imines formed are interesting since they contain a cyclic β-aminocarbonyl motif. Catalysis of this reaction using basic additives allowed milder reaction conditions with electron-rich C=C bonds such as enol ethers. Efforts have also been made towards the derivatization of these azomethine imines into useful products. It was discovered that upon reduction and aromatization of azomethine imines, pyrazolones could be obtained. This is providing a novel modular approach to these compounds, which have relevance in pharmaceuticals and agrochemicals. This reactivity was extended to include imino-isothiocyanates.