Isoprene-mediated lithiation of imidazole derivatives: mechanism considerations

The isoprene-mediated lithiation, with lithium metal, of different imidazole derivatives is an interesting methodology for their functionalization. Studies of different possible intermediates involved in the reaction employing density functional theory calculations, at the B3LYP/6-311++G(d,p) level are considered. A plausible mechanism is described, in which isoprene is reduced, to the corresponding radical anion, in the presence of Li(s), acting then as a base deprotonating N-methylimidazole (NMI) and producing the 1,1-dimethylallyl radical. This radical is further reduced by the excess of lithium proceeding once more as a base. This final step produces stable final products that compensate the previous equilibriums, making favourable the whole process.

1,2-Functionalized Imidazoles as Palladium Ligands: An Efficient and Robust Catalytic System for the Fluorine-Free Hiyama Reaction

A variety of hydroxy- and amino-functionalized imidazoles were prepared from 1-methyl- and 1-(diethoxymethyl)imidazole by means of isoprene-mediated lithiation followed by reaction with an electrophile. These compounds in combination with palladium acetate were screened as catalyst systems for the Hiyama reaction under fluorine-free conditions using microwave irradiation. The systematic study of the catalytic system showed 1-methyl-2-aminoalkylimidazole derivative L1 to be the best ligand, which was employed under solvent-free conditions with a 1:2 Pd/ligand ratio and TBAB (20 mol-%) as additive. The study has revealed an interaction between the Pd/ligand ratio and the amount of TBAB. The established catalytic system presented a certain degree of robustness, and it has been successfully employed in the coupling of a range of aryl bromides and chlorides with different aryl siloxanes. Furthermore, both reagents were employed in an equimolecular amount, without an excess of organosilane.

Stereoselective synthesis of substituted hexahydro-3a,4a-diazacyclopentaphenanthren-4-ones and aminoferrocenes

This thesis explored the development of several methodologies for the stereoselective construction of ligand frameworks and some of their applications. The first segment concerns the application of an enantioselective lithiation at an Sp3_ hybridized position adjacent to nitrogen by means of the widely used and typically highly effective enantioselective lithiation with ( -)-sparteine. This investigation was intended to develop a method to install chirality into a system that would be converted into a family of diaminoylidenes for use as phosphine mimics in transition metal catalysis or as nucleophilic reagents. Molecular modeling of the system revealed some key interactions between the substrate and (-)-sparteine that provided general insight into the diamine's mode of action and should lend some predictive value to its future applications. The second portion focuses on the development of methods to access 1,2- disubstituted aminoferrocenes, an underexplored class of metallocenes possessing planar chirality. Two routes were examined involving a diastereoselective and an enantioselective pathway, where the latter method made use of the first BF3-mediated lithiation-substitution to install planar chirality. Key derivatives such as 1,2- aminophosphines, made readily accessible by the new route, were evaluated as ligands for Pd(II), Pt(II) and Ir(I). These complexes show activity in a number of transformations with both achiral and prochiral substrates. Optimization experiments were conducted to prepare enantiomerically enriched 2-substituted-I-aminoferrocenes by direct asymmetric lithiation of BF3-coordinated tertiary aminoferrocenes. A predictive computational model describing the transition state of this reaction was developed in collaboration with Professor Travis Dudding's group (Department of Chemistry, Brock University). The predicted stereochemistry of the process was confirmed by single-crystal X-ray analysis of a 2-phosphino-l-dimethylaminoferrocene derivative. Enantiomerically pure samples of the aminophosphine ligands derived from this new process have given promising preliminary results in the enantioselective hydrogenation of prochiral alkenes and warrant further stUdy in metal-mediated catalysis.

Understanding the Origin of Selectivity in the Asymmetric Lithiation Reaction of N-Heterocycles: A Theoretical Study

The natural abundance of the N-heterocycle containing compounds has pushed the synthetic community toward the invention of new synthetic methods that result in the structural diversity of N-heterocycles. Among this, is the efficient and highly selective diamine mediated asymmetric lithiation process. Amongst the diamine chiral ligands, (-)-sparterine, which is a naturally occurring alkaloid proved to be an efficient one. Many successful, good yielding and highly selective lithiation reactions have been accomplished with the mediation by this chiral diamine base. Although, there are some examples of experimental and theoretical mechanistic studies in the literature, there is a lack of detailed understanding as to how it exactly induces the chirality. In this thesis is described a systematic investigation of how (-)-sparteine influences the stereoselectivity in the course of asymmetric lithiation reaction. This led us to the establishment of the function of A-ring’s β-CH2 effect and D-ring effect. Consequently, the importance of the A-ring and D-ring portions of (-)-sparteine in the stereoselectivity is unraveled. Another part of this thesis deals with the asymmetric lithiation of BF3-activated N,N- dimethylaminoferrocene in the presence of (1R, 2R)-N1,N2-bis(3,3-dimethylbutyl)-N1,N2-dimethylcyclohexane-1,2-diamine ( a (R,R)-TMCDA surrogate) with i-PrLi. Computational findings were in full accord with the experimental observations. Subsequently, the theoretically provided insights into the mechanism of the reaction were exploited in computational design of a new ligand. Unfortunately, the outcome of this design was not experimentally robust and an updated approach towards a successful design was explained.

Implication of 3D culture system for study of prostate cancer (CaP) mediated bone metastasis

Introduction : For the past decade, three dimensional (3D) culture has served as a foundation for regenerative medicine study. With an increasing awareness of the importance of cell-cell and cell-extracellular matrix interactions which are lacking in 2D culture system, 3D culture system has been employed for many other applications namely cancer research. Through development of various biomaterials and utilization of tissue engineering technology, many in vivo physiological responses are now better understood. The cellular and molecular communication of cancer cells and their microenvironment, for instance can be studied in vitro in 3D culture system without relying on animal models alone. Predilection of prostate cancer (CaP) to bone remains obscure due to the complexity of the mechanisms and lack of proper model for the studies. In this study, we aim to investigate the interaction between CaP cells and osteoblasts simulating the natural bone metastasis. We also further investigate the invasiveness of CaP cells and response of androgen sensitve CaP cells, LNCaP to synthetic androgen.----- Method : Human osteoblast (hOB) scaffolds were prepared by seeding hOB on medical grade polycaprolactone-tricalcium phosphate (mPLC-TCP) scaffolds and induced to produce bone matrix. CaP cell lines namely wild type PC3 (PC3-N), overexpressed prostate specific antigen PC3 (PC3k3s5) and LNCaP were seeded on hOB scaffolds as co-cultures. Morphology of cells was examined by Phalloidin-DAPI and SEM imaging. Gelatin zymography was performed on the 48 hours conditioned media (CM) from co-cultures to determine matrix metalloproteinase (MMP) activity. Gene expression of hOB/LNCaP co-cultures which were treated for 48 hours with 1nM synthetic androgen R1881 were analysed by quantitative real time PCR (qRT-PCR).----- Results : Co-culture of PCC/hOB revealed that the morphology of PCCs on the tissue engineered bone matrix varied from homogenous to heterogenous clusters. Enzymatically inactive pro-MMP2 was detected in CM from hOBs and PCCs cultured on scaffolds. Elevation in MMP9 activity was found only in hOB/PC3N co-culture. hOB/LNCaP co-culture showed increase in expression of key enzymes associated with steroid production which also corresponded to an increase in prostate specific antigen (PSA) and MMP9.----- Conclusions : Upregulation of MMP9 indicates involvement of ECM degradation during cancer invasion and bone metastases. Expression of enzymes involved in CaP progression, PSA, which is not expressed in osteoblasts, demonstrates that crosstalk between PCCs and osteoblasts may play a part in the aggressiveness of CaP. The presence of steroidogenic enzymes, particularly, RDH5, in osteoblasts and stimulated expression in co-culture, may indicate osteoblast production of potent androgens, fuelling cancer cell proliferation. Based on these results, this practical 3D culture system may provide greater understanding into CaP mediated bone metastasis. This allows the role of the CaP/hOB interaction with regards to invasive property and steroidogenesis to be further explored.

Inhibition of myeloperoxidase-mediated hypochlorous acid production by nitroxides

Tissue damage resulting from the extracellular production of HOCl (hypochlorous acid) by the MPO (myeloperoxidase)-hydrogen peroxide-chloride system of activated phagocytes is implicated as a key event in the progression of a number of human inflammatory diseases. Consequently, there is considerable interest in the development of therapeutically useful MPO inhibitors. Nitroxides are well established antioxidant compounds of low toxicity that can attenuate oxidative damage in animal models of inflammatory disease. They are believed to exert protective effects principally by acting as superoxide dismutase mimetics or radical scavengers. However, we show here that nitroxides can also potently inhibit MPO-mediated HOCl production, with the nitroxide 4-aminoTEMPO inhibiting HOCl production by MPO and by neutrophils with IC50 values of approx. 1 and 6 μM respectively. Structure–activity relationships were determined for a range of aliphatic and aromatic nitroxides, and inhibition of oxidative damage to two biologically-important protein targets (albumin and perlecan) are demonstrated. Inhibition was shown to involve one-electron oxidation of the nitroxides by the compound I form of MPO and accumulation of compound II. Haem destruction was also observed with some nitroxides. Inhibition of neutrophil HOCl production by nitroxides was antagonized by neutrophil-derived superoxide, with this attributed to superoxide-mediated reduction of compound II. This effect was marginal with 4-aminoTEMPO, probably due to the efficient superoxide dismutase-mimetic activity of this nitroxide. Overall, these data indicate that nitroxides have considerable promise as therapeutic agents for the inhibition of MPO-mediated damage in inflammatory diseases.

Discovery and characterization of IGFBP-mediated endocytosis in the human retinal pigment epithelial cell line ARPE-19

Insulin-like growth factor binding proteins (IGFBPs) are prime regulators of IGF-action in numerous cell types including the retinal pigment epithelium (RPE). The RPE performs several functions essential for vision, including growth factor secretion and waste removal via a phagocytic process mediated in part by vitronectin (Vn). In the course of studying the effects of IGFBPs on IGF-mediated VEGF secretion and Vn-mediated phagocytosis in the RPE cell line ARPE-19, we have discovered that these cells avidly ingest synthetic microspheres (2.0 μm diameter) coated with IGFBPs. Given the novelty of this finding and the established role for endocytosis in mediating IGFBP actions in other cell types, we have explored the potential role of candidate cell surface receptors. Moreover, we have examined the role of key IGFBP structural motifs, by comparing responses to three members of the IGFBP family (IGFBP-3, IGFBP-4 and IGFBP-5) which display overlapping variations in primary structure and glycosylation status. Coating of microspheres (FluoSpheres®, sulfate modified polystyrene filled with a fluorophore) was conducted at 37 °C for 1 h using 20 μg/mL of test protein, followed by extensive washing. Binding of proteins was confirmed using a microBCA assay. The negative control consisted of microspheres treated with 0.1% bovine serum albumin (BSA), and all test samples were post-treated with BSA in an effort to coat any remaining free protein binding sites, which might otherwise encourage non-specific interactions with the cell surface. Serum-starved cultures of ARPE-19 cells were incubated with microspheres for 24 h, using a ratio of approximately 100 microspheres per cell. Uptake of microspheres was quantified using a fluorometer and was confirmed visually by confocal fluorescence microscopy. The ARPE-19 cells displayed little affinity for BSA-treated microspheres, but avidly ingested large quantities of those pre-treated with Vn (ANOVA; p < 0.001). Strong responses were also observed towards recombinant formulations of non-glycosylated IGFBP-3, glycosylated IGFBP-3 and glycosylated IGFBP-5 (all p < 0.001), while glycosylated IGFBP-4 induced a relatively minor response (p < 0.05). The response to IGFBP-3 was unaffected in the presence of excess soluble IGFBP-3, IGF-I or Vn. Likewise, soluble IGFBP-3 did not induce uptake of BSA-treated microspheres. Antibodies to either the transferrin receptor or type 1 IGF-receptor displayed slight inhibitory effects on responses to IGFBPs and Vn. Heparin abolished responses to Vn, IGFBP-5 and non-glycosylated IGFBP-3, but only partially inhibited the response to glycosylated IGFBP-3. Our results demonstrate for the first time IGFBP-mediated endocytosis in ARPE-19 cells and suggest roles for the IGFBP-heparin-binding domain and glycosylation status. These findings have important implications for understanding the mechanisms of IGFBP actions on the RPE, and in particular suggest a role for IGFBP-endocytosis.

Poly-immunoglobulin receptor-mediated transport of IgA into the male genital tract is important for clearance of chlamydia muridarum infection

Problem: Chlamydia trachomatis is the most common sexually transmitted infection worldwide. While infection in females requires a Th1 response for clearance, such a response in males may disrupt the immune privileged nature of the male reproductive tract, potentially contributing to infertility. Method of study: We investigated the role of IgA in protection against an intrapenile Chlamydia muridarum infection of C57BL/6 and pIgR−/− mice. Results: Here, we show that the poly immunoglobulin receptor is the main pathway for IgA transport into the male reproductive tract. The high levels of IgA seen in prostatic fluid of wild-type mice correlate with reduction in chlamydial infection both in vitro and in vivo. Conclusion: These findings indicate that a Chlamydia vaccine that induces neutralizing IgA in the prostate will aid in the protection against infection in males.

Medienvermittelte transnationale Öffentlichkeiten im Zeitalter praktischer Hybridisierung (English title: Mediated transnational publics in environments of practical hybridisation)

This article is concerned with the repercussions of societal change on transnational media. It offers a new understanding of multilingual programming strategies by examining “Radio MultiKulti” (RM), a public service radio station discontinued from 1/1/2009 by Rundfunk Berlin-Brandenburg. In its fourteen years of existence, “RM” had to implement a well-intended and politically-motivated logic of ‘multiethnic, intercultural service station’. However, as we demonstrate, such a direction, despite some achievements, has resulted in the constraints to RM’s journalistic activities and language policy, drawing criticism for the station’s economic viability. This paper proposes that multilingual media services are to be framed by the concept of practical hybridity that allows a necessary responsiveness towards an ever-changing media environment, at the moment within digital culture. Our approach draws on Mikhail Bakhtin’s and Yuri Lotman’s theoretical approaches to hybridity, as well as in-depth interviews conducted with “RM” staff from 2005 onwards, further interviews with key agents outside RM and a continuous monitoring of the public debate which culminated at the end of 2008 in the controversial decision to close the radio station. Against this background, the concluding remarks are meant to contribute to the scholarly debate on hybridization as well as to inform multilingual media policy in the 21st century.

Enhancing learning in dance technique through on-line-mediated reflective practice

‘Practice makes perfect’ expresses the common misconception that repetitive practice without appropriate feed-back will deliver improvement in tasks being practised. This paper explores the implementation of a student-driven feed-back mechanism and shows how functional and aesthetic understanding can be progressively enhanced through reflective practice. More efficient practice of clearly understood tasks will enhance dance training outcomes. We were looking for ways to improve teaching efficiency, effectiveness of the students’ practice in the studio and application of safe dance practices. We devised a web-based on-line format, ‘Performing Reflective Practice’, designed to augment and refine studio practice. Only perfect practice makes perfect!