Cell density control of staphylococcal virulence mediated by an octapeptide pheromone.

Some bacterial pathogens elaborate and secrete virulence factors in response to environmental signals, others in response to a specific host product, and still others in response to no discernible cue. In this study, we have demonstrated that the synthesis of Staphylococcus aureus virulence factors is controlled by a density-sensing system that utilizes an octapeptide produced by the organism itself. The octapeptide activates expression of the agr locus, a global regulator of the virulence response. This response involves the reciprocal regulation of genes encoding surface proteins and those encoding secreted virulence factors. As cells enter the postexponential phase, surface protein genes are repressed by agr and secretory protein genes are subsequently activated. The intracellular agr effector is a regulatory RNA, RNAIII, whose transcription is activated by an agr-encoded signal transduction system for which the octapeptide is the ligand.

Peptide synthesis using unprotected peptides through orthogonal coupling methods.

We describe an approach to the synthesis of peptides from segments bearing no protecting groups through an orthogonal coupling method to capture the acyl segment as a thioester that then undergoes an intramolecular acyl transfer to the amine component with formation of a peptide bond. Two orthogonal coupling methods to give the covalent ester intermediate were achieved by either a thiol-thioester exchange mediated by a trialkylphosphine and an alkylthiol or a thioesterification by C alpha-thiocarboxylic acid reacting with a beta-bromo amino acid. With this approach, unprotected segments ranging from 4 to 37 residues were coupled to aqueous solution to give free peptides up to 54 residues long with high efficiency.

Involvement of the double-stranded-RNA-dependent kinase PKR in interferon expression and interferon-mediated antiviral activity.

The signaling mechanisms responsible for the induced expression of interferon (IFN) genes by viral infection or double-stranded RNA (dsRNA) are not well understood. Here we investigate the role of the interferon-induced dsRNA-dependent protein kinase PKR in the regulation of IFN induction. Biological activities attributed to PKR include regulating protein synthesis, mediating IFN actions, and functioning as a possible tumor suppressor. Since binding of dsRNA is required for its activation, PKR has been considered as a candidate signal transducer for regulating IFN expression. To examine this role of PKR, loss-of-function phenotypes in stable transformants of promonocytic U-937 cells were achieved by two different strategies, overexpression of an antisense PKR transcript or a dominant negative PKR mutant gene. Both types of PKR-deficient cells were more permissive for viral replication than the control U-937 cells. As the result of PKR loss, they also showed impaired induction of IFN-alpha and IFN-beta genes in response to several inducers--specifically, encephalomyocarditis virus, lipopolysaccharide, and phorbol 12-myristate 13-acetate. Interestingly, while IFN-alpha induction by dsRNA was impaired in PKR-deficient cells, IFN-beta induction remained intact. Loss of PKR function also resulted in decreased antiviral activity as elicited by IFN-alpha and, to a greater extent, by IFN-gamma. These results implicate PKR in the regulation of several antiviral activities.

Potentiation of epidermal growth factor receptor-mediated oncogenesis by c-Src: implications for the etiology of multiple human cancers.

c-Src is a nontransforming tyrosine kinase that participates in signaling events mediated by a variety of polypeptide growth factor receptors, including the epidermal growth factor receptor (EGFR). Overexpression and continual ligand stimulation of the EGFR results in morphological transformation of cells in vitro and tumor development in vivo. Elevated levels of c-Src and the EGFR are found in a variety of human malignancies, raising the question of whether c-Src can functionally cooperate with the EGFR during tumorigenesis. To address this issue, we generated c-Src/EGFR double overexpressors and compared their proliferative and biochemical characteristics to those of single overexpressors and control cells. We found that in cells expressing high levels of receptor, c-Src potentiated DNA synthesis, growth in soft agar, and tumor formation in nude mice. Growth potentiation was associated with the formation of a heterocomplex between c-Src and activated EGFR, the appearance of a distinct tyrosyl phosphorylation on the receptor, and an enhancement of receptor substrate phosphorylation. These findings indicate that c-Src is capable of potentiating receptor-mediated tumorigenesis and suggest that synergism between c-Src and the EGFR may contribute to a more aggressive phenotype in multiple human tumors.

Hypoxia-mediated induction of acidic/basic fibroblast growth factor and platelet-derived growth factor in mononuclear phagocytes stimulates growth of hypoxic endothelial cells.

Wound repair and tumor vascularization depend upon blood vessel growth into hypoxic tissue. Although hypoxia slows endothelial cell (EC) proliferation and suppresses EC basic fibroblast growth factor (bFGF) expression, we report that macrophages (MPs) exposed to PO2 approximately 12-14 torr (1 torr = 133.3 Pa) synthesize and release in a time-dependent manner platelet-derived growth factor (PDGF) and acidic/basic FGFs (a/bFGFs), which stimulate the growth of hypoxic ECs. Chromatography of hypoxic MP-conditioned medium on immobilized heparin with an ascending NaCl gradient resolved three peaks of mitogenic activity: activity of the first peak was neutralized by antibody to PDGF; activity of the second peak was neutralized by antibody to aFGF; and activity of the third peak was neutralized by antibody to bFGF. Metabolically labeled lysates and supernatants from MPs exposed to hypoxia showed increased synthesis and release of immunoprecipitable PDGF and a/bFGF in the absence of changes in cell viability. Possible involvement of a heme-containing oxygen sensor in MP elaboration of growth factors was suggested by the induction of bFGF and PDGF by normoxic MPs exposed to nickel or cobalt, although metabolic inhibitors such as sodium azide were without effect. These results suggest a paracrine model in which hypoxia stimulates MP release of PDGF and a/bFGF, inducing EC proliferation and potentially promoting angiogenesis in hypoxic environments.

Intramolecular Bromoamination of Conjugated N-Tosylaminodienes Using N-Bromosuccinimide. Synthesis of Bromoallyl-Substituted N-Heterocycles and Their Applications as Building Blocks

The bromonium-promoted cyclization of conjugated aminodienes is described. The reaction proceeds smoothly in the presence of N-bromosuccinimide as halonium promoter, and using N-tosyl-protected aminodienes as substrates, to give the corresponding halocyclization products in high yields and with high diastereoselectivities. It can be envisaged that the formation of these products is the result of an SN2′-type ring-opening of a terminal bromonium intermediate in a 5-exo-trig or 6-exo-trig cyclization mode. The presence of an allyl bromide moiety in the haloamination products makes these molecules highly attractive from a synthetic point of view, as it opens the way for further transformations. Thus, allylic substitution reactions with different nucleophiles (acetate, azide, cyanide, and malonate), palladium-catalysed Suzuki coupling, and silver-mediated bromine displacement reactions were carried out successfully.

THE DESIGN AND SYNTHESIS OF SILOXANE PRECURSORS FOR CHIRAL PERIODIC MESOPOROUS ORGANOSILICA MATERIALS

The development of cost-effective and reliable methods for the synthesis and separation of asymmetric compounds is paramount in helping to meet society’s ever-growing demand for chiral small molecules. Of these methods, chiral heterogeneous supports are particularly appealing as they allow for the reuse of the chiral source. One such support, based on the synergy between chiral organic units and structurally stable inorganic silicon scaffolds are periodic mesoporous organosilicas (PMOs). In the work described herein, I examine some of the factors governing the transmission of chirality between chiral dopants and prochiral bulk phases in chiral PMO materials. In particular, the exploration of 1,1’-binaphthalene-bridged chiral dopants with a focus on the point of attachment into the materials. Moreover, the effects of ordering in the materials are examined and reveal that chirality transfer is more facile in materials with molecular-scale order then those containing amorphous walls. Secondly, the issues surrounding the synthesis and purification of aryl-triethoxysilanes as siloxane precursors are addressed. Both the introduction of a two-carbon linker and the direct attachment of allyl and mixed allyldiethoxysilane species are explored. This work demonstrates that allyldiethoxysilanes are ideal, in that they are stable enough to permit facile synthesis, while still being able to hydrolyze completely to produce well-ordered materials. Lastly, the production of new bulk phases for chiral PMO materials is examined by introducing new prochiral nitrogen-containing siloxane precursors. Biphenyldiamine and bipyridine-bridged siloxane precursors are readily synthesized on reasonable scales. Their use as the bulk siloxane precursor in the production of PMO materials however, is precluded by insufficient gelation and additional siloxane precursors are necessary for the production of ordered materials. In addition to the research detailed above that forms the body of this thesis, two short works are appended. The first details the production of polythiophene assemblies mediated through coordination nanospaces, while the second explores the production of N-heterocyclic carbene functionalized gold nanoparticles through ligand exchange.

Synthesis, Modification, and Application of Siloxane Materials for Environmental Sensing

As human populations and resource consumption increase, it is increasingly important to monitor the quality of our environment. While laboratory instruments offer useful information, portable, easy to use sensors would allow environmental analysis to occur on-site, at lower cost, and with minimal operator training. We explore the synthesis, modification, and applications of modified polysiloxane in environmental sensing. Multiple methods of producing modified siloxanes were investigated. Oligomers were formed by using functionalized monomers, producing siloxane materials containing silicon hydride, methyl, and phenyl side chains. Silicon hydride-functionalized oligomers were further modified by hydrosilylation to incorporate methyl ester and naphthyl side chains. Modifications to the siloxane materials were also carried out using post-curing treatments. Methyl ester-functionalized siloxane was incorporated into the surface of a cured poly(dimethylsiloxane) film by siloxane equilibration. The materials containing methyl esters were hydrolyzed to reveal carboxylic acids, which could later be used for covalent protein immobilization. Finally, the siloxane surfaces were modified to incorporate antibodies by covalent, affinity, and adsorption-based attachment. These modifications were characterized by a variety of methods, including contact angle, attenuated total reflectance Fourier transform infrared spectroscopy, dye labels, and 1H nuclear magnetic resonance spectroscopy. The modified siloxane materials were employed in a variety of sensing schemes. Volatile organic compounds were detected using methyl, phenyl, and naphthyl-functionalized materials on a Fabry-Perot interferometer and a refractometer. The Fabry-Perot interferometer was found to detect the analytes upon siloxane extraction by deformation of the Bragg reflectors. The refractometer was used to determine that naphthyl-functionalized siloxanes had elevated refractive indices, rendering these materials more sensitive to some analytes. Antibody-modified siloxanes were used to detect biological analytes through a solid phase microextraction-mediated enzyme linked immunosorbent assay (SPME ELISA). The SPME ELISA was found to have higher analyte sensitivity compared to a conventional ELISA system. The detection scheme was used to detect Escherichia coli at 8500 CFU/mL. These results demonstrate the variety of methods that can be used to modify siloxanes and the wide range of applications of modified siloxanes has been demonstrated through chemical and biological sensing schemes.

Design and synthesis of constrained azacyclic pyrrolidine analogues of FTY720 as anticancer agents & metal coordination-controlled and bifunctional catalysis toward tertiary β-Ketols

Cette thèse se compose en deux parties: Première Partie: La conception et la synthèse d’analogues pyrrolidiniques, utilisés comme agents anticancéreux, dérivés du FTY720. FTY720 est actuellement commercialisé comme médicament (GilenyaTM) pour le traitement de la sclérose en plaques rémittente-récurrente. Il agit comme immunosuppresseur en raison de son effet sur les récepteurs de la sphingosine-1-phosphate. A fortes doses, FTY720 présente un effet antinéoplasique. Cependant, à de telles doses, un des effets secondaires observé est la bradycardie dû à l’activation des récepteurs S1P1 et S1P3. Ceci limite son potentiel d’utilisation lors de chimiothérapie. Nos précédentes études ont montré que des analogues pyrrolidiniques dérivés du FTY720 présentaient une activité anticancéreuse mais aucune sur les récepteurs S1P1 et S1P3. Nous avons soumis l’idée qu’une étude relation structure-activité (SARs) pourrait nous conduire à la découverte de nouveaux agents anti tumoraux. Ainsi, deux séries de composés pyrrolidiniques (O-arylmethyl substitué et C-arylmethyl substitué) ont pu être envisagés et synthétisés (Chapitre 1). Ces analogues ont montré d’excellentes activités cytotoxiques contre diverses cellules cancéreuses humaines (prostate, colon, sein, pancréas et leucémie), plus particulièrement les analogues actifs qui ne peuvent pas être phosphorylés par SphK, présentent un plus grand potentiel pour le traitement du cancer sans effet secondaire comme la bradycardie. Les études mécanistiques suggèrent que ces analogues de déclencheurs de régulation négative sur les transporteurs de nutriments induisent une crise bioénergétique en affamant les cellules cancéreuses. Afin d’approfondir nos connaissances sur les récepteurs cibles, nous avons conçu et synthétisé des sondes diazirine basées sur le marquage d’affinité aux photons (méthode PAL: Photo-Affinity Labeling) (Chapitre 2). En s’appuyant sur la méthode PAL, il est possible de récolter des informations sur les récepteurs cibles à travers l’analyse LC/MS/MS de la protéine. Ces tests sont en cours et les résultats sont prometteurs. Deuxième partie: Coordination métallique et catalyse di fonctionnelle de dérivés β-hydroxy cétones tertiaires. Les réactions de Barbier et de Grignard sont des méthodes classiques pour former des liaisons carbone-carbone, et généralement utilisées pour la préparation d’alcools secondaires et tertiaires. En vue d’améliorer la réaction de Grignard avec le 1-iodobutane dans les conditions « one-pot » de Barbier, nous avons obtenu comme produit majoritaire la β-hydroxy cétone provenant de l’auto aldolisation de la 5-hexen-2-one, plutôt que le produit attendu d’addition de l’alcool (Chapitre 3). La formation inattendue de la β-hydroxy cétone a également été observée en utilisant d’autres dérivés méthyl cétone. Étonnement dans la réaction intramoléculaire d’une tricétone, connue pour former la cétone Hajos-Parrish, le produit majoritaire est rarement la β-hydroxy cétone présentant la fonction alcool en position axiale. Intrigué par ces résultats et après l’étude systématique des conditions de réaction, nous avons développé deux nouvelles méthodes à travers la synthèse sélective et catalytique de β-hydroxy cétones spécifiques par cyclisation intramoléculaire avec des rendements élevés (Chapitre 4). La réaction peut être catalysée soit par une base adaptée et du bromure de lithium comme additif en passant par un état de transition coordonné au lithium, ou bien soit à l’aide d’un catalyseur TBD di fonctionnel, via un état de transition médiée par une coordination bidenté au TBD. Les mécanismes proposés ont été corroborés par calcul DFT. Ces réactions catalytiques ont également été appliquées à d’autres substrats comme les tricétones et les dicétones. Bien que les efforts préliminaires afin d’obtenir une enantioselectivité se sont révélés sans succès, la synthèse et la recherche de nouveaux catalyseurs chiraux sont en cours.

Design and synthesis of constrained azacyclic pyrrolidine analogues of FTY720 as anticancer agents & metal coordination-controlled and bifunctional catalysis toward tertiary β-Ketols

Cette thèse se compose en deux parties: Première Partie: La conception et la synthèse d’analogues pyrrolidiniques, utilisés comme agents anticancéreux, dérivés du FTY720. FTY720 est actuellement commercialisé comme médicament (GilenyaTM) pour le traitement de la sclérose en plaques rémittente-récurrente. Il agit comme immunosuppresseur en raison de son effet sur les récepteurs de la sphingosine-1-phosphate. A fortes doses, FTY720 présente un effet antinéoplasique. Cependant, à de telles doses, un des effets secondaires observé est la bradycardie dû à l’activation des récepteurs S1P1 et S1P3. Ceci limite son potentiel d’utilisation lors de chimiothérapie. Nos précédentes études ont montré que des analogues pyrrolidiniques dérivés du FTY720 présentaient une activité anticancéreuse mais aucune sur les récepteurs S1P1 et S1P3. Nous avons soumis l’idée qu’une étude relation structure-activité (SARs) pourrait nous conduire à la découverte de nouveaux agents anti tumoraux. Ainsi, deux séries de composés pyrrolidiniques (O-arylmethyl substitué et C-arylmethyl substitué) ont pu être envisagés et synthétisés (Chapitre 1). Ces analogues ont montré d’excellentes activités cytotoxiques contre diverses cellules cancéreuses humaines (prostate, colon, sein, pancréas et leucémie), plus particulièrement les analogues actifs qui ne peuvent pas être phosphorylés par SphK, présentent un plus grand potentiel pour le traitement du cancer sans effet secondaire comme la bradycardie. Les études mécanistiques suggèrent que ces analogues de déclencheurs de régulation négative sur les transporteurs de nutriments induisent une crise bioénergétique en affamant les cellules cancéreuses. Afin d’approfondir nos connaissances sur les récepteurs cibles, nous avons conçu et synthétisé des sondes diazirine basées sur le marquage d’affinité aux photons (méthode PAL: Photo-Affinity Labeling) (Chapitre 2). En s’appuyant sur la méthode PAL, il est possible de récolter des informations sur les récepteurs cibles à travers l’analyse LC/MS/MS de la protéine. Ces tests sont en cours et les résultats sont prometteurs. Deuxième partie: Coordination métallique et catalyse di fonctionnelle de dérivés β-hydroxy cétones tertiaires. Les réactions de Barbier et de Grignard sont des méthodes classiques pour former des liaisons carbone-carbone, et généralement utilisées pour la préparation d’alcools secondaires et tertiaires. En vue d’améliorer la réaction de Grignard avec le 1-iodobutane dans les conditions « one-pot » de Barbier, nous avons obtenu comme produit majoritaire la β-hydroxy cétone provenant de l’auto aldolisation de la 5-hexen-2-one, plutôt que le produit attendu d’addition de l’alcool (Chapitre 3). La formation inattendue de la β-hydroxy cétone a également été observée en utilisant d’autres dérivés méthyl cétone. Étonnement dans la réaction intramoléculaire d’une tricétone, connue pour former la cétone Hajos-Parrish, le produit majoritaire est rarement la β-hydroxy cétone présentant la fonction alcool en position axiale. Intrigué par ces résultats et après l’étude systématique des conditions de réaction, nous avons développé deux nouvelles méthodes à travers la synthèse sélective et catalytique de β-hydroxy cétones spécifiques par cyclisation intramoléculaire avec des rendements élevés (Chapitre 4). La réaction peut être catalysée soit par une base adaptée et du bromure de lithium comme additif en passant par un état de transition coordonné au lithium, ou bien soit à l’aide d’un catalyseur TBD di fonctionnel, via un état de transition médiée par une coordination bidenté au TBD. Les mécanismes proposés ont été corroborés par calcul DFT. Ces réactions catalytiques ont également été appliquées à d’autres substrats comme les tricétones et les dicétones. Bien que les efforts préliminaires afin d’obtenir une enantioselectivité se sont révélés sans succès, la synthèse et la recherche de nouveaux catalyseurs chiraux sont en cours.

Synthesis in the plakortone series: Plakortone E

A Pd(II)-mediated hydroxycyclisation-carbonylation-lactonisation sequence has operated efficiently with racemic enediol (8) to furnish (four) separable diastereomers of the bicyclic lactone system assigned to the sponge-derived, bioactive plakortone E. All four are cis ring-fused, and one is identical, on the basis of H-1 and C-13 NMR spectroscopic comparisons, with plakortone E, thus confirming its constitution and relative stereochemistry about the bicyclic lactone core. This synthetic approach, when applied to stereoisomer (13), will establish the absolute stereochemistry of plakortone E, likely to be that shown for (14).

A Two-Step Sol–Gel Method for Synthesis of Nanoporous TiO2

This article reports a study of the effects of synthesis parameters on the preparation and formation of mesoporous titania nanopowders by employing a two-step sol-gel method. These materials displayed crystalline domains characteristic of anatase. The first step of the process involved the hydrolysis of titanium isopropoxide in a basic aqueous solution mediated by neutral surfactant. The solid product obtained from step 1 was then treated in an acidified ethanol solution containing the same titanium precursor to thicken the pore walls. Low pH and higher loading of the Ti precursor in step 2 produced better mesoporosity and crystallinity of titanium dioxide polymorphs. The resultant powder exhibited a high surface area (73.8 m(2)/g) and large pore volume (0.17 cm(3)/g) with uniform mesopores. These materials are envisaged to be used as precursors for mesoporous titania films as a wide band gap semiconductor in dye-sensitized nanocrystalline TiO2 solar cells.

Inhibition of in vitro VEGF expression and choroidal neovascularization by synthetic dendrimer peptide mediated delivery of a sense oligonucleotide

Ocular neovascularisation is the leading cause of blindness in developed countries and the most potent angiogenic factor associated with neovascularisation is vascular endothelial growth factor (VEGF). We have previously described a sense oligonucleotide (ODN-1) that possesses anti-human and rat VEGF activity. This paper describes the synthesis of lipid-lysine dendrimers and their subsequent ability to delivery ODN-1 to its target and mediate a reduction in VEGF concentration both in vitro and in vivo. Positively charged dendrimers were used to deliver ODN-1 into the nucleus of cultured D407 cells. The effects on VEGF mRNA transcription and protein expression were analysed using RT-PCR and ELISA, respectively. The most effective dendrimers in vitro were further investigated in vivo using an animal model of choroidal neovascularisation (CNV). All dendrimer/ODN-1 complexes mediated in a significant reduction in VEGF expression during an initial 24 hr period (40-60%). Several complexes maintained this level of VEGF reduction during a subsequent, second 24 hr period, which indicated protection of ODN-1 from the effects of endogenous nucleases. In addition, the transfection efficiency of dendrimers that possessed 8 positive charges (chi = 81(.)51%) was significantly better (P = 0(.)0036) than those that possessed 4 positive charges (chi = 56(.)8%). RT-PCR revealed a correlation between levels of VEGF protein mRNA. These results indicated that the most effective structural combination was three branched chains of intermediate length with 8 positive charges such as that found for dendrimer 4. Dendrimer 4 and 7/ODN-1 complexes were subsequently chosen for in vivo analysis. Fluorescein angiography demonstrated that both dendrimers significantly (P < 0(.)0001) reduced the severity of laser mediated CNV for up to two months post-injection. This study demonstrated that lipophilic, charged dendrimer mediated delivery of ODN-1 resulted in the down-regulation of in vitro VEGF expression. In addition, in vivo delivery of ODN-1 by two of the dendrimers resulted in significant inhibition of CNV in an inducible rat model. Time course studies showed that the dendrimer/ODN-1 complexes remained active for up to two months indicating the dendrimer compounds provided protection against the effects of nucleases. (C) 2004 Elsevier Ltd. All rights reserved.

Modeling the molecular weight distribution of block copolymer formation in a reversible addition-fragmentation chain transfer mediated living radical polymerization

Block copolymers have become an integral part of the preparation of complex architectures through self-assembly. The use of reversible addition-fragmentation chain transfer (RAFT) allows blocks ranging from functional to nonfunctional polymers to be made with predictable molecular weight distributions. This article models block formation by varying many of the kinetic parameters. The simulations provide insight into the overall polydispersities (PDIs) that will be obtained when the chain-transfer constants in the main equilibrium steps are varied from 100 to 0.5. When the first dormant block [polymer-S-C(Z)=S] has a PDI of 1 and the second propagating radical has a low reactivity to the RAFT moiety, the overall PDI will be greater than 1 and dependent on the weight fraction of each block. When the first block has a PDI of 2 and the second propagating radical has a low reactivity to the RAFT moiety, the PDI will decrease to around 1.5 because of random coupling of two broad distributions. It is also shown how we can in principle use only one RAFT agent to obtain block copolymers with any desired molecular weight distribution. We can accomplish this by maintaining the monomer concentration at a constant level in the reactor over the course of the reaction. (c) 2005 Wiley Periodicals, Inc.

Design strategies for controlling the molecular weight and rate using reversible addition-fragmentation chain transfer mediated living radical polymerization

Living radical polymerization has allowed complex polymer architectures to be synthesized in bulk, solution, and water. The most versatile of these techniques is reversible addition-fragmentation chain transfer (RAFT), which allows a wide range of functional and nonfunctional polymers to be made with predictable molecular weight distributions (MWDs), ranging from very narrow to quite broad. The great complexity of the RAFT mechanism and how the kinetic parameters affect the rate of polymerization and MWD are not obvious. Therefore, the aim of this article is to provide useful insights into the important kinetic parameters that control the rate of polymerization and the evolution of the MWD with conversion. We discuss how a change in the chain-transfer constant can affect the evolution of the MWD. It is shown how we can, in principle, use only one RAFT agent to obtain a poly-mer with any MWD. Retardation and inhibition are discussed in terms of (1) the leaving R group reactivity and (2) the intermediate radical termination model versus the slow fragmentation model. (c) 2005 Wiley Periodicals, Inc.