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1
Easton, Christopher J. "Aspects of biological and organic chemistry, particularly amino acid, cyclodextrin, and free radical chemistry /." Title page, contents and abstract only, 1997. http://web4.library.adelaide.edu.au/theses/09S.D/09s.de13.pdf.
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Ding, Xiao Dong. "Synthesis and characterization of heme models and spectroelectrochemical studies of heme proteins." Diss., The University of Arizona, 1997. http://hdl.handle.net/10150/282523.
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To better understand the relationship between the structure and electronic properties of the iron center and the functions of heme proteins, both naturally occurring heme proteins and synthetically prepared heme models have been studied. The reduction potential (E°') and the pH dependence of E°' of nitrophorin 1 (NP1) and myoglobin (Mb) were determined by spectroelectrochemical techniques. The difference in the electrostatic interaction of the Fe(III) center with buried charged groups in the heme pocket of Mb and NP1 is the major factor that causes the 300 mV difference in E°. The pH dependence of the E°' determined between pH 5.5 and 7.5 is small for both Mb and NP1 because they have the same axial ligands. Three meso-ortho-phenyl substituted porphyrins, (o-F), (o-CF₃) and (2,6-Cl₂)(p-OCH₃)₃TPP and one meso-para-phenyl substituted porphyrin, (p-OCH₃)₄TPP, were synthesized as models of cytochrome b₅ . Cyclic voltammetry was used to measure their reduction potentials. The overall formation constants, logβ₂III and logβ₂II, have been calculated based on the reduction potentials of the iron(III)/(II) couple as a function of N-methylimidazole concentration. The values of logβ₂III are in the order of o-F > o-CF₃ > p-OCH₃ ≈ 2,6-Cl₂, indicating that the electron-donating ability is in the order of o-F < o-CF₃ < p-OCH₃ ≈ 2,6-Cl₂. The overlap and direct transfer of electron density from the halogen to the iron in the product reduces the Lewis acidity of iron(III), resulting in decreased logβ₂III. The order of logβ₂II for N-methylimidazole complexed Fe(II) porphyrinates is similar to that of the Fe(III) complexes, indicating no major difference in the Lewis acidity of Fe(II) as compared to Fe(III). Basket handle porphyrinates with covalently bound methionine and aliphatic amine model ligands (RCH₂SCH₃, RCH₂SCH₃ and RNH₂, RNH₂) were chosen as precusor of cytochrome c and f. The Fe(III) complexes were to be prepared and investigated by electrochemical and spectroscopic techniques. The precursor porphyrin was synthesized. Several schemes were investigated for the synthesis of the handles having methylthioether and aliphatic amine without success, and it was decided not to continue this project. Therefore, no final basket handle porphyrin was available for further characterization.
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Agha, Kazim Ally. "Synthesis, physicochemical and biochemical properties of C3'-modified 2',5'-linked oligonucleotides." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=84457.
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Oligoribonucleotides comprising of 2',5' -linked internucleotide linkages are known to bind selectively to RNA over DNA. The ability to bind to RNA renders them suitable as probes for many biological applications, such as 'antisense technology'. Little is known about the effect of sugar structure (and conformation) on the binding properties of 2',5'-linked oligonucleotides. To get insight into the role of sugar conformation, 2 ',5'-linked oligonucleotides modified at the C3'-position of the furanose ring were synthesized via solid phase synthesis and their binding to complementary single stranded DNA and RNA was studied. Their application as antisense oligonucleotides was also evaluated.<br>The first analogue studied was the C3'-epimer of 2',5'-linked ribonucleic acids (2 ',5'-RNA), that is, an oligonucleotide in which the ribofuranose sugar is replaced by xylofuranose (2' ,5'-XNA). This was followed by the synthesis and analysis of the C3'-fluorinated xylofuranose analogue (2',5'-FXNA). The sugar conformation in these oligonucleotides are believed to have a very high population of the C3'-endo ('extended') conformation. Consistent with this notion, CD structural studies indicated that 2 ',5,'-linked XNA and FXNA show structural similarities to the 'extended' C2'- endo form of DNA. We found that both 2',5 '-XNA and 2',5'-FXNA bound weakly to complementary single stranded DNA and RNA. Neither of the xylooligomers resulted in RNaseH activated degradation of RNA.<br>The last modified oligonucleotide to be studied was the C3' -fluorinated-2',5'-linked ribonucleic acids (2',5'-FRNA), which has a compact C2'-endo sugar conformation. In contrast to 2',5'-FXNA, 2 ',5'-FRNA bound strongly to complementary oligonucleotides and showed structural similarities to RNA (CD spectroscopy). Our studies showed that it did not cause RNaseH based degradation of RNA.<br>These studies are consistent with the notion that the effect of sugar conformation in 2',5'-oligonucleotides is opposite to that of 3',5'-oligonucleotides. In other words, a C3'-endo sugar conformation in 2',5'-oligomers renders the oligonucleotide as 'extended' and portrays itself equivalent to the 'extended' DNA conformation (which has C2'-endo conformation), whereas a C2'-endo 2 ',5'-oligonucleotide adopts a 'compact' conformation that is equivalent to that seen in 3',5 '-oligonucleotides adopting the C3'- endo pucker (e.g. RNA).
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Morley, Krista Louise. "Focusing mutagenesis into the active site to improve hydrolase selectivity." Thesis, McGill University, 2005. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111830.
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Occasionally, researchers need to modify enzymes through amino acid substitutions to make them more efficient catalysts for organic synthesis. There is still debate over the best protein engineering strategy for improving enzyme enantioselectivity: rational design or directed evolution. Rational design experiments focus mutations close to the active site, while directed evolution experiments often find mutations far from the active site.<br>In this thesis, a combination of the two strategies improved Pseudomonas fluorescens esterase (PFE) for production of a useful synthetic building block for organic synthesis. Random mutagenesis within the active site increased enantioselectivity more effectively (up to 5-fold reaching E = 61) than random mutagenesis of the entire protein (only 1.5-fold reaching E = 19). A general survey of previously published enzyme improvements showed that closer mutations were more effective than distant mutations for improving enantioselectivity. On this basis, we proposed that random mutagenesis focused in the active site may dramatically increase the success rate in future directed evolution experiments. The X-ray crystal structures of three improved PFE mutants showed that mutations directly in the active site can increase enantioselectivity without significantly altering the shape of the binding pocket. For rationalizing the improved enantioselectivity, a crystal structure of a transition state analogue-complex provided the conformation of the fast reacting enantiomer and computer modeling determined the conformation of the slow reacting enantiomer.<br>When novel esterases are discovered from directed evolution experiments, they are screened with libraries of esters to identify their preferred substrates. A convenient method for the parallel synthesis of esters was developed by using solid-supported reagents to eliminate traditional purification.<br>Acetyl xylan esterase (AxeA) was examined as a potential catalyst for the production of chitosan, a biopolymer with many commercial applications. Screening for chitin deacetylase activity showed that AxeA preferentially deacetylates chitosan oligosaccharides over alkali-treated chitin and crystalline chitin.
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Roman, Miguel Angel. "Synthesis of branched nucleosides and oligonucleotides containing flexible alkylamine linkers on the heterocyclic bases." Thesis, McGill University, 1995. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=22797.
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The N3 position of thymidine was alkylated with different sized bromoalkyl phthalimide linkers in generally good yields, with no alkylation observed on the sugar ring. The free amino group, available after methylamine deprotection of the phthalimide linker, was protected with the levulinyl protecting group (e.g., 20). Branched 'Y'-shaped nucleic acids containing branching monomer 20 were synthesized using standard solid phase synthetic methodology and complexes of these branched nucleic acids with one and two mole-equivalents of linear complement dA$ sb{10}$ are investigated by thermal melting. At the branching point the levulinic amide exhibited poor lability to Letsinger's hydrazine solution and proved to be incompatible with the solid phase synthesis of branched nucleic acids. The primary amino group provided a handle which was used to extend the size of the linker incorporating a primary hydroxyl group at the site of levulinic protection (e.g., 37, 38). The levulinic ester showed much better lability to hydrazinolysis rendering it more compatible with the solid phase synthesis of branched nucleic acids. An extensive one and two dimensional NMR characterization of the functionalized nucleosides is reported.
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Park, Seongsoon. "Enhancing hydrolase activity and selectivity by medium, substrate, and protein engineering." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=83088.
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Researchers use enzymes for enantio- and regioselective reactions because of their high selectivity and activity toward natural substrates. However, researchers sometimes need to modify the reaction system or the enzyme itself to get reliable selectivity and activity when they deal with unnatural substrates. To obtain researcher's need, one can change the solvent, modify the substrates, or alter the enzyme itself. These processes are called medium, substrate, and protein engineering, respectively.<br>This thesis deals with hydrolases, which are classified by EC 3. We applied the proper approach to improve their activity and selectivity depending on the reactions. For the first approach, highly polar ionic liquids were applied to lipase-catalyzed acylation. Ionic liquids worked reliably in enantio- and regioselective lipase-catalyzed reactions. In particular, ionic liquids dissolved polar substrates such as glucose and L-ascorbic acid, thereby facilitating their acylations. In the second approach to improving enantioselectivity of CAL-B (Candida antarctica lipase B) in beta-lactam ring opening reactions, we changed the nucleophile from water to a range of alcohols. Longer, secondary alcohols increased the reaction rate as well as the enantioselectivity. Molecular modeling revealed that the high enantioselectivity of CAL-B and the critical role of alcohols. For the last approach, structure-guided random mutagenesis was applied to increase the enantioselectivity of PFE ( Pseudomonas fluorescens esterase) toward MBMP (methyl 3-bromo-2-methylpropionate). The homology model was used to select amino acid residues for mutagenesis near the stereocenter of the docked tetrahedral intermediate of the substrate. Randomization of these residues yielded a Val122Ser mutant with E increased to 61 (from 12 of wild type enzyme), as well as a Val122Met mutant to 36.
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Zhou, Wen-Qiang. "The synthesis of amide-linked 2',3'-cyclopropanated dinucleosides and the effect of their incorporation into DNA-strands on duplexation." Thesis, McGill University, 1995. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=40024.
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In order to establish if binding affinity could be improved by conformationally restricting the amide backbone, exo-amide-linked and endo-amide-linked 2$ sp prime$,3$ sp prime$-cyclopropanated dinucleoside analogs (e.g. dimers 35, 54, and 55) have been studied. The dimers were synthesized by coupling the corresponding cyclopropyl acids (5$ sp prime$-end building unit) with aminothymidines (3$ sp prime$-end building unit) by standard peptide synthesis methodology. After proper functionalizations, the dimers were incorporated into DNA sequences, and the effects of their incorporation into DNA-strands on binding to complementary DNA and RNA were evaluated.<br>Different strategies were explored to prepare the carboxylic acid-functionalized 2$ sp prime,3 sp prime$-cyclopropanated nucleoside analogs as the 5$ sp prime$-end building block. It was found that reaction of the $ alpha, beta$-unsaturated selenonyl uridine with the anions of 2-substituted acetates could efficiently yield the ester-functionalized cyclopropanes (e.g. 32 and 49) in a stereoselective manner, through a Michael-type cyclopropanation mechanism. Proper transformations of the ester precursor successfully provided the desired acid derivatives (e.g. 33, 52 and 53).
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Mangos, Maria M. "Factors governing the design, selection and cleavage of sugar-modified duplexes by ribonuclease H." Thesis, McGill University, 2005. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=85579.
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The antisense principle bases its premise in the exquisite complementarity of a synthetic, chemically-modified oligonucleotide to tightly bind with a unique target RNA sequence. Rapid and selective genetic discrimination, as driven by the formation of multiple points of target contact, constitutes a central goal of oligonucleotide therapies. Most synthetic designs have, however, provided little structural insight on the role of the antisense oligonucleotide (AON) in triggering RNA cleavage of preformed hybrids, as catalyzed by a ubiquitous, intracellular enzyme known as ribonuclease H. The use of RNase H to assist AON inhibition of gene expression is crucial to mainstream antisense technologies, yet the precise mode by which this enzyme acts on AON/RNA duplexes remains unclear.<br>To address the role of substrate structure on enzyme activation, a dominant theme of this thesis highlights the design, synthesis and structural studies of novel AONs comprised of rigid 2'-deoxy-2'-fluoroarabino (2'F-ANA) or native (DNA) nucleotides, containing interspersed flexible (e.g. "2',3'-seconucleotides") or anucleosidic (e.g. butyl) residues. This unique AON class combines both pre-organization & flexibility within the hosting heteroduplex, which on their own usually prove detrimental towards enzyme trigger. Their combination, however, synergistically activates both E. coli and human RNases H, leading to potent destruction of duplexed RNA. These compounds thus represent the first examples of modified AONs lacking deoxyribose sugars that elicit RNase H activity comparably to the native (DNA) systems. DNA-derived AONs with acyclic residues also amplify enzyme-catalyzed target degradation, suggesting the added flexibility imparted to the substrate structure to be vital for ameliorating the protein/nucleic acid interaction. Melting and circular dichroic experiments have revealed that the enhanced dynamics associated with a particular acyclic modification remain globally undetectable, indicating the acyclic residues induce only local structural deformations to the helix architecture.<br>Intricate comparisons of the structural and biological properties of various acyclic residues (e.g. butyl, propyl and ethyl interresidue spacers) designed to locally compress or expand the AON helix backbone at a defined axial site has enabled a deeper understanding of the conformational factors that underlie the observed enhancements.
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Kutterer, Kristina M. K. "Urea and carbamate backbone modified DNA & work towards a synthesis of polyoxin L and analogues." Thesis, McGill University, 1995. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=28808.
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Urea, N-methylurea and carbamate linked thymidine dimers, 38, 37 and 39 respectively, were synthesized efficiently and incorporated into 12- and 19-mer DNA oligonucleotide strands, via solid-phase synthesis. Thermal denaturation studies utilizing complementary single stranded DNA, RNA and duplex DNA, indicated selectivity of binding of all three backbone modified oligomers to single stranded DNA. The oligomer containing dimer 37 exhibited the strongest binding to single stranded DNA. Carbamate and 5$ sp prime$-N-methylurea modified dimers suitable for the preparation of longer nucleoside homopolymers were efficiently prepared from either thymidine or 3$ sp prime$-azido-3$ sp prime$-deoxythymidine.<br>Three efficient syntheses of precursors (7, 33, 36) of carbamoylpolyoxamic acid starting from L-arabinose were developed. Utilizing uridine as the starting material, an expedient synthesis of a precursor (44) to the nucleoside moiety of polyoxin L was also achieved. The conversion of cyanides to carboxylic acids was extensively investigated.<br>Protected polyoxamic acid 17 was synthesized in 11 steps from L-arabinose.* ftn*Please refer to the dissertation for diagrams.
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Chefson, Amandine. "Towards the use of P450 enzymes in synthesis : cofactor replacement and activity of CYP3A4 in non-aqueous media." Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=100784.
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Enantioselective synthesis is one of the most important challenges of today's synthetic chemists. In particular, the hydroxylation of non-activated C-H bonds remains a significant challenge that few chemical catalysts have succeeded to overcome. The P450 enzymes, a family of heme-containing monooxygenases including more than 5000 known isoforms, are gaining considerable attention due to their ability to catalyze the very difficult regio- and stereo-selective oxidation of inactivated C-H bonds. The use of such enzyme is however limited by their functional complexity, low activity, need for cofactors, and poor stability. In this thesis, we elected to study the human P450 CYP3A4, because of its high substrate promiscuity. The first part of the project involved the replacement of the required cofactors (NADPH and cytochrome P450 reductase) by some cheap hydrogen peroxide donors or organic peroxides. Several surrogates, such as sodium percarbonate and cumene hydroperoxide, were found to be efficient at replacing the natural cofactor, without a significant loss of stability and activity. The second part of this thesis deals with optimization of the lyophilization conditions. Among the numerous additives tested, some sugars led to significant lyoprotection during the freeze-drying process. Finally, in the third part, the effect of the presence of organic solvents and ionic liquids on CYP3A4 activity was evaluated.
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Krupa, Joanne C. (Joanne Christine). "Coenzyme analogs in dehydrogenase-catalyzed reactions." Thesis, McGill University, 1995. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=39934.
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To enhance the performance of dehydrogenases in synthetic organic chemistry, coenzyme analogs of NAD(P)$ sp+$/NAD(P)H were examined. Our study shows that dehydrogenases are more efficient with NAD(P)$ sp+$ than with the APAD(P)$ sp+,$ FPAD$ sp+$ or SPAD(P)$ sp+$ under non-regenerating oxidative conditions in terms of yields. Under regenerating conditions, APAD$ sp+$ often exceeded NAD$ sp+$ in terms of product formation. Lower yields were observed with SPAD(P)$ sp+$ and FPAD$ sp+.$ These coenzyme analogs also displayed reduced reactivity, but APAD$ sp+$ was the most promising of the three. In reduction experiments, the analogs are as equally or slightly less efficient as NAD(P)H in terms of yields, but react slower. Electropotentials could not explain all of the variation in yields. Therefore, to better understand the interactions made by the 3-substituent of the nicotinamide ring in the dehydrogenase active site, the binding properties of 1-methyl-3-substituted pyridinium salts (iodide and chloride) to 8 different dehydrogenases were examined. The salts were R = CONH$ sb2,$ COONa, CN, CH$ sb3,$ CONHCH$ sb3,$ CH$ sb2$OH, CH$ sb2$CN, Cl, H, CH(OH)$ sb2,$ COOCH$ sb3$ and COCH$ sb3$ did not bind well into the active site. However, the five compounds that did bind, in order of their observed increased inhibitory effects are R = CH=NOH, CONHNH$ sb2,$ CSNH$ sb2,$ NH$ sb2$ and ONa. The ONa chloride salt binds on average 336 times more tightly than CONH$ sb2.$ It is proposed that the tightly bound compounds with electron-withdrawing substituents (CH=NOH, CONHNH$ sb2,$ CSNH$ sb2)$ and pKa's in the range of 9.4-10.4 form stronger hydrogen bonds and the two with strong electron-donating groups (ONa and NH$ sb2)$ with low pKa's in their protonated forms bound in a geometry optimized for efficient hydrogen bond exchange.<br>The difference in binding between the nicotinamide and thio derivatives can be geometry related. The X-ray crystal structure of 1-methyl-thionicotinamide shows a different ground state orientation (s-cis) than that previously reported for 1-methyl-nicotinamide (s-trans). From ab initio calculations, the thio derivative has a global and local minima at $ approx$30$ sp circ$ and 135$ sp circ$ as compared to 0$ sp circ$ and 180$ sp circ$ respectively for the oxygen compound. However, both compounds have an out-of-plane conformation in crystal. The enzyme favored trans out-of-plane conformation may also be more easily accessible for the thio than the oxygen derivative since the equilibrium constant for the cis to trans process is favored by a factor of 5.4.<br>By extension, the geometry that SPAD(P)$ sp+$ adopts to maximize hydrogen bond formation may cause it to have a slow turnover rate and to be an inefficient hydride acceptor, while APAD(P)$ sp+$ which is not as tightly bound to dehydrogenases, is a more efficient coenzyme.
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Qiu, Wei. "Design and synthesis of conformationally and topographically constrained amino acids as peptidomimetics." Diss., The University of Arizona, 2001. http://hdl.handle.net/10150/280486.
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A major goal of peptide research has been to elucidate or understand the relationships between a peptide's three-dimensional structure and its biological activity. De Novo design of peptide mimetics requires assembling all components necessary for molecular recognition and transduction, which needs the proper choice of a template that can place the key side chain residues in 3D space. Two widely used methods are novel β-substituted amino acids and conformationally constrained secondary structure mimetics. In this thesis, we report our efforts to fulfill the aforementioned criteria in synthesizing β-isopropyl aromatic amino acids and constrained reverse turn dipeptide mimetics. Through asymmetric Michael addition reaction, highly topographically constrained β-isopropyl aromatic amino acids have been synthesized. In order to develop a general approach to synthesize these novel amino acids, we re-examined the reaction conditions for Evans' diastereoselective 1,4-addition, and found conditions which gave excellent diastereoselectivities and good chemical yields. A concise and straightforward five-step synthesis of [5.5]-bicyclic reverse turn dipeptide mimetic scaffolds with side chain functionality at the i+1 and i+2 positions has been developed. In the bicyclic structure, two dihedral angles (ψ₂ and φ₃) are greatly restricted. Further development of this synthesis will enable us to prepare various types of reverse turns with different backbone geometry and side chain topography. Enantiomerically pure (S)-trans-cinnamylglycine and (S)-α-trans-cinnamyl-α-alanine have been prepared via reaction of chiral Ni (II)-complexes of glycine and alanine respectively, with cinnamyl halides. Inexpensive and readily available reagents and solvents are used, including a recyclable chiral ligand. The simplicity of the experimental procedures and high stereochemical outcome make this method synthetically attractive for preparing the target amino acids on multi-gram scales. Further studies by incorporating these mimetics into potent peptide analogues will greatly help us to understand the bioactive conformation of the parent peptides.
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Labell, Rachel. "Synthesis and characterization of galactosyl lipids that bind HIV-1 gp120." Diss., The University of Arizona, 2001. http://hdl.handle.net/10150/289764.
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The HIV-1 virus has a protein, gp120, on its surface that is responsible for the initial recognition between the virus and human cells by binding to the CD4 receptor, which is found on many types of human cells. An alternative receptor, galactosylceramide (GalCer), has also been identified. It binds to HIV-1 gp120 and facilitates the infection of human cells via a CD4 independent mechanism. The goal of this research project was to design, synthesize, and test the effectiveness of galactosyl lipids that bind to gp120. A versatile synthesis was developed and used to synthesize five different GalCer analogs. Professor Scott Saavedra and coworkers used total internal reflection fluorescence microscopy (TIRF) to measure quantitative binding affinities to gp120 at equilibrium for each glycolipid analog. A GalCer analog with octadecyl lipid chains and a tetraethylene glycol spacter group had the highest binding affinity of the analogs tested. Monolayers of lipid mixtures were investigated for phase behavior using epifluorescence microscopy. It was determined that GalCer analogs with saturated tails formed domains in monolayers with DOPC. GalCer analogs were also incorporated into liposomes and were subjected to an HIV-1 inhibition assay in Dr. Ahmad's lab. The GalCer analog liposomes showed similar inhibition as GalCer liposomes.
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Bowen, Martina E. "I. Use of 9-methylfluorene as an indicator for organometallic titrations II. Synthesis of a branched polyethylene glycol linker for peptide ligands III. Synthesis of a linker for peptide ligands and a study of its pH sensitivity." Diss., The University of Arizona, 2004. http://hdl.handle.net/10150/290107.
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This work details the synthesis of some useful organic molecules: (I) 9-Methylfluorene was tested as an indicator in the titration of commonly used organometallic reagents. This indicator is readily prepared in three steps from fluorenone. In THF solution the deprotonated indicator is red and exhibits a sharp endpoint. The highly basic reagents sec-butyllithium and tert -butyllithium can be titrated in ether solution, where the color of the deprotonated indicator is yellow. (II) A branched, polyethylene glycol based linker for peptide ligands was designed and synthesized. The linker needed to be water soluble, to be stable under both acidic and basic conditions, to have amine and carboxyl termini for use in solid phase peptide synthesis, and to have an attachment site for a fluorescent marker. The polyethylene glycol linker was designed with a carboxyl terminus and two amine termini that can be differentially protected to facilitate selective deprotection and reaction with three peptides or two peptides and a fluorescent tag. (III) A molecule containing a linear polyethylene glycol linker with beta-alkoxyamide moiety was synthesized and its pH stability determined using an HPLC method. Stability was assayed in 5 mM buffers at pH 4, 7, and 9 over 24 hours. No decomposition in these solutions was detected. The linker was then subjected to 10 mM acid or base solution and analyzed over 24 hours. Again, no decomposition was observed.
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Wallace, Clare E. "Investigating Conformational Changes in Sialylations by using a C-3 Labeled Sialyl Donor." Thesis, Southern Illinois University at Edwardsville, 2014. http://pqdtopen.proquest.com/#viewpdf?dispub=1560926.
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<p> The synthesis of complex carbohydrates containing sialic acid is very challenging by chemical methods, in part due to the competing elimination reaction, as well as poor stereocontrol. One major disadvantage in the field is the limited knowledge of the mechanism of sialylations. The mechanism is believed to go through an oxacarbenium ion intermediate. Recent findings suggest the existence of an all-axial conformation of the oxacarbenium ion that might be important in controlling the overall efficiency of sialylations. As a part of a general research goal to gain a better understanding of sialylations, we describe herein the synthesis of a C-3 deuterium-labeled substrate that might give insight on the existence of such conformation.</p>
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Simpson, Levi Samuel. "The development and application of methodologies for the study of sulfate monoesters in biological systems." [Bloomington, Ind.] : Indiana University, 2007. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3297086.
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Thesis (Ph. D.)--Indiana University, Dept. of Chemistry, 2007.<br>Title from dissertation home page (viewed Sept. 26, 2008). Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 1019. Adviser: Theodore S. Widlanski.
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Amato, Nicholas J. "Impact of DNA Structure and Aeropyrum pernix Single-Strand DNA Binding Protein on Oxidative Damage to DNA." University of Toledo / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1372296254.
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Mukherjee, Herschel. "On the Biological Activity of the Natural Product (+)-Avrainvillamide." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:17467289.
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Nucleophosmin (NPM1) is a multifunctional phosphoprotein localized predominantly within the nucleoli of eukaryotic cells. Mutations within its C-terminal domain are frequently observed in patients with acute myeloid leukemia (AML), are thought to play a key role in the initiation and progression of the disease, and result in aberrant, cytoplasmic localization of the mutant protein. It has previously been demonstrated that the electrophilic antiproliferative natural product (+)-avrainvillamide binds to proteins, including nucleophosmin, by ¬S-alkylation of cysteine residues. In this thesis we report that the biological activity of avrainvillamide is mediated by NPM1 and the nuclear export receptor exprtin-1 (Crm1). Using mass spectrometry, we demonstrate that the antiproliferative activity of a series of avrainvillamide analogs correlates with their ability to bind C-terminal NPM1 truncation constructs; it is also observed that the interaction between avrainvillamide and the C-terminal domain of NPM1 is fully reversible under our experimental conditions. We report that avrainvillamide restores nucleolar localization of certain AML-associated mutant forms of NPM1 and provide evidence that this relocalization is mediated by interactions of avrainvillamide with mutant NPM1 and Crm1. Immunofluorescence and mass spectrometric experiments employing a series of different NPM1 constructs suggest that a specific interaction between avrainvillamide and cysteine-275 of certain NPM1 mutants mediates the relocalization of these proteins to the nucleolus. Avrainvillamide is further shown to inhibit nuclear export of Crm1 cargo proteins, including AML-associated NPM1 mutants; this marks the first evidence that avrainvillamide directly influences the biology of a cellular target other than NPM1. We also observe that avrainvillamide treatment displaces thr199-phosphorylated NPM1 from duplicated centrosomes, leads to an accumulation of supernumerary centrosomes, and causes mitotic defects in vitro. Finally, we show that avrainvillamide treatment increases levels of thr199-phosphorylated NPM1 by inhibiting the action of protein phosphatase 1 beta on phosphorylated NPM1, thereby indirectly displacing NPM1 from nucleoli and destabilizing nucleolar and nuclear structure.<br>Chemistry and Chemical Biology
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Tahira, Ambreen. "Synthesis of Bivalent Organothiophosphate Inhibitors and Their Inhibition of Butyrylcholinesterase| Studies towards a Potential Treatment of Cognitive Loss Associated with Alzheimer's Disease." Thesis, California State University, Long Beach, 2017. http://pqdtopen.proquest.com/#viewpdf?dispub=10636600.
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<p> Recently, it has been shown that a decrease in activity of acetylcholinesterase (AChE) in AD patients is compensated by an increase in butyrylcholinesterase (BuChE) activity. Therefore, BuChE also becomes a significant target in the treatment of cognitive loss associated with Alzheimer’s disease. While the majority of drug development so far has centered on AChE inhibitors in order to increase the acetylcholine level in AD patients, the development of specific and potent BuChE inhibitors have also begun to attract attention. We recently synthesized and assayed a library of bisthiophosphates as potential BuChE inhibitors. To evaluate if the analogs were selective for BuChE, their inhibitory properties against both BuChE and AChE were determined. </p><p>
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Priem, Jessica. "Main Group Elements Supported by pi-Conjugated, Nitrogen-Rich Ligand Frameworks and the Catalytic Formation of Guanidines." Thesis, University of Ottawa (Canada), 2010. http://hdl.handle.net/10393/28656.
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This thesis investigated the structure and reactivity of group 13 elements boron, aluminum and gallium supported by nitrogen rich ligand systems. The majority of this work deals with N,N',N"-trisubstituted guanidinate ligands and N,N'-disubstituted-1,8-diaminonaphthalene dianionic ligand frameworks. New methods for the catalytic formation of guanidines have also been explored.
Chapter 1 outlines the basics of using nitrogen rich compounds as ligands. This includes the introduction of guanidinates as supporting ligands and the description of using a rigid backbone system such as an N,N'-disubstituted-1,8-diaminonaphthalene dianionic ligand.
Chapter 2 presents the synthetic routes taken to form a variety of aluminum amide, alkyl and halide complexes using N, N',N"-trisubstituted triisopropyl guanidinates as supporting ligands. The formation of dinuclear guanidinate species was also observed.
Chapter 3 investigates the catalytic formation of guanidines using inexpensive, commercially available aluminum compounds as catalysts for the guanylation of various amines with carbodiimide. A full catalytic cycle was calculated using DFT studies for both guanylation of amines and phosphines catalyzed by aluminum amides.
Chapter 4 presents the application of N,N'-disubstituted-1,8-diaminonaphthalene ligands for the stabilization of coordinately unsaturated group 13 elements. The formation of boron halides with both diaryl and diisopropyl 1,8-diaminonaphthalene ligands as well as aluminum halides and a dinuclear aluminum complex is presented.
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Kaye, P. T. """Of molecules and men"" : inaugural lecture delivered at Rhodes University." Rhodes University, Grahamstown, 1989. http://hdl.handle.net/10962/d1020712.
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Lamparski, Henry Gennady. "Polymerization in two-dimensional assemblies of sorbyl-containing lipids." Diss., The University of Arizona, 1993. http://hdl.handle.net/10150/186492.
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Mono- and bis-substituted phosphatidylcholines (PC) containing a polymerizable sorbyl ester group at the acyl chain terminus were synthesized. The thermotropic phase behavior, two-dimensional polymerizability, and polymerized assemblies stability were investigated. The thermotropic phase behavior of mono- and bis-sorbylPCs was examined by differential scanning calorimetry. Each SorbPC exhibited a single endotherm which occurred at temperatures below the main phase transition (T(m)) of the corresponding linear saturated PC. Variations in the chain length of bis-SorbPC resulted in a pronounced odd/even alternation of the T m. The interaction of the sn-2 chain sorbyl ester carbonyl with neighboring methylene chains appears to be predominantly intermolecularly or intramolecularly depending on whether the chain length is even or odd, respectively. Intermolecular orientation of the sorbyl ester carbonyl decreased the T(m) to a greater extent than intramolecular orientation. The magnitude of the odd/even effect diminished as the chain length increased and the van der Waals interchain interactions increased. Lipid bilayers composed of either mono- or bis-SorbPCs were thermally polymerized (60°C) to high conversion with the radical initiator AIBN. Transesterification of poly-(SorbPC), resulting in removal of the lipid headgroup, yielded a soluble polymer which was analyzed by size exclusion chromatography relative to PMMA standards. The relative number-average degree of polymerization (X(n)) varied from 50 to nearly 600, and was proportional to [I]⁻¹. The Xn was identical for both mono- and bis-substituted SorbPCs at constant [I]. These results suggest that at high conversion the chain termination of the growing polymer occurs by primary radical termination, rather than bimolecular chain termination. UV-photopolymerization of mono-Sorb PC yielded oligomers. The stability of poly-(SorbPC) vesicles to detergent dissolution was examined by quasielastic light scattering. Poly-vesicles consisting of linear polymers having a X(n) of 50 did not undergo lysis with detergent, whereas UV-polymerized vesicles with X(n) of 3-5 were lysed. Prolonged UV-irradiation of bis-SorbPC vesicles resulted in stabilization to detergent, a result of extensive crosslinking of the short polymer chains. UV-polymerized vesicles of mono- and bisSorbPC were only stabilized if the mole fraction of bis-SorbPC was ≥ 0.4.
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Colla, Alexander J. "Development of an efficient approach for the incorporation of a series of fluorotyrosines in peptides." Thesis, California State University, Long Beach, 2015. http://pqdtopen.proquest.com/#viewpdf?dispub=1603748.
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<p> Site-directed mutagenesis provides a powerful tool in the study of enzyme function. Residues suggested to be important for catalysis can be readily mutated and the energetic effects measured. However, the limited repertoire of naturally occurring amino acids constrains the substitutions that can be made. To obtain a deeper understanding of how enzymes work requires using unnatural amino acids to systematically perturb enzymatic residues. For example, hydrogen bonds in an ‘oxyanion hole’ are a common feature of enzyme active sites and often suggested to be important for catalysis. However, water can form hydrogen bonds, so for enzyme-mediated hydrogen bonds to be catalytic the energetics of these hydrogen bonds must be different than those made with water. A previous study in the enzyme ketosteroid isomerase (KSI) used a series of fluorotyrosine analogs to perturb the p<i>K<sub>a</sub></i> of the tyrosine hydrogen bond donor and results suggested a modest catalytic contribution of oxyanion hole hydrogen bonds. However, challenges in synthesis limited the set of fluorotyrosine analogs used. To overcome these challenges and extend the series of fluorotyrosines used in enzymatic studies, we developed an approach to selectively incorporate fluorotyrosines in peptides using silyl-based protecting groups. The fluorotyrosine must be protected on the amino and phenol groups and then incorporated in a peptide using solid phase synthesis. More so, the protection chemistry must be friendly in such a way that it does not have drastic side effects on any other part of the system. We tested the stability of silyl groups including TBDMS-Cl, TIPS-Cl, and TBDPS-Cl for their use in peptide synthesis. With the doubly protected fluorotyrosines, solid phase peptide synthesis will occur in order to place the new tyrosine into the protein of interest where these analogs will be used to investigate the energetics of enzymatic hydrogen bonds.</p>
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Onaran, Mehmet B. "Design and synthesis of bioactive small molecules and high-throughput identification of enantiomeric excess." View abstract/electronic edition; access limited to Brown University users, 2008. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3319114.
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Cavagnero, Silvia 1962. "Structure-activity studies of delta-selective opioid analogues." Thesis, The University of Arizona, 1990. http://hdl.handle.net/10150/278183.
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The two structurally different peptides DPDPE and Dermenkephalin show a similar remarkably high affinity and selectivity for the delta opioid receptor subtype. An effort has been made to gain some insight into the factors responsible for the recognition ability of these two molecules by synthesizing some DPDPE-Dermenkephalin peptide hybrids and some conformationally restricted Dermenkephalin analogues. The results of the binding and the in-vitro bioassays have been compared with those of the parent peptides. A general decrease in receptor affinity has been observed in the peptide hybrids while the dermenkephalin analogues have shown a wider range of affinities and selectivities. The above findings contribute to the understanding of the structural requirements of the delta receptor, provide information about the sensitivity of Dermenkephalin to enzymatic degradation, and indicate directions for future research.
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Ferguson, Ronald Dale 1966. "Design, synthesis and biological screening of combinatorial chemical libraries." Thesis, The University of Arizona, 1996. http://hdl.handle.net/10150/278584.
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Although combinatorial libraries owe their inception to applications in peptide and bacteriophage libraries, the breadth of current applications include solution phase chemical reaction optimization, material science investigation, natural products modifications, and agricultural research. As a conceptual application, combinatorial library techniques can enhance a researcher's ability to transcend beyond the examination of one or several compounds to that of thousands or millions of these species simultaneously. The work described here, limited to scaffolded combinatorial chemical libraries, focuses primarily on the design and synthesis of these systems and how they have been analyzed against biological targets. Of the three scaffolded libraries, two were developed from aromatic templates (3,5-diaminobenzoic acid and 1,2,4-benzenetricarboxylic acid) while the last was built upon the cyclohexyl, Kemp's triacid platform. Although these libraries did not provide compounds with high affinity for the receptors investigated, they served to improve the understanding of combinatorial chemistry as a practice.
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Joshi, Hemant K. "Synthetic, structural, spectroscopic and computational studies of metal-dithiolates as models for pyranopterindithiolate molybdenum and tungsten enzymes: Dithiolate folding effect." Diss., The University of Arizona, 2003. http://hdl.handle.net/10150/280480.
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Coordination by an axial oxo and an equatorial ene-dithiolate group is a salient feature of the active sites of the mononuclear pyranopterin Mo/W enzymes. Discrete mononuclear model complexes encompassing these features are important in understanding the metal-ligand interactions in these active sites. The compounds (Tp*)ME(S-S) (M = Mo, W; E = O, NO) and Cp₂M(S-S) (M = Ti, Mo, W) (where Tp* is hydrotris(3,5-dimethyl-1-pyrazolyl)borate, Cp is η⁵-cyclopentadienyl, S-S represents a generic ene-1,2-dithiolate ligand for example 1,2-benzenedithiolate and 3,6-dichloro-1,2-benzenedithiolate) provide access to three different electronic configurations of the metal, formally d¹, d² and d⁰, respectively. These compounds also allow the study of two metal, two axial ligand and two equatorial ene-dithiolate perturbations. X-ray crystallography, density functional theory and photoelectron spectroscopy are utilized to understand the metal-sulfur interaction in the above complexes. Subtle differences in the geometry of these compounds are observed, including the metal-dithiolate fold angle which is sensitive to the electronic occupation of the metal in-plane orbital. This orbital is presumably the "host" orbital to the electrons during catalysis. The work in this area has resulted in the development of a dithiolate-folding-effect. This effect relates to the experimental verification of the Lauher and Hoffmann bonding model for the metal-dithiolate interaction in these complexes. This "dithiolate-folding-effect" is proposed to account for the electronic buffering at the metal center. This effect may provide a regulatory mechanism for the metal-sulfur interactions and could be a factor in the electron transfer reactions that regenerate the active sites of molybdenum and tungsten enzymes. The structure and properties of these compounds are correlated with those of the enzyme active sites.
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Clapp, Paula Jean 1968. "Visible-light induced reactions in lamellar phospholipid assemblies." Diss., The University of Arizona, 1996. http://hdl.handle.net/10150/282185.
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Cyanine photosensitizers are visible-light absorbing dyes commercially useful in photography and available in a variety of structures with tunable redox, absorptivity and excited state properties. The areas of research described in this dissertation all utilize cyanine dyes to photosensitize various processes to visible-light within the confines of lamellar phospholipid assemblies. In Chapter III, an efficient three component, liposome-bound photochemical molecular device for transfer of energy and electrons is described. A porphyrin (free base or metallated) serves as the energy donor, a cyanine functions as the energy acceptor/electron acceptor and Ph₃BnB- acts as an electron donor. In Chapter IV, the first example of visible-light sensitized bilayer polymerization is presented. Sensitizing the polymerization of two-dimensional lipid assemblies to visible-light is part of a current research effort to exploit the properties of polymerized supramolecular structures. Extending the polymerization sensitivity of such amphiphile aggregates to lower energy visible-light creates possibilities for applications where UV photolysis would not be useful. Applications of liposomes as drug delivery vehicles thus far have relied upon such release mechanisms as pH sensitivity or target specific interactions to empty the liposome contents to the cell. Chapter V describes a new system for the successful destabilization of liposomes at pH 4.5 via visible-light sensitive polymerization of lipids. Visible-light sensitized polymerization induces the release of liposome aqueous contents providing temporal and spatial control over the release event and lower energy irradiation with more tissue penetration than UV photolysis.
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Bondurant, Bruce. "Photoinduced release of contents from sterically stabilized liposomes." Diss., The University of Arizona, 2000. http://hdl.handle.net/10150/284207.
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The design of sterically stabilized liposomes with surface grafted poly(ethylene glycol) (PEG-liposomes) for drug delivery requires a very low rate of leakage so that drugs will remain encapsulated during the 24 to 48 hr that these liposomes remain in circulation. This design can interfere with the rapid release of the encapsulated drugs at the site of action. An effective technique for the photodestabilization of PEG-liposomes is described. Photosensitive PEG-liposomes containing the polymerizable lipid 1,2-bis[10-(2',4 '-hexadienoyloxy)decanoyl]-sn-glycero-3-phosphatidylcholine (bis-SorbPC₁₇,₁₇) were prepared in the presence of a water soluble fluorescent probe and were polymerized with UV light (254 nm). Liposomes composed of PEG-DOPE/bis-SorbPC₁₇,₁₇/cholesterol/DOPC (15/30/40/15) showed a 200-fold increase in the rate of leakage at very high conversion of monomer (>98%). Liposomes composed of PEG-DSPE/bis-SorbPC₁₇,₁₇/cholesterol/DSPC (5/30/35/30) showed increases in the rate of leakage that were: 90-fold at only 20% conversion of monomer, 9-fold at 80 to 90% conversion, and 150-fold at greater than 95% conversion. These liposomes also exhibited a 17-fold increase in the rate of leakage near the main phase transition temperature (T(M)) of bis-SorbPC₁₇,₁₇ (28.8°C). These observations suggest that the polymerization of phase-separated domains of bis-SorbPC₁₇,₁₇ causes defects in the membrane that result in increased permeability. While the leakage kinetics at high conversion followed a first order rate law, leakage at low conversion did not. This, as well as the biphasic dependence of the rate of release on irradiation time in liposomes exhibiting enhanced release at low conversion, indicate that the mechanisms of leakage at low and high conversion are different. Syntheses of neutral and polymerizable PEG-lipids as well as polymerizable phosphatidylcholines are described here. Neutral PEG-lipids were used to study the effect of lipid charge on mechanical properties of PEG-lipids and on the interactions of PEG-liposomes with cells. The compressibility of a neutral PEG grafted supported bilayer could be fitted most closely with the MWC mean field model for a polymer brush (Efremova et al. Biochemistry 2000 39: 3441--3451). Neutral liposomes were endocytosed at a significantly higher rate than anionic liposomes by HeLa ovarian carcinoma cells, while murine macrophage cells showed no preference (Miller et al. Biochemistry 1998 37: 12875--12883).
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Oliver, John William Kidder. "Design and Improvement of the Biosynthesis of 2,3--Butanediol from CO2 by Metabolic Engineering of Cyanobacterium Synechococcus elongatus PCC7942." Thesis, University of California, Davis, 2015. http://pqdtopen.proquest.com/#viewpdf?dispub=3685273.
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<p> This dissertation describes metabolic engineering of cyanobacterium <i> Synechococcus elongatus</i> PCC7942 as a photosynthetic host for the conversion of CO<sub>2</sub> into 2,3-butanediol. Current advances in pathway design, genetic tool development, and yield improvement are described (Chapter 1). A pathway for the synthesis of 2,3-butanediol is designed based on collective concepts of pathway strength, robustness, and irreversibility, and extensively tested through the generation of mutants (Chapter 2). This pathway is then optimized through modulation of translation by combinatorial mixing of ribosome binding sites (Chapter 3). Finally, photosynthetic productivity is investigated through expression of an exogenous pathway targeting every step between fixation and product (Chapter 4). All materials and methods are given separately for easy reference (Chapter 5).</p>
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Houghton, Stephen Richard. "Improving access to biologically and pharmaceutically relevant molecules by understanding mechanisms of biosynthesis and improving chemical synthesis." Related electronic resource: Current Research at SU : database of SU dissertations, recent titles available full text, 2008. http://wwwlib.umi.com/cr/syr/main.
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Menegatti, Stefano. "Design, Selection, and Development of Novel Peptide Ligands for Bioseparations and Diagnostics." Thesis, North Carolina State University, 2013. http://pqdtopen.proquest.com/#viewpdf?dispub=3575894.
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<p> The relevance of protein-based biopharmaceuticals has increased dramatically in the past decades and a variety of products are now available for human therapy. Antibodies in particular are currently the most heavily consumed protein therapeutics, with a current market volume expected to reach 1 trillion US$ in 2015 and a compound annual growth rate (CAGR) of 3-6%. Meeting the increasing demand for these therapeutics at lower prices while complying with increasingly stringent regulatory environments, calls for the development of new technologies and platform approaches for efficient downstream protein purification. Extended use of affinity chromatography holds great promise in meeting the urgent demand for affordable high-quality biological products. This technology, however, is still dependent on the use of biological ligands, such as Protein A, Protein G, and Protein L, that have significant issues associated with their high cost, harsh elution conditions, narrow specificity, low chemical stability, and immunogenicity in patients if they leach into the product stream. Small, robust, synthetic ligands may offer an effective alternative to protein ligands. Peptides in particular combine levels of affinity and specificity similar to those of biological ligands with high chemical and biochemical stability, broader specificity, low immunogenicity and ease of synthesis that can reduce costs.</p><p> The work in this thesis aims to discover and characterize novel peptide ligands to produce efficient, robust, and affordable affinity adsorbents for improved downstream purification of biologics. Two main areas have been investigated: (a) the development of linear hexapeptide – based adsorbents for the purification of human antibodies and (b) the design and screening of novel libraries of cyclic peptides for the discovery of novel ligands.</p><p> The research conducted on the characterization and development of competitive peptide-based affinity adsorbents comprises: (a.1) testing existing peptide ligands for the purification of antibodies from a variety of sources; (a.2) optimizing the protocol of ligand coupling on chromatographic resins to increase adsorbent binding capacity; (a.3) a method of modification of the resin’s surface chemistry to increase the adsorbent's chemical stability in harsh alkaline conditions; and (a.4.) a combined computational and chemical strategy for the design of protease-stable peptide ligands. The resulting peptide affinity adsorbents compete well with advanced Protein A – based adsorbents in terms of product yield and purity, dynamic binding capacity (~ 50 – 60 g/L), resistance to alkaline cleaning and sanitization, and biochemical stability in the presence of proteolytic enzymes.</p><p> In the second part of this work, two methods are presented for the design, synthesis, and screening of libraries of cyclic peptides for the identification of novel affinity ligands. The first method involves the generation and screening of (b.1) a biological mRNA-display library of cyclic peptides, and the second method (b.2) uses a synthetic solid-phase library of “reversible cyclic peptides”. Both libraries have been screened for the identification of ligands for human antibodies. The results of these studies indicate that these libraries are very promising tools for the discovery of robust, selective and affordable peptide ligands.</p><p> The methods presented herein offer a new set of tools, not only for affinity ligand discovery, but also for finding new drugs and diagnostic methods. Besides their technological value, these studies also offer insights into the mechanisms of non-covalent interaction that underlie the phenomena of biorecognition and protein activity.</p>
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Kriss, Caroline Theresa. "Conformational analysis of O-linked glycopeptides related to enkephalin and nuclear pore proteins." Diss., The University of Arizona, 1999. http://hdl.handle.net/10150/284116.
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The effect of O-linked glycosylation on peptide conformation has been studied in pursuit of understanding one of the natural roles of carbohydrates, and with an interest in drug design. Solution conformations were analyzed using a combination of NMR, molecular modeling, and kinetics techniques. Variations in peptide sequence, carbohydrate, and stereochemistry of linkage were made. Glycosylation of enkephalin analogs at the Ser⁶ position of the sequence Tyr-c-[D-cys-Gly-Phe-D-cys]-Ser-Gly-CONH₂ with a glucose moiety affected only the exocyclic portion of the molecule. An α-linkage constrained the carbohydrate and lessened the impact on peptide conformation, when compared to a β-linkage. Glycosylation of nuclear pore protein models of the sequence c-[Cys-Ser-Pro-Ser-Thr-Cys] at the Ser⁴ position increased turn formation irrespective of carbohydrate identity or linkage. Glycosylation of the uncyclic form of this sequence at Ser⁴ demonstrated no conformational differences. Flexibility of the unglycosylated sequence is paramount in determining the potential impact of glycosylation since two completely different effects are observed for glycosylation of the cyclic and uncyclic sequence.
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Brotherton, Carolyn Adams. "Investigations of the Biosynthesis and Structure of Colibactin, a Cytotoxin Made by Human-Associated Escherichia Coli." Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:26718729.
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Humans exist in symbiosis with trillions of bacteria that are collectively referred to as the human microbiota. While commensal microbes are essential for health, some resident microbes can promote disease. Certain strains of human-associated Escherichia coli cause double-strand breaks in host DNA through the production of colibactin, a genotoxin of unknown structure. To broaden our understanding of the chemistry of the human microbiota, we sought to elucidate the structure of colibactin and characterize its biosynthetic pathway.
We first characterized the self-resistance mechanism in colibactin biosynthesis (Chapter 2). We found that the enzyme that initiates the assembly line pathway is ClbN, a non-ribosomal peptide synthetase (NRPS). Biochemical assays showed that ClbN biosynthesizes an N-terminal prodrug motif consisting of an N-myristoyl-D-asparagine residue that is proposed to mask the reactivity of colibactin. We performed bioinformatic analyses to identify the enzyme that acts after ClbN in the assembly line. In vitro reconstitution assays revealed that ClbB, a NRPS/ polyketide synthase (PKS) hybrid, elongates the prodrug motif produced by ClbN. In addition, we demonstrated that the periplasmic peptidase ClbP cleaves the prodrug motif from synthesized model substrates and that the membrane domain of ClbP is required for full activity.
Next, we sought to isolate precolibactin, the prodrug-containing precursor to the active genotoxin (Chapter 3). Metabolite profiling of the extracted metabolome of ΔclbP and wild-type colibactin-producing strains led to the identification of several candidate precolibactins. We isolated one of these metabolites, which we named Metabolite B. This metabolite contains an unusual azaspiro[2.4] bicyclic ring system that has not been observed previously in a natural product scaffold. The structure of Metabolite B suggested that the colibactin assembly line pathway may utilize novel biosynthetic logic and pointed to a possible mechanism through which colibactin damages DNA.
Finally, we describe the isolation of other pks-associated metabolites and provide biosynthetic hypotheses for these pathway intermediates (Chapter 4). We also present our attempts to characterize the next enzymatic steps in the colibactin biosynthetic pathway. As part of these efforts, the PKS module of ClbB was characterized in vitro and several other colibactin biosynthetic enzymes were examined using genetic studies. The challenges associated with studying a biosynthetic pathway for which the final product is unknown are highlighted. Overall, the work presented here contributes significantly to our knowledge of the biosynthesis and structure of a microbial genotoxin.<br>Chemistry and Chemical Biology
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Stein, Natalia. "Spectroscopic and electrochemical studies of Shewanella oneidensis cytochrome c nitrite reductase, and improving c-heme expression systems." Thesis, The University of Wisconsin - Milwaukee, 2015. http://pqdtopen.proquest.com/#viewpdf?dispub=3685085.
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<p> In this work the redox properties of cytochrome c nitrite reductase (CcNiR), a decaheme homodimer that was isolated from <i>S. oneidensis,</i> were determined in the presence and absence of the strong-field ligands cyanide and nitrite. Four hemes per CcNiR protomer are hexa-coordinate with tightly bound axial histidines, while the fifth (active site) has one tightly bound lysine and a distal site that can be open, or contain exogenous ligands such as the substrate nitrite. Controlled potential electrolysis in combination with UV/visible absorption (UV-vis) and electron paramagnetic resonance (EPR) spectroscopies allowed for assignment of all heme midpoint potentials under each set of conditions. The studies show that the active-site heme is the first to be reduced under all conditions. The midpoint redox potential of that heme shifts approximately 70mV to the positive upon binding a strong field ligand such as nitrite or cyanide. When controlled potential electrolysis was carried out in the presence of nitrite, a concerted two electron reduction was observed by UV-vis, and a {Fe(NO)}<sup>7</sup> reduced product was revealed in EPR. In addition, an asymmetry in ligand binding between active sites was revealed. This information is relevant for the interpretation of planned and ongoing mechanistic studies of CcNiR. </p><p> Over-expression, partial purification and characterization of another <i> S. oneidensis</i> multiheme enzyme, known as octaheme tetrathionate reductase (OTR), is also described herein. Though of unknown cellular function, OTR was previously reported to have tetrathionate reductase activity, in addition to nitrite and hydroxylamine reductase activities. The new results indicate that the expression of OTR has no effect on tetrathionate or nitrite reductase activities in the whole cell lysate, and only hydroxylamine reductase activity was substantially elevated in the overexpressing bacteria. OTR was stable in buffered solutions, but substantial activity loss during all attempts at column chromatography was a major obstacle to the complete purification. OTR also proved quite hydrophobic, so possible membrane association should be considered in future attempts to purify this protein. </p><p> Finally, this dissertation also reports attempts to improve <i> S. oneidensis'</i> ability to express foreign proteins. Though ideally suited to expressing c-hemes, it proved difficult to express carboxy his-tagged proteins in <i>S. oneidensis</i> because of persistent tag degradation. Attempts to knock out lon protease, a cytoplasmic carboxypeptidase, as well as the result of redirecting ccNiR from the SecA to the possibly more protected signal particle recognition (SRP) secretion pathway, are described. </p><p> Iron heme cofactors are single-electron transport moieties that play a crucial role in respiration. While oxygen is the electron acceptor of choice in aerobic atmospheres, microorganisms that live in anaerobic environments utilize other molecules with similarly high reduction potentials. <i> S. oneidensis</i> can utilize numerous terminal electron acceptors, including nitrite, dimethylsulfoxide and even uranium, thanks to a particularly rich array of multi c-heme respiratory proteins. Understanding of how the midpoint potentials and heme arrangements within the proteins influence these exotic respiratory processes is of interest in the fields of bioremediation and fuel development.</p>
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Armitage, Bruce Alan. "Photoinduced electron transfer, energy transfer and polymerization reactions in phospholipid membranes." Diss., The University of Arizona, 1993. http://hdl.handle.net/10150/186212.
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The differential physical properties found at different depths of a phospholipid membrane permit design of systems for vectorial reactions (which are not possible in isotropic solution). In the system described in Chapter IV, a hydrophobic electron donor (triphenylbenzylborate) binds to the membrane interior while a hydrophilic electron acceptor (a cyanine dye) binds to the surface. Irradiation with red light leads to vectorial electron flow via photoinduced electron transfer (PET), as monitored by fluorescence quenching and photobleaching techniques. The PET reaction efficiency is enhanced over the case where the donor and acceptor are present in water without membranes. In that case, strong dimeric complexes are formed which reduce the efficiency of PET by enhancing nonradiative decay modes for the dye (Chapter III). Addition of a porphyrin to the membrane surface extends the sensitivity of the system to blue light (Chapter V). Excitation of the porphyrin at 417 nm and subsequent energy transfer to the cyanine trigger the same PET chemistry observed with red-light irradiation. Furthermore, the electron- and energy-transfer reactions are enhanced on polymerized, phase-separated membranes (Chapter VI). In these applications, membranes are media for chemical reactions. Membranes also make interesting substrates for chemical reactions, in this case, polymerization. A system consisting of a membrane-bound, amphiphilic cyanine dye and molecular oxygen is described in Chapter VII which effectively initiates the polymerization of vesicles upon irradiation with visible light. Potential utility in drug delivery applications is discussed.
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Lin, Ying. "Design and synthesis of conformationally constrained glucagon analogues to study the conformational features important for glucagon bioactivity." Diss., The University of Arizona, 1993. http://hdl.handle.net/10150/186227.
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We have synthesized ten glucagon analogues that are either conformationally constrained systematically in the middle portion of the molecule, or modified from the known superagonist analogue [Lys¹⁷,¹⁸, Glu²¹]glucagon to study the structure-activity relationships of glucagon. The analogues were prepared using the solid-phase peptide synthesis method. Cyclizations were accomplished by forming the side chain lactam (amide) bridges on the resin. All peptide analogues were cleaved from the solid support, deprotected by the low-high HF procedure, and purified by a combination of gel filtration chromatography and dialysis followed by reverse-phase high performance liquid chromatography. A new characterization method for cyclic glucagon analogues using fast atom bombardment mass spectrometry with endoproteinase Asp-N peptide mapping has been developed that has provided unequivocal confirmation of the presence and site of the rings as well as the amino acid compositions. Receptor binding and adenylate cyclase activity assays and circular dichroism spectroscopy have been used to reveal the role of the structure and conformation of the middle portion of the molecule. The effects of the modification of the 17, 18 and 21 positions on the superagonist activity have also been examined. Several key features of the peptide backbone conformation responsible for binding and transduction have been further studied by theoretical calculations and computer modeling (energy minimization) using the Sybyl program.
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Malasi, Wilhelm S. "Synthesis and complexation of functionalized mixes thia-aza-macrocyclic and medium sized ligands." Akron, OH : University of Akron, 2009. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=akron1239210466.
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Thesis (Ph. D.)--University of Akron, Dept. of Chemistry, 2009.<br>"May, 2009." Title from electronic dissertation title page (viewed 11/25/2009). Advisor, Michael J. Taschner; Committee members, David Modarelli, Jun Hu, Wiley J. Youngs, Amy Milsted; Department Chair, Kim C. Calvo; Dean of the College, Chand Midha; Dean of the Graduate School, George R. Newkome. Includes bibliographical references.
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Xiong, Chiyi. "Asymmetric synthesis of conformationally and topographically constrained amino acids as peptidomimetics: An approach to design and synthesis of opioid receptor selective ligands." Diss., The University of Arizona, 2003. http://hdl.handle.net/10150/280393.
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As part of continuing efforts to obtain backbone and side chain conformationally constrained, novel amino acids,¹⁻⁷ we have successfully developed the asymmetric synthesis of β-phenyl-substituted cysteine, tryptophan, and serene derivatives. In this approach, the key intermediate, enantiomerically pure 3-phenylaziridine-2-carboxylic ester, was prepared from an α, β-unsaturated ester by employing the Sharpless asymmetric dihydroxylation. The aziridine was treated with 4-methoxybenzylthiol, indole, and acetic acid to give β-phenyl-substituted cysteine, tryptophan, and serine, respectively, in a clean S(N)2 type ring opening at the C3 position. This methodology was readily extended to provide a general approach to the synthesis of optically pure anti- and syn-β-substituted cysteines. We also described an effective means to synthesize 8-phenyl-substituted thiaindolizidinone amino acids through a convergent strategy. β-Phenyl-substituted cysteine benzyl esters were prepared according to our new protocol developed above. The doubly protected glutamic acid gamma-aldehyde was prepared according to a known procedure. The key step was the condensation of the aldehyde with β-phenyl-substituted cysteines to afford novel 8-phenylthiaindolizidinone amino acids as epimers at the bridgehead, which can be readily separated. These novel 8-phenylthiaindolizidinone amino acids, which are constrained β-turn dipeptide mimetics, were incorporated into Leu-enkephalin peptides as a replacement of the dipeptide Gly³-Phe⁴ to afford individual isomers of Leu-enkephalin analogues. The conformationally restricted nature of these analogues rendered them amenable to conformational analysis in solution because they are less subject to dynamic averaging than are more the flexible linear compounds.
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Bonner, Grady Gregg 1965. "Synthesis, pharmacology, and structural analysis of opioid peptides: Cyclic somatostatin analogs which include unusual amino acids with conformationally restricted side-chain groups." Diss., The University of Arizona, 1997. http://hdl.handle.net/10150/282318.
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This work relates the activities of Somatostatin-derived opioid peptides to their 3-dimensional structures. Due to the flexability of peptides, they adopt multiple conformations. This flexibility leads to ambiguity in the development of structure-activity profiles. Here flexibility is reduced by incorporation of amino acids with conformationally restricted side-chain groups. These groups are restricted to certain topographies by cyclization or biased through substitution at the beta carbon. The opioid activities of these peptides are determined by brain binding radioligand competition assay, in vitro activity in the mouse vas deferens (MVD) and guinea pig ileum (GPI) smooth muscle twitch-height inhibition assays, and in vivo in the mouse warm water tail-flick latency assay. Their 3-dimensional structures were determined through a variety of 2D 1H NMR including TOCSY, ROESY, DQF-COSY, and COSY-35, as well as molecular modeling including energy minimization and molecular dynamics. By knowing the activity of the peptides, and their preferred conformations, a reasonably confident structure-activity profile was generated.
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Shokeer, Abeer. "Design of Glutathione Transferase Variants for Novel Activities with Alternative Substrates." Doctoral thesis, Uppsala universitet, Institutionen för biokemi och organisk kemi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-119768.
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Glutathione transferases (GSTs) play a pivotal role in cellular defense, since they are main contributors to the inactivation of genotoxic compounds of exogenous and endogenous origins. Directed evolution was used to improve the catalytic activities of Theta class GST T1-1 toward different substrates. The library was constructed by recombination of cDNA coding for human GST T1-1 and rodent Theta class GSTs, resulting in the F2-F5 generations. The clones were heterologously expressed in Escherichia coli and screened for variants with enhanced alkyltransferase activity. A mutant, F2:1215, with a 70-fold increased catalytic efficiency with 4-nitrophenethyl bromide (NPB) compared to human GST T1-1, was isolated from the second generation. NPB was used as a surrogate substrate of the anticancer drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in order to facilitate the screening process. The catalytic efficiency of the F2:1215 with BCNU had improved 170-fold compared to wild-type human GST T1-1, suggesting that NPB is a suitable model substrate for the anticancer drug BCNU. The sequence of the F2:1215 mutant differs from wild-type human GST T1-1 by three residues; one of these differences is Arg234, which corresponds to Trp in the human enzyme. Upon replacing the Trp234 in the human GST T1-1 with Arg, the resulting mutant (hTrp234Arg) showed enhanced alkyltransferase activity with a wide range of substrates (e.g. haloalkanes and other typical GSTs substrates). The three-dimensional structures of both wild-type human GST T1-1 and hTrp234Arg mutant help to explain the higher activity showed by of hTrp234Arg mutant compared to wild-type enzyme. The reciprocal mutation of the residue 234 in mouse GST T1-1 to that found in human, mArg234Trp, caused a dramatic decrease in the activity of the mouse enzyme to be similar to human GST T1-1. This indicates that residue 234 can be considered as a master switch of activities between human and rodent GST T1-1. Another important residue in the C-terminal helix of GST T1-1 is Met232. Although residue 232 points away from the H-site, it influences the catalytic activity and substrate selectivity of the mouse GST T1-1. A minor modification of Met232 induces major changes in the substrate-activity profile of the mouse GST T1-1 to favor novel substrates such as isothiocyanates and hydroperoxides and decreases the activity toward substrates that catalyzed by the wild-type enzyme.
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Chen, Chou-Hsiung. "Total synthesis of argyrin A and analogues thereof." Thesis, University of Nottingham, 2013. http://eprints.nottingham.ac.uk/13296/.
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The cyclin dependent kinase inhibitor p27 is one of the most frequently dysregulated tumour suppressor protein in human cancers. A reduction in the level of cellular p27 is frequently due to increased proteasome-dependent degradation. Recently, studies show that the macrocyclic octapeptide argyrin A induced an increase in cellular p27 levels by preventing the turnover of the protein via inhibition of proteasome function. In order to investigate this interesting biological property, this project embarked on the total synthesis of argyrin A, a naturally occurring macrocyclic peptide originally isolated from myxobacteria Archangium gephyra. Argyrin A is a non-ribosomal octapeptide containing four standard amino acids and three unusual amino acid-based subunits. The synthesis of these three unusual amino acid components was established. In particular, a novel generic synthetic route to access the optically pure N-Fmoc-4-methoxy-tryptophan and analogues thereof was developed. Key features of the synthetic route include the use of chiral Strecker amino acid synthesis and mild conditions to hydrolyse α-amino nitrile to α-amino acid. Furthermore, the total synthesis of argyrin A and analogues was accomplished by the application of modern solid-phase chemistry and macrocyclisation strategies. This platform technology will enable the robust total chemical synthesis of a focused library of argyrin analogues, which will facilitate a comprehensive SAR study. Additionally, the synthesised argyrin A and analogues thereof comprising unique tryptophan analogues were tested in a cytotoxicity assay against HCT-117 human colon cell line. The results showed that all synthetic argyrin derivatives display growth inhibitory effects at nanomolar concentrations. The best result was obtained for the argyrin A and (5-methoxy-Trp4)argyrin with GI50 value at 1.8 and 3.8 nM, respectively. In summary, it became apparent that the methoxy group at 4- or 5-position of tryptophan-5 residue is essential for the biological activity of argyrin.
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Restituyo, Rosario Elizabeth. "Chemical Tools for the Selective Control of Proteins: Protein Kinases and Acetyl Transferases." Diss., The University of Arizona, 2015. http://hdl.handle.net/10150/594650.
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Post-translational modifications (PTMs) of proteins are now known to increase the complexity of biological signaling networks. The ability to selectively control the activity of individual proteins and enzymes involved in PTMs enables studies of biological systems and in designing therapeutic agents. The phosphorylation of serine, threonine, and tyrosine catalyzed by protein kinases play an important role in signaling and when deregulated are involved in different types of cancer amongst other diseases. Therefore, selective regulation of kinase activity by inhibition could provide a means to understand signaling and also aid in developing therapies. The design of specific kinase inhibitors is challenging because most inhibitors target the common ATP-binding site, leading to several off-target hits with adverse effects. With this in mind, a fragment-based bivalent strategy was developed to potentially increase affinity and selectivity. Our approach seeks to target the ATP-binding site with a small molecule while simultaneously targeting any available site on the kinase with a cyclic peptide that potentially increases selectivity and affinity. The bivalent approach has produced effective and selective inhibitors for cAMP-dependent protein kinase (PKA). Herein, we extended this approach to Aurora kinase (Aurora A), RSK1 and MSK1. Using our optimized selection conditions, we have successfully selected several cyclic peptide ligands to later generate bivalent ligands for these kinases. This strategy may prove a robust method to discover new allosteric sites on kinases as well as other proteins, furthermore it also has the potential to provide insight towards the design of new ATP targeted approaches. A second area of research focuses on PTMs involved in the acetylation of lysine residues by enzymes dubbed histone acetyl transferases (HATs) or lysine acetyl transferases (KATs). Recent studies have shown that a large fraction of the proteome may be modifies by KATs but it is challenging to develop uniquely selective inhibitors as the KATs like Kinases have similar active sites. Towards the goal of developing a method to control a single KAT in the presence of other KATs, we designed ligand-gated split-proteins based on a sequence dissimilarity based approach. Potential fragmentation sites were identified by insertion of a 25-residue loop insertion. Successful loop insertion mutants provided guidance for the dissection of KATs into fragments tethered to FKBP and FRB that cannot spontaneously assemble, but that do upon the addition of a ligand, rapamycin to generate catalytically active. The method was successfully applied to design the very first split-KAT, GCN5,which only showed activity in the presence of an added small molecule. The method was shown to be potentially general by application to a second KAT, PCAF. The above studies provide potentially new approaches for selectively targeting and studying PTMs catalyzed by kinases and lysine acetyl transferase.
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Mahmood, Raza. "Synthesis of benzofurans and benzopyrans." Thesis, University of Hertfordshire, 2002. http://hdl.handle.net/2299/14039.
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Syntheses of 2,3-dihydrobenzofurans and 3,4-dihydrobenzopyrans normally involve several stages, therefore efficient syntheses of these compounds are desirable. A number of 3,4-dihydrobenzopyrans derivatives were prepared in one pot syntheses. The reaction is thought to proceed through cation intermediates, involving allylic cations generated by the reaction of allyl alcohols, 1,3-dienes, or diols in the presence of acids such as trifluoroacetic acid (TFA), or in a solution of glacial acetic and a metal catalyst or glacial acetic acid and sulphuric acid, followed by reaction with nucleophiles such as phenols or hydroquinones. This leads to the formation of allyl aryl ethers which rearrange via a [3,3]-sigmatropic reanangement and acid-catalysed intramolecular cyclisation, to give the conesponding 3,4-dihydrobenzopyrans. 5-Formyl-2,3-dihydrobenzofuran and 6-formylamino-3,4-dihydrobenzopyran were synthesized by several routes and the isolated products exhibited cis-trans isomerism. It was found that the introduction of an alkyl substituent at carbon-4 in the model benzopyran locks the 2,3,4-alkyl substituents into axial and equatorial orientations. This may influence the peroxyl-radical scavenging activity of these compounds by altering the extent of orbital overlap between the 2p-type lone pair on the pyran oxygen and the aromatic 1t-electron system. 2,3-Dihydrobenzofurans such as 5-hydroxy-2,3-dihydrobenzofuran have been shown to have better antioxidant activity than alpha-tocopherol due to the influence of the smaller, more strained ring, which allows better overlap of the oxygen lone pair and the 1t-electrons in the aromatic system. 5-Hydroxy-2,3-dihydrobenzofuran was efficiently synthesised in an improvement on the yields previously reported in the literature. A series of substituted 2,3-dihydrobenzofurans were synthesised by the reaction of phenols with allylic alcohols or aldehydes in the presence of trifluoroacetic acid or catalytic amounts of sulphmic acid, which also promoted the acid-catalysed intramolecular cyclisation.
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Ametsetor, Ebenezer, and Ismail Kady. ""Hydroxytyrosol Derivatives: Synthesis and Biomedical Assessment"." Digital Commons @ East Tennessee State University, 2019. https://dc.etsu.edu/asrf/2019/schedule/170.
Full textAbstract:
Hydroxytyrosol is one of the most powerful known antioxidants. It is a naturally occurring polyphenol, most commonly produced in Olive tree, (Olea europaea). The remarkable antioxidant and pharmacological properties of hydroxytyrosol has made it an outstanding compound in the polyphenol family and of great interest to many researchers. Hydroxytyrosol has the ability to scavenge free radicals produced during cellular oxidative stress and helps to protect the integrity of cells in living systems. Despite its numerous biological and pharmacological uses, it is found in very low concentration in olive oil, this limits its biomedical applications. A novel method for synthesizing pure hydroxytyrosol from commercially less expensive precursor catechol was successfully developed in our lab. However, it is believed that the cellular uptake of hydroxytyrosol is slow because of its high hydrophilicity. Therefore, we plan in this ongoing research to synthesize less hydrophilic derivatives of hydroxytyrosol by introducing some hydrophobic groups (such as alkyl, acyl, …) to its molecular skeleton.
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Whitwood, Jennifer. "Using mating-type switching to investigate Smc5/6 function in Schizosaccharomyces pombe." Thesis, University of Sussex, 2014. http://sro.sussex.ac.uk/id/eprint/48308/.
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The essential Smc5/6 complex is structurally related to cohesin and condensin. It is required for homologous recombination (HR), rDNA stability and telomere maintenance. In Schizosaccharomyces pombe, two hypomorphic smc6 mutants, smc6-X and smc6-74, haven been shown to be deficient in HR-dependent processing of collapsed replication forks. Collapsed replication forks can generate single-ended DNA double strand breaks (se-DSB) which require HR to restore replication. In this study the requirement for Smc5/6 at a site-specific se-DSB at the mating-type locus and in the mating-type switch process were analysed. In S.pombe mating-type switching occurs over two S phases; in the first S phase replication fork stalling at mat1 leads to an imprint, which is converted to an se-DSB during the next S phase. This initiates the copying of the donor cassette using HR. In the absences of donors the sister chromatid is used for repair. Mating-type switching analysis showed that snc6-74 had a defect in switching dependent on the genotype of the smc6-74 parent. Both smc6 mutants had reduced viability in the absence of donors, consistent with a defect in HR repair of an se-DSB. analysis in an inducible system (Holmes et al., 2005) showed that in response to a se-DSB Rad52 foci appeared with wild type kinetics but the smc6 mutants delayed entry into mitosis for approximately 2hrs, dependent on the DNA damage checkpoint kinase Chk1. In order to test whether this delay facilitated rescue by a converging replication fork a novel inducible converged fork (cf) DSB system was developed. The cf-DSB required HR and the RecQ helicase Rqh1 for repair but did not require Mus81. The converging fork rescued smc6-74 but not smc6-X showing Smc5/6 to be required for repair of both types of replication-associated DSBs.
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Kler, Rantej Singh. "Metal oxide nanomaterials and their application in solar photoelectrolysis of water." Thesis, University of Sussex, 2014. http://sro.sussex.ac.uk/id/eprint/48904/.
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Solar generated hydrogen as an energy source is green, sustainable, with a high energy density. One day the majority of current fossil fuel based technology could be replaced with hydrogen technology reducing CO2 emission drastically. The goal in this research is to explore hybrid metal oxide photocatalysts in the pursuit of achieving highly efficient photoanodes for use in photoelectrochemical cells (PEC). Achieving high efficiencies of hydrogen production in photoelectrochemical cells is the key challenge for the commercialisation of PEC technology as a viable, sustainable, hydrogen source; limited only by the lifetime of the sun and the resources of the metal oxide materials. In this research TiO2, Fe-Ti-O, ZnO, and Zn2TiO4 are the photocatalysts explored. Alloys of Ti-Fe-O showed improvement over TiO2, whilst a hybrid heterostructure of ZnO/Zn2TiO4/TiO2 enhanced photocurrent densities significantly. A barrier layer in the photoanode achieved localised exciton separation and reduction of recombination rates by inhibiting back flow of electrons after injection into the TiO2 layer. Nanotubes are created by the simple electrochemical process of anodisation. The nanotube composition depends on the anode material. To control the composition ofthe anode, iron and titanium are co-deposited onto a substrate using electron beam evaporation. The introduction of iron into titania nanotubes engineered the band gap, lowering the band gap energy to that of iron oxide whilst the positions of the conduction and valence bands with respect to the oxidation and reduction potentials of water remained favourable. Fe-Ti-O nanotubes showed remarkable photocurrent density improvement compared to TiO2 nanotubes. ZnO nanostructures deposited by vapour transport mechanisms showed variability in the morphology of the structures, as governed by the growth dynamics. Herein, it is shown that an electronically favourable situation arises by the formation of a ZnO-Zn2TiO4-TiO2 heterostructure and a high photocatalytic activity is reported. The structure is composed of a large surface area ZnO nanorod photoabsorber formed on a Ti foil which forms a Zn2TiO4 barrier layer between ZnO and TiO2. The Zn2TiO4 layer inhibits electron transport toward the surface of the photoanode whilst encouraging charge transport to the hydrogenation electrode. The heterostructure interfacial surface area is extended through the utilisation of TiO2 nanotubes, which demonstrated a 20.22 % photoelectrochemical efficiency under UV illumination. Surface modification of ZnO nanorods with aerosol assisted chemical vapour deposited TiO2 nanoparticles enhanced photocurrent densities of the ZnO rods, improving charge separation of excitons created within the TiO2 nanoparticles. ZnO nanotubes formed via a novel route using chemical bath deposition of ZnO is investigated, an annulus ZnO seed layer facilitated the site specific growth of ZnO nanotubes whilst a uniform seed layer formed ZnO nanorods.
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Li, Zhuo. "Theoretical study of the circular dichroism spectroscopy of proteins." Thesis, University of Nottingham, 2018. http://eprints.nottingham.ac.uk/49128/.
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Circular dichroism (CD) spectroscopy is an important technique in studying protein structure, especially for protein secondary structures and conformational changes during biological processes. A fully quantitative theory of the relationship between protein conformation and optical spectroscopy would facilitate deeper interpretation and insight into biophysical and simulation studies of protein dynamics and folding. Vibrational structure in the electronic CD spectra of proteins is an important source of information on protein conformation and can be exploited to study structure and folding. We employ the state-averaged complete active space (CAS) method to calculate the ab initio electronic ground and excited states of N-methylacetamide (NMA), toluene, p-cresol and 3-methylindole (3-MI), which represent chromophores that are significant in the CD spectroscopy of proteins in the far- and near-ultraviolet (UV) regions. The results of these calculations are used to incorporate vibronic levels of the excited states into first principles calculations of CD using an exciton approach. The far-UV CD spectra of a set of 49 proteins, comprising a range of structural types, are calculated to assess the influence of the vibrational structure. The calculated spectra of -helical proteins are better resolved using the vibronic parameters and correlation between the experimental and the calculated intensity of less regular structure proteins improves over most wavelengths in the far-UV. No obvious improvement is observed in the calculated spectra of regular -sheet proteins. The near-UV CD spectra of 40 proteins are calculated with the new parameter set and the correlation between the computed and the experimental intensity from 270 to 290 nm is much improved. The contribution of individual chromophores to the CD spectra has been calculated for several mutants and in many cases helps rationalize changes in their experimental spectra. Considering conformational flexibility by using families of NMR structures leads to further improvements for some proteins and illustrates an informative level of sensitivity to side chain conformation. In several cases, the near-UV CD calculations can distinguish the native protein structure from a set of computer-generated misfolded decoy structures. CD spectra of proteins are better reproduced in both far- and near-UV by considering vibrational structures in electronic transitions of chromophores. This improvement can provide more details in connecting the spectroscopic data to the conformations of proteins and will encourage a broader use of CD in protein studies. Besides shedding light on the importance of vibronic transition, results in this thesis also show other aspects that may further improve CD calculations, such as developing parameters of disulfide bond, calculating CD using molecular dynamics (MD) trajectories and taking into account the influence of surroundings of chromophores.
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Almulathanon, Ammar. "Efficacy and toxicity of linear polyamidoamine polymers used in gene delivery." Thesis, University of Nottingham, 2018. http://eprints.nottingham.ac.uk/49894/.
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Introduction: Interestingly, polyamidoamines (PAAs) are a group of biodegradable cationic polymers that exhibit a transfection efficiency comparable to that obtained with PEI but with a low cytotoxicity compared to other polycationic vectors. However, their cellular adverse effects have only been probed in assays indirectly measuring the cell metabolic activity based on the reduction of tetrazolium salts into coloured formazan derivatives by mitochondrial enzymes such as the MTT, MTS or XTT assays. There is therefore a need for a deeper insight into their cytotoxic profile before their potential as delivery agents in gene therapy can be fully appreciated. Furthermore, the intracellular fate of PAA polyplexes remains unclear to date, prompting questions around the mechanisms underlying their ability to mediate gene transfer. The present work explored cellular events induced by DNA polyplexes prepared using a linear PAA with a methylenebisacrylamide/dimethylethylenediamine (MBA-DMEDA) backbone of a molecular weight over 10kDa with the aim to confirm and better understand the absence of relationship between cytotoxicity and transfection efficiency reported with those vectors. Methods: Complexes were formed between pDNA and branched polyethyleneimine (BPEI, 25 kDa) or various PAAs including a homopolymer of a molecular weight >10 kDa (HP->10), a PEGylated Triblock Copolymer (CP) and a blend of CP and HP->10 to form a bioreducible cross-linked delivery system. Physicochemical properties of pDNA polyplexes were analysed by using simple techniques including gel electrophoresis, dynamic light scattering (DLS) and zeta potential measurement. The transfection efficiency of pDNA complexes was compared in A549 cells using a luciferase reporter gene assay. The impact of endo/lysosomal escape on transfectionefficiency of pDNA polyplexes was investigated by transfecting cells in presence of bafilomycin A1 or chloroquine, whereas the role of reactive oxygen species in transgene expression was evaluated by using hydroquinone as antioxidant. Cytotoxicity caused by the vectors was evaluated by measuring cell metabolic activity, lactate dehydrogenase release, formation of reactive oxygen species and changes in mitochondrial membrane potential. Results: Gel electrophoresis showed complete incorporation of pDNA by individual and cross linked PAA polyplexes at a similar polymer to DNA (RU:Nt) ratio of (2:1). In comparison, pDNA incorporation was achieved by BPEI at a 3:1 RU:Nt ratio. All polymers were able to condense pDNA into nanoparticles of ≤200 nm in diameter with a positive surface charge. Non-PEGylated PAA polyplexes showed higher luciferase activity than the PEGylated counterparts, but lower than that of BPEI. However, in contrast to BPEI vectors, PAA polyplexes caused negligible cytotoxic effects. The transfection efficiency of PAA polyplexes was significantly reduced in presence of bafilomycin A1 while chloroquine either enhanced or decreased transgene expression depending on the RU:Nt. No changes were found in the transfection of PAA polyplexes in the presence of hydroquinone compared to BPEI. Conclusions: PAA polyplexes displayed a pH-dependent endo/lysosomal escape which was not associated with cytotoxic events, unlike observed with BPEI polyplexes. This is likely due to their greater interactions with biological membranes at acidic than neutral pH. The presence of excess polymer is considered necessary for improving transfection efficiency of PAA polyplexes. PAA was identified as promising and safe candidate for in vitro gene delivery and has potential for future in vivo gene therapy.
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Powell, Kimberley Jade. "Synthetic and biophysical studies on the tridachiahydropyrone family of natural products." Thesis, University of Nottingham, 2014. http://eprints.nottingham.ac.uk/14228/.
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This thesis primarily details synthetic and biophysical studies on the tridachiahydropyrone family of natural products. The general aim of this work was to explore an hypothesis regarding the location and function of these metabolites, isolated from sacoglossan molluscs. Specifically, it was hypothesised that tridachiahydropyrone is synthesised photochemically from linear polyene precursors via a selective double bond isomerisation-6pi electrocyclisation sequence which occurs within the cell membrane of the producing organism. Furthermore, it was postulated that this reaction sequence, and subsequent photochemical transformations of tridachiahydropyrone into the related products phototridachiahydropyrone and oxytridachiahydropyrone, serve to protect the producing mollusc from the damaging effects of UV radiation. Firstly, the proposed polyene precursors were synthesised using a convergent strategy dependent upon a late-stage Suzuki coupling. Their photochemical, biomimetic conversion into tridachiahydropyrone, phototridachiahydropyrone and oxytridachiahydropyrone, was then accomplished. The interactions of tridachiahydropyrone and its biomimetic precursors with model membrane systems were next explored, using a fluorescence spectroscopic technique. This work demonstrated that the molecules bind to phospholipid vesicles (PLVs) of varying compositions. The synthesis of tridachiahydropyrone within the PLVs was also achieved The propensity of the compounds to act as sunscreens was lastly investigated, by measuring the degree of protection against photochemically-induced lipid peroxidation they conferred on irradiated PLVs, using the thiobarbituric acid reactive substances assay. At high compound concentrations the compounds were found to act as sunscreens, whilst at lower concentrations pro-oxidant activity was observed. In addition to this main work, methodology for the palladium-catalysed cyanation of vinyl halides with acetone cyanohydrin was developed. Conditions were optimised using beta-bromostyrene, and shown to be applicable to a range of diverse substrates. The protocol proved chemoselective for vinyl bromides in the presence of aryl bromides, which were left unaffected and available for further chemical transformations, adding to the synthetic utility of the reaction.