Work was undertaken to examine the antibacterial mechanism of allyl isothiocyanate (AITC), as well as explore the hydrolysis of sinigrin by bacteria and sinigrin metabolism in Escherichia coli O157:H7. Glucosinolates are found in cruciferous plants and react with the plant enzyme myrosinase to form antimicrobial compounds called isothiocyanates (ITCs). The mechanism of bactericidal action of ITCs is not well understood. Inhibition experiments against 12 bacteria including foodborne pathogens and starter cultures indicated that AITC exhibited a minimum inhibitory concentration (MIC) profile most similar to antibiotics ciprofloxacin and polymyxin B. Similarities in mechanisms of action between groups were identified. The sinigrin degradation capacity of a range of bacteria was examined. The effect of glucose in combination with other sugars on the sinigrin degradation capacity of E. coli O157:H7 was assessed. No statistically significant differences in sinigrin utilisation were observed. Results indicated that the metabolism of sinigrin was simultaneous with glucose consumption. / February 2016

The oncogenic protein Bcl-2 is overexpressed in many cancers and prevents cells from undergoing apoptosis in response to traditional chemotherapeutic agents. Recent research has focussed on the development of novel agents that can disrupt the function of Bcl-2 and trigger apoptosis in cancer cells. The isothiocyanates are a class of naturally-occurring phytochemical with potential for development as both chemopreventive and chemotherapeutic agents. This thesis investigated the ability of the isothiocyanates to induce apoptosis in cells that overexpressed Bcl-2. Initially, phenethyl isothiocyanate was shown to be cytotoxic to the Jurkat Tlymphoma cell line with an LD50 of 7.4 µM. Bcl-2 expression had little protective effect, and even greater than 50-fold overexpression only increased the LD50 to 15.1 µM. Morphological and biochemical assays indicated that death still occurred by apoptosis despite overexpression of Bcl-2. A variety of other isothiocyanates were also screened for cytotoxic activity. While the isothiocyanate moiety was crucial for induction of apoptosis, the chemistry of the side chain attached to the isothiocyanate moiety also profoundly influenced the ability of an isothiocyanate to kill Bcl-2 overexpressing cells. The aromatic isothiocyanates were generally far more cytotoxic than aliphatic isothiocyanates. However, within the aromatic isothiocyanates tested in this study the length of the carbon linker group, between the phenyl ring and the isothiocyanate moiety, also influenced cytotoxic activity. Phenethyl isothiocyanate was identified as the most promising compound when targeting cells that overexpressed Bcl-2. Given that minor structural alterations significantly altered cytotoxic activity it is hypothesised that specific interactions with cellular targets may mediate induction of apoptosis by the isothiocyanates. Finally, using a sensitive proteomic technique to label oxidised thiol proteins a preliminary investigation of the targets of the isothiocyanates was performed. A number of thiol proteins were selectively modified following exposure to phenethyl isothiocyanate. One thiol protein that consistently changed was identified as mitochondrial peroxiredoxin-3. Changes to the oxidation state of peroxiredoxin-3 occurred well before activation of apoptosis and may play a role in mediating induction of apoptosis in cells that overexpress Bcl-2. The results of this thesis have provided a platform to permit further investigation of the chemotherapeutic potential of the isothiocyanates and investigation of the mechanisms that allow the isothiocyanates to induce apoptosis in cells that overexpress the oncogene Bcl-2. In the future, the identification of primary targets of the isothiocyanates may aid the design and testing of novel anticancer drugs, and it will also provide novel insight into the regulation of apoptosis.

München, Techn. Universiẗat, Diss., 2006.

The oncogenic protein Bcl-2 is overexpressed in many cancers and prevents cells from undergoing apoptosis in response to traditional chemotherapeutic agents. Recent research has focussed on the development of novel agents that can disrupt the function of Bcl-2 and trigger apoptosis in cancer cells. The isothiocyanates are a class of naturally-occurring phytochemical with potential for development as both chemopreventive and chemotherapeutic agents. This thesis investigated the ability of the isothiocyanates to induce apoptosis in cells that overexpressed Bcl-2. Initially, phenethyl isothiocyanate was shown to be cytotoxic to the Jurkat Tlymphoma cell line with an LD50 of 7.4 µM. Bcl-2 expression had little protective effect, and even greater than 50-fold overexpression only increased the LD50 to 15.1 µM. Morphological and biochemical assays indicated that death still occurred by apoptosis despite overexpression of Bcl-2. A variety of other isothiocyanates were also screened for cytotoxic activity. While the isothiocyanate moiety was crucial for induction of apoptosis, the chemistry of the side chain attached to the isothiocyanate moiety also profoundly influenced the ability of an isothiocyanate to kill Bcl-2 overexpressing cells. The aromatic isothiocyanates were generally far more cytotoxic than aliphatic isothiocyanates. However, within the aromatic isothiocyanates tested in this study the length of the carbon linker group, between the phenyl ring and the isothiocyanate moiety, also influenced cytotoxic activity. Phenethyl isothiocyanate was identified as the most promising compound when targeting cells that overexpressed Bcl-2. Given that minor structural alterations significantly altered cytotoxic activity it is hypothesised that specific interactions with cellular targets may mediate induction of apoptosis by the isothiocyanates. Finally, using a sensitive proteomic technique to label oxidised thiol proteins a preliminary investigation of the targets of the isothiocyanates was performed. A number of thiol proteins were selectively modified following exposure to phenethyl isothiocyanate. One thiol protein that consistently changed was identified as mitochondrial peroxiredoxin-3. Changes to the oxidation state of peroxiredoxin-3 occurred well before activation of apoptosis and may play a role in mediating induction of apoptosis in cells that overexpress Bcl-2. The results of this thesis have provided a platform to permit further investigation of the chemotherapeutic potential of the isothiocyanates and investigation of the mechanisms that allow the isothiocyanates to induce apoptosis in cells that overexpress the oncogene Bcl-2. In the future, the identification of primary targets of the isothiocyanates may aid the design and testing of novel anticancer drugs, and it will also provide novel insight into the regulation of apoptosis.

In Brassicaceae werden bei einer Gewebszerstörung unreaktive Glukosinolate durch das Enzym Myrosinase hydrolysiert. Es entstehen reaktive Substanzen wie Isothiocyanate (ITCs). Da diese Reaktion sehr schnell erfolgt wird sie auch als Senföl-Glukosid-Bombe bezeichnet. In Arabidopsis thaliana erfolgt nach Verwundung und Pathogeninfektion eine massive Akkumulation des ITCs Sulforaphan (SF), welches eine reaktive elektophile Spezies (RES) darstellt. Zu der Gruppe der RES zählen auch einige Oxylipine mit einer α,β-ungesättigten Carbonylgruppen wie 12-oxo-Phytodiensäure (OPDA) oder Phytoprostan A1 (PPA1). Die Fähigkeit der kovalenten Modifikation von Peptiden und Proteinen gilt als essentiell sowohl für die toxischen als auch die Gen-induzierenden Eigenschaften der RES. Neben ihrer Reaktivität spielt auch die Lipophilie eine Rolle für die Fähigkeit über Membranen zu diffundieren und unspezifisch an Proteine zu binden. Die in der vorliegenden Arbeit durchgeführten Transkriptomanalysen an Arabidopsis-Keimlingen mit sub-toxischen Konzentrationen von SF, Benzylisothiocyanat (BITC) und dem Oxylipin Prostaglandin A1 (PGA1) zeigten, dass strukturell sehr verschiedene RES einen gemeinsamen Satz von 55 Genen induzieren. Unter diesen befanden sich verschiedene Hitzeschock-, Stressassoziierte- und Detoxifizierungsgene. Diese Ergebnisse deuten darauf hin, dass die Aktivierung über eine Muster-spezifische Erkennung der RES erfolgt. Als einen möglichen Mechanismus der RES-vermittelten Geninduktion wird die Regulation durch die Veränderung des zellulären Redox-Potentials als Folge kovalenter Modifikation von GSH durch RES diskutiert. Die Untersuchung der GSH-Gehalte sowie des Redox-Potential nach Behandlung mit sub-toxischen RES-Konzentrationen in Arabidopsis-Keimlingen zeigte jedoch unter den getesteten Bedingungen keine Veränderung. Neben dem Erkennungs- und Signaltransduktionsmechanismus ist auch die biologische Bedeutung von RES für die Vermittlung einer Stresstoleranz noch weitgehend unklar. Durch die Untersuchung der Genexpression in Arabidopsis-Pflanzen nach Verwundung konnte gezeigt werden, dass eine wundinduzierte Akkumulation von SF zur Induktion einiger Gene der Hitzeschockreaktion (HSR) im Wildtyp, jedoch nicht in der myrosinase-defiziten tgg1tgg2-Mutante führte. Auch in der Transkriptomanalyse war nach RES-Gabe ebenfalls eine starke Induktion hitze-responsiver Gene, deren Regulation über den Masterregulator dem Hitzeschock-TF A1 vermittelt wird, zu beobachten. Besonders die Induktion der HSPs, welche als Chaperone fungieren und damit Thiolgruppen von Proteinen vor Modifikation schützen können, haben vermutlich bei chemischer Intoxikation protektive Eigenschaften für die Zellen. Tatsächlich zeigte sich unter den gewählten Bedingungen die hsfa1a,b,d,e-Mutante empfindlicher gegenüber ITCs als der Wildtyp. Die Fähigkeit, eine HSR ausbilden zu können, scheint in Arabidopsis bei chemischer Intoxikation eine bedeutende Rolle zu spielen. Eine Vorbehandlung mit RES wie SF, BITC oder dem HSP90-Inhibitor Radicicol in Arabidopsis-Keimlingen konnte eine Schutzwirkung vor chemischer Intoxikation vermitteln. Dies erfolgte jedoch nicht nach Behandlung mit moderater Hitze (zwei Stunden, 37 °C). Somit scheint die HSR alleine nicht ausreichend für den Aufbau eines effektiven Schutzes vor BITC-Intoxikation zu sein. Als metabolische Antwort von Arabidopsis-Keimlingen auf Intoxikation mit RES konnte eine konzentrationsabhängige Senkung der maximalen Quantenausbeute am Photosystem II (PSII), sowie gleichzeitig eine Akkumulation an TAG-Spezies beobachtet werden. Diese metabolische Reaktion ist in der Literatur bereits als Schutz gegen Hitzestress beschrieben. Die Bedeutung der TAG-Akkumulation nach chemischem ITC-Stress ist noch unklar. / In Brassicaceae, unreactive glucosinolates are hydrolyzed by the enzyme myrosinase during tissue destruction. Reactive substances such as isothiocyanates (ITCs) are formed. Since this reaction takes place very quickly, it is also called mustard oil bomb. In Arabidopsis thaliana, after wounding and pathogen infection, a massive accumulation of the ITC Sulforaphan (SF) occurs, which is a reactive electophilic species (RES). The group of RES also includes some oxylipins with a α,β-unsaturated carbonyl group such as 12-oxo-phytodienoic acid (OPDA) or phytoprotane A1 (PPA1). The ability to covalently modify peptides and proteins is considered essential for the toxic and gene inducing properties of RES. In addition to their reactivity, lipophilia also plays a role in the ability to diffuse across membranes and bind unspecifically to proteins. The transcriptome analyses performed on Arabidopsis-seedlings with sub-toxic concentrations of SF, benzylisothiocyanate (BITC) and oxylipin prostaglandin A1 (PGA1) showed that structurally very different RES induce a common set of 55 genes. Among the induced genes were several heat shock, stress associated and detoxification genes. These observations suggest that activation is via pattern-specific recognition of RES. As a possible mechanism of RES-mediated gene induction, regulation by alteration of the cellular redox potential as result of covalent modification of GSH by RES is discussed. However, the study of GSH levels and redox potential after treatment with sub-toxic RES concentrations in Arabidopsis-seedlings showed no change under the tested conditions. In addition to the recognition and signal transduction mechanism for RES, the biological significance of RES for the mediation of stress tolerance is still largely unclear. By studying the gene expression of Arabidopsis-plants after wounding, it was shown that wound induced accumulation of SF led to the induction of some genes of heat shock response (HSR) in the wild type, but not in the myrosinase-deficient tgg1tgg2-mutant. Also in transcriptome analysis a strong induction of heat-responsive genes could be observed after RES administration, whose regulation is mediated by the master regulator of the heatshock-TF A1 (HSFA1). In particular, the induction of HSPs, which act as chaperones and can thus protect thiol groups of proteins from modification, presumably have protective properties for the cells during chemical intoxication. In fact, under the conditions tested, the hsfa1a,b,d,e-mutants were more sensitive to ITCs than the wild type. The ability to form HSR seems to play an important role in chemical intoxication in Arabidopsis. Pretreatment with RES such as SF, BITC or the HSP90 inhibitor radicicol in Arabidopsis-seedlings could mediate a protective effect against chemical intoxication. However, this was not done after treatment with moderate heat (two hours, 37 °C). Thus, HSR alone does not seem to be sufficient to provide effective protection against BITC intoxication. As a metabolic response of Arabidopsis-seedlings to intoxication with RES, a concentration-dependent reduction of the maximum quantum yield at the photosystem II (PSII), as well as an accumulation of TAG species could be observed. This metabolic reaction is already described in the literature as protection against heat stress. The significance of TAG accumulation after chemical stress in the form of ITC intoxication is still unclear.

In this work, the [3,3] sigmatropic rearrangement of different substituted propargyl thiocyanates and double [3,3] sigmatropic rearrangement of enynyl isothiocyanates either by flash vacuum pyrolysis or by thermolysis in solution are studied. Additionally, the intramolecular reactions of the resulting allenyl isothiocyanates are studied, and the reaction mechanisms for the generation of the final products, such as [1,5] sigmatropic migrations or electrocyclic ring closures, are explained. These highly reactive allenyl isothiocyanates are used as appropriate electrophilic precursors for the preparation of novel examples of thiazoles substituted at C-2 position using different types of nucleophiles. For the formation of these substituted thiazoles, the necessary nucleophilicity as well as the regioselectivity, the stereoselectivity, and the reaction mechanisms are investigated. / In der vorliegenden Arbeit sind die [3,3]-sigmatrope Umlagerung von verschiedenen Propargylthiocyanaten und die doppelte [3,3]-sigmatrope Umlagerung von Eninylisothiocyanaten entweder durch Blitzvakuumthermolyse oder Thermolyse in Lösung untersucht worden. Zusätzlich wurden die intramolekularen Reaktionen der resultierten Allenylisothiocyanate studiert. Außerdem sind die Reaktionsmechanismen zur Bildung der Thermolyseprodukte wie z.B. die [1,5]-sigmatropen Umlagerungen und die elektrocyclischen Ringschlüsse erklärt. Die hochreaktiven Allenylisothiocyanate sind als geeignet elektrophile Vorläufer zur Synthese von neuen Thiazolen verwendbar, die an der C-2 Position substituiert sind. Dabei kommen verschiedene Nucleophile zum Ansatz. Für die Bildung dieser substituierten Thiazole sind die Regioselektivität, Stereoselektivität, Reaktionsmechanismen und der Bereich der einsetzbaren Nucleophile untersucht worden.

Les glucosinolates (GLs) sont des métabolites secondaires très présents dans la famille des Brassicaceae, une famille de végétaux connus dans l’alimentation humaine pour leurs propriétés gustatives et aussi pour leurs effets chimio-préventifs. La myrosinase (E.C. 3.2.1.147) est la seule enzyme capable d’hydrolyser les GLs. Nous nous sommes intéressés aux produits obtenus lors de cette dégradation - les isothiocyanates (ITCs) mais aussi à la fonction thiohydroximate, et plus particulièrement à la synthèse de nitrones thiofonctionnalisées. La synthèse d’analogues d’ITC ainsi que de certains de leurs thio-conjugués a conduit à une large gamme de molécules permettant de cerner les implications chimio-préventives et les potentiels en bio-activité (notamment contre Plasmodium falciparum). Partant d’observations analytiques relatives à certains GLs comme la glucoraphénine, il a été découvert qu’un thiohydroximate pouvait présenter un caractère nucléophile comparable à celui d’une oxime dans un processus cyclisant du type Grigg. Nous avons dès lors cherché à développer de nouvelles voies d’accès à un système fonctionnel très peu décrit dans la littérature : les N-oxydes de thioimidate (OTI). Une étude de réactivité d’un OTI-modèle a révélé des comportements originaux, en particulier dans des réactions de couplage pallado-catalysées qui ouvrent un accès à des nitrones cycliques originales et peu aisées à préparer par des voies plus classiques. / Glucosinolates (GLs) are widespread secondary metabolites in Brassicaceae, a family of vegetables known in human feeding for their tastes and also for their chemo-prevention effects. Myrosinase (E.C. 3.2.1.147) is the only enzyme able to hydrolyze GLs. We have been interested in products acquired during this degradation - isothiocyanates (ITCs) but also in the thiohydroximate function, and more particularly in the synthesis of thiofunctionalized nitrones. The synthesis of ITC analogues as well as some of their thio-conjugates has led to a broad range of molecules allowing to surround chemo-preventive involvements and potentials in bio-activity (namely against Plasmodium falciparum). Starting from analytical observations related to some GLs such as glucoraphenin, it has been disclosed that a thiohydroximate could display a nucleophilic character comparable to that of an oxime in a Grigg-type cyclic process. We have developed new pathways to a scarcely described functional system : thioimidate N-oxides (OTI). Reactivity studies on a model-OTI have revealed an atypical behaviour, notably in palladium-catalyzed coupling reactions : those open the way to original cyclic nitrones which are not easily available via standard methods.

A variety of heterocyclic alcohols were coupled with silyl ketene acetals and other [pi]-nucleophiles in the presence of trimethylsilyl trifluoromethanesulfonate to provide an array of substituted [beta]-heteroaryl propionates, including those with contiguous quaternary centers. This reaction also proceeds with high diastereoselectivity when the [pi]-nucleophile bears a chiral auxiliary. The formal synthesis of N-methylwelwitindolinone C isothiocyanate, a densely functionalized alkaloid with the ability to reverse multiple drug resistance, was completed featuring several key transformations. The first key transformation consisted of coupling a heterocyclic alcohol with a silyl ketene acetal to give a highly functionalized intermediate. Next, a palladium catalyzed enolate arylation followed by an intramolecular allylic alkylation successfully constructed the bicyclo(4.3.1)decane backbone of the N-methylwelwitindolinone C isothiocyanate. / text

Ready-to-eat meat products such as dry-fermented sausages have been associated with foodborne outbreaks despite the multiple hurdles used in the manufacturing process to prevent growth of pathogens. As a result, new strategies such as natural products with antimicrobial activity are being used to control pathogens of importance like Escherichia coli O157:H7. This study investigated how different concentrations and sources of mustard can influence its antimicrobial activity against E. coli O157:H7 in dry-fermented sausage, as well as the contribution of residual myrosinase enzyme in mustard to this process. The genetic basis for the degradation of mustard glucosinolate by E. coli O157:H7, which is associated with the antimicrobial action of mustard, was also characterized. The ability of E. coli O157:H7 to withstand inhibitory allyl isothiocyanate (AITC) concentrations and the role of the two-component BaeSR system as a defense mechanism against AITC was also investigated. Results showed that 4% (w/w) deodorized yellow mustard powder was effective to control E. coli O157:H7 in dry-fermented sausage at 28 d. The presence of endogenous plant myrosinase in the mustard powder or meal enhanced E. coli O157:H7 reduction rates. Fully-deodorized, deoiled, yellow mustard meal as low as 2% (w/w) containing either 0.1% or 0.2% of residual plant myrosinase achieved the same results as 4% (w/w) mustard powder also containing similar residual myrosinase. Regardless of the type of mustard, the antimicrobial activity of yellow mustard derivatives were more pronounced than those of Oriental mustard. The initial genetic assessment through in silico analysis found similarity between plant myrosinase and enzymes encoded by genes (bglA, ascB, and chbF) from β-glucosidase families in E. coli O157:H7 strains. After disruption of these genes using lambda-red replacement, single (∆bglA, ∆ascB, ∆chbF) and double (∆bglAascB, ∆chbFascB, ∆chbFbglA) mutant strains were created and assessed for glucosinolate degradation. The comparison of the gene expression profiles and changes in the extent of sinigrin degradation by different mutants suggested that ascB have a prominent role in the degradation of this β-glucoside by E. coli O157:H7. E. coli O157:H7 did not develop resistance to AITC, the essential oil formed from sinigrin degradation that is responsible for the antimicrobial activity of Oriental mustard.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22494号 / 農博第2398号 / 新制||農||1076(附属図書館) / 学位論文||R2||N5274(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 小川 順, 教授 栗原 達夫, 教授 加納 健司 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM