The reaction mechanisms followed by ethylene polymerization and selective oligomerization (tri- and tetramerization) are conceptually very different, being a non-redox chain growth and a redox metallacycle ring expansion pathway respectively. With chromium being the metal of choice, metal oxidation states and the variation of ancillary ligand able to support specific metal oxidation states responsible for selective trimerization, tetramerization or nonselective oligomerization/polymerization were varied. In this research project we have explored a broad range of novel pyridine containing modified PN ligand scaffolds with the aim of probing the role of the pyridine donor substituent in stabilizing lower oxidation states and ultimately affecting the selectivity of the ethylene in the catalytic cycle. In this study, pyridine PNP pincer ligands in conjunction with chromium salts and alkylaluminium activators have been explored. Their catalytic activities toward ethylene oligomerization were rationalized through the isolation of different chromium-aluminate intermediates in different oxidation states during the catalytic reaction. Moreover, we explored other pyridine modification of NNP type ligand. Its anionic ligand shows a rare example of a Cr(II) hydride cluster which shows a high activity as a nonselective ethylene oligomerization catalyst. Finally, a cyclic PNPN type ligand was explored. The ligand enabled the isolation of a mix-valent Cr(I)/Cr(II) species which was found to be inactive toward ethylene oligomerization due to its geometric constraint.

The reaction mechanisms followed by ethylene polymerization and selective oligomerization (tri- and tetramerization) are conceptually very different, being a non-redox chain growth and a redox metallacycle ring expansion pathway respectively. With chromium being the metal of choice, metal oxidation states and the variation of ancillary ligand able to support specific metal oxidation states responsible for selective trimerization, tetramerization or nonselective oligomerization/polymerization were varied. In this research project we have explored a broad range of novel pyridine containing modified PN ligand scaffolds with the aim of probing the role of the pyridine donor substituent in stabilizing lower oxidation states and ultimately affecting the selectivity of the ethylene in the catalytic cycle. In this study, pyridine PNP pincer ligands in conjunction with chromium salts and alkylaluminium activators have been explored. Their catalytic activities toward ethylene oligomerization were rationalized through the isolation of different chromium-aluminate intermediates in different oxidation states during the catalytic reaction. Moreover, we explored other pyridine modification of NNP type ligand. Its anionic ligand shows a rare example of a Cr(II) hydride cluster which shows a high activity as a nonselective ethylene oligomerization catalyst. Finally, a cyclic PNPN type ligand was explored. The ligand enabled the isolation of a mix-valent Cr(I)/Cr(II) species which was found to be inactive toward ethylene oligomerization due to its geometric constraint.

No description available.

No description available.

Daptomycin is a lipopeptide antibiotic that contains 13 amino acids and an N-terminally attached fatty acyl residue. The antibiotic kills Gram-positive bacteria by membrane depolarization. It has long been assumed that the mode of action of daptomycin involves the formation of oligomers on the bacterial cell membrane; however, at the outset of my studies, this had not been experimentally demonstrated. In the work described in this thesis, I have used fluorescence energy transfer (FRET) between native daptomycin and an NBD-labeled daptomycin derivative to demonstrate that the antibiotic indeed forms oligomers on bacterial cell membranes. In a liposome model, oligomer formation depends on calcium and on phosphatidylglycerol (PG). The oligomer forms rapidly and is stable for a length of time longer than required for the bactericidal effect. Through variation of the ratio of FRET donor (native daptomycin) and acceptor (NBD-daptomycin), I have determined that the oligomer consists of approximately 6–7 molecules, or, depending on the structure of the oligomer, possibly up to twice that number. Oligomer formation on liposomes and on bacterial membranes was confirmed using excimer fluorescence of a perylene-labeled daptomycin derivative. Excimer fluorescence was also used to demonstrate a stoichiometric interaction between daptomycin and PG. It has previously been shown that the bactericidal activity of daptomycin requires calcium and correlates with the concentration of PG in the bacterial cell membrane; these requirements mirror those observed here for oligomer formation. Furthermore, membrane permeabilization is selective, and electron microscopy of bacterial membranes exposed to daptomycin has revealed no discontinuities or accretions of electron density. Both of these findings suggest formation of a small membrane lesion, which is compatible with the small size of the oligomer that was determined here. In conjunction with these previous findings, the experiments contained in my thesis strongly suggest that the oligomer is the bactericidal form of daptomycin.

Thermolysis of Cp*W(NO)(CH ₂CMe₃) ₂,CpW(NO)(CH ₂CMe₃)₂, Cp*W(NO)(CH₂SiMe₃)(η²-CPhCH₂), or Cp*W(NO)[CH(Ph)CH₂CH(nPr)CH₂]in cyclic olefins results in the formation of ring-retaining oligomers having lengths up to dodecamers. The main cyclohexene dimer is 3-cyclohexylcyclohexene. A small percentage of oligomers contain neopentyl or CH=CHPh end groups. Turnover frequencies for the Cp*-tungsten precatalysts range from 5.5 to 6.5 mol/h at 100 °C. In room temperature solutions, Cp*Mo(NO)(CH₂CMe₃)₂ generates the alkylidene intermediate [Cp*Mo(NO)(=CHCMe₃)], which couples with cyclic olefins to form cismetallacycles. The isolable cyclopentene-derived cis-metallacycle, Cp*Mo(NO)[cis-η² CH(CH₂)₃CHCHCMe₃], converts in the solid state to the allyl-hydride complex Cp*Mo(NO)(H)(η³-CH(CH₂)3CCHCMe₃). With larger cyclic olefins (cyclohexene through cyclooctene) the initial cis-metallacycles isomerize to trans-metallacycles of the form Cp*Mo(NO)[trans-η²-CH(CH₂)nCHCHCMe₃] (n = 4, 5, 6), and these subsequently convert with loss of dihydrogen to η⁴-diene complexes, Cp*Mo(NO)[η⁴-CHCH(CH₂)n-₁CCHCMe₃]. Thermolysis of the η⁴-diene complexes in cyclohexene results in decomposition of the organometallic complex with small amounts of oligomer formation. Thermolysis of Cp*W(NO)CH₂CMe₃)₂ in cyclic-olefm substrates generates the alkylidene intermediate [Cp*W(N0)(=CHCMe₃)], which couples with cyclic olefins in a manner analogous to the Cp*Mo-system. Tungsten trans-metallacycles are observed by ¹H NMR spectroscopy, but the organometallic subsequently reacts further with loss of the coupled neopentyl-cyclic olefin and coordination of two substrate molecules to form the putative Cp*W(NO)(cyclic olefm)₂ complex. Two additional cyclooctene products are isolated, the 1,4- diene Cp*W(N0)[η⁴-CHCH(CH₂)₅CHCCH(CH₂)₆] and the allyl hydride Cp*W(NO)(H)[η³- CH(CH₂)₆CCCHCH(CH₂)₅], both containing two coupled cyclooctene molecules. A tungsten cis-metallacycle forms with 2,5-dihydrofiiran, but a ring-opened alkoxy-allyl complex forms with 3,4-dihydro-2H-pyran, and 1,2,3,6-tetrahydro-pyridine undergoes N -H bond activation to afford an amido product. CpW(NO)(CH₂CMe₃)₂ produces some oligomers of cyclohexene, but in all other reactions bimetallic decomposition pathways predominate.

The bacterial toxin pyolysin (PLO) is a member of the family of Cholesterol-Dependent Cytolysins, which form large, oligomeric pores in cholesterol-containing membranes. The general CDC structure has an elongated shape and consists of four domains rich in β-sheet structure. Upon binding to a membrane, molecules diffuse laterally on the surface and oligomerize to form a pre-pore complex, then insert into the membrane yielding pores of unusually large size, approximately 30 nm in diameter. In this work, the oligomerization properties of PLO were investigated. In particular, the role of the C-terminal domain in the oligomerization process, the effects of a disulphide-tethered mutant on the activity of the wild type toxin, and the pore-forming ability of oligomers pre-formed in solution were characterized. Chapter 2 characterizes the functional properties of a recombinant fragment that corresponds to the C-terminal domain 4 of PLO. It is shown that this fragment can form hybrid oligomers with intact PLO toxin molecules, and is also capable of self-oligomerization. The fragment has no haemolytic activity of its own; nevertheless, it can to some degree increase the haemolytic activity of the wild type toxin. In addition, in a mixture domain 4 and wild type interact in such a way as to form unusual shapes on cholesterol crystals that have not been previously observed. Chapter 3 describes the effects of a disulphide bond linking domain 2 to a membrane-inserting region of domain 3 on the oligomerization process. The disulphide mutant was not able to oligomerize on its own, and when combined with active PLO toxin, the haemolytic activity of wild type was significantly inhibited. Also, the combination of the disulphide-tethered mutant with intact toxin resulted in the formation of hybrid oligomers. This, in turn, caused an increase in incomplete ring formations on cholesterol surfaces which correlate to a reduction in functional pore size, suggesting that insertion of subunits is partially cooperative. The results of the investigation of the pore-forming ability of solution-derived oligomers (SDO) are described in Chapter 4. Here, the fluorescence emission of an environmentally-sensitive probe on the SDO after membrane insertion was a fraction of that observed with the monomeric control, which was supported by hydrophobic quenching analyses. This suggests that the formation of SDO may block necessary conformational changes in the intact toxin to allow membrane insertion.

Abstract KChIPs are Kv channel-interacting proteins that bind to the cytoplasmic N-terminus of Kv4

Thermolysis of Cp*W(NO)(CH ₂CMe₃) ₂,CpW(NO)(CH ₂CMe₃)₂, Cp*W(NO)(CH₂SiMe₃)(η²-CPhCH₂), or Cp*W(NO)[CH(Ph)CH₂CH(nPr)CH₂]in cyclic olefins results in the formation of ring-retaining oligomers having lengths up to dodecamers. The main cyclohexene dimer is 3-cyclohexylcyclohexene. A small percentage of oligomers contain neopentyl or CH=CHPh end groups. Turnover frequencies for the Cp*-tungsten precatalysts range from 5.5 to 6.5 mol/h at 100 °C. In room temperature solutions, Cp*Mo(NO)(CH₂CMe₃)₂ generates the alkylidene intermediate [Cp*Mo(NO)(=CHCMe₃)], which couples with cyclic olefins to form cismetallacycles. The isolable cyclopentene-derived cis-metallacycle, Cp*Mo(NO)[cis-η² CH(CH₂)₃CHCHCMe₃], converts in the solid state to the allyl-hydride complex Cp*Mo(NO)(H)(η³-CH(CH₂)3CCHCMe₃). With larger cyclic olefins (cyclohexene through cyclooctene) the initial cis-metallacycles isomerize to trans-metallacycles of the form Cp*Mo(NO)[trans-η²-CH(CH₂)nCHCHCMe₃] (n = 4, 5, 6), and these subsequently convert with loss of dihydrogen to η⁴-diene complexes, Cp*Mo(NO)[η⁴-CHCH(CH₂)n-₁CCHCMe₃]. Thermolysis of the η⁴-diene complexes in cyclohexene results in decomposition of the organometallic complex with small amounts of oligomer formation. Thermolysis of Cp*W(NO)CH₂CMe₃)₂ in cyclic-olefm substrates generates the alkylidene intermediate [Cp*W(N0)(=CHCMe₃)], which couples with cyclic olefins in a manner analogous to the Cp*Mo-system. Tungsten trans-metallacycles are observed by ¹H NMR spectroscopy, but the organometallic subsequently reacts further with loss of the coupled neopentyl-cyclic olefin and coordination of two substrate molecules to form the putative Cp*W(NO)(cyclic olefm)₂ complex. Two additional cyclooctene products are isolated, the 1,4- diene Cp*W(N0)[η⁴-CHCH(CH₂)₅CHCCH(CH₂)₆] and the allyl hydride Cp*W(NO)(H)[η³- CH(CH₂)₆CCCHCH(CH₂)₅], both containing two coupled cyclooctene molecules. A tungsten cis-metallacycle forms with 2,5-dihydrofiiran, but a ring-opened alkoxy-allyl complex forms with 3,4-dihydro-2H-pyran, and 1,2,3,6-tetrahydro-pyridine undergoes N -H bond activation to afford an amido product. CpW(NO)(CH₂CMe₃)₂ produces some oligomers of cyclohexene, but in all other reactions bimetallic decomposition pathways predominate.

Neuropathic (NP) pain is a debilitating form of chronic pain that can result from a variety of disease states that target the sensory nervous system. NP pain can manifest as burning or shooting pain, and is commonly refractory to traditional analgesics including opioids. Clinically used opioids target the mu opioid receptors (MORs); preclinical reports, however, suggest that the delta opioid receptor (DOR) may be a valid target. It is now accepted that MORs and DORs oligomerize in heterologous expression systems and exhibit novel pharmacology distinct from their monomers. This research aimed to utilize both functional and molecular evidence to identify if a heteromeric mu-delta opioid receptor oligomer (M/DOR) forms in vivo and whether it is upregulated in NP pain. In an animal model of NP pain, animals displayed characteristic behaviours including protecting of the ipsilateral hindpaw from environmental stimuli, and mechanical allodynia in this paw. Behavioural studies reported that acute injection of DOR- selective agonists that bind M/DOR produced enhanced thermal antinociception and reversed mechanical allodynia in NP rats. DOR agonists that have low binding affinity for M/DOR did not produce enhanced thermal antinociception but did reverse mechanical allodynia in NP rats. Molecular studies were employed to characterize the molecular species of ORs in the lumbar spinal cord. Isolated spinal cord membranes were subjected to coimmunoprecipitation with a M/DOR antibody. Co-immunoprecipitation was unable to conclusively identify changes in M/DOR levels in the dorsal horn but did confirm that such a species exists in vivo. Furthermore, antibody characterization was completed to determine if the commercial antibodies used were labeling the appropriate OR proteins. HEK293T cells transfected with MOR and/or DOR plasmids were used in Western blotting and immunocytochemistry protocols to test commercially available antibodies. These studies determined that MOR and DOR antibodies do label their respective OR type, but also recognize additional proteins (non-specifically) in Western blotting protocols. In conclusion, behavioural studies revealed a putative role for M/DOR agonists in the treatment of NP pain; however, more sensitive tools and protocols must be developed before molecular experiments are able to identify quantifiable changes with endogenous M/DORs. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2009-09-24 12:43:58.472