Thesis (Ph. D.)--Michigan State University. Dept. of Chemistry, 2008. / Title from PDF t.p. (viewed July 31, 2009). Includes bibliographical references (p. 179-187). Also issued in print.

Thesis (M.S.)--Youngstown State University, 2008. / Includes bibliographical references (leaves 77-83). Also available via the World Wide Web in PDF format.

Thesis (Ph. D.)--Oregon State University, 2008. / Printout. Includes bibliographical references (leaves 80-103). Also available on the World Wide Web.

Sialic acids are involved in many biological processes. In glycoproteins and glycolipids they are essential for signalling and mediate molecular interactions as well as being targets for many pathogens such as influenza virus. The synthesis of sialylated glycoconjugates is of great importance. The incorporation of sialic acid through chemical synthesis carries several difficulties, enzymatic strategies using glycosyltransferases are very attractive alternative strategy, and have been used on a broad range of substrates forming glycosidic linkages with regio-and stereo-specificity. The work presented herein shows the study and application of two enzymes, Photobacteriumdamselae alpha2,6-sialyltransferase (Pd2,6ST) and Trypanosoma cruzi trans-sialidase (TcTS) which are used in the synthesis of sialyloligosaccharides. Both enzymes were expressed in E.coli and purified for biotransformations. In the first application new sialylated chromogenic compounds were generated through this enzymatically by using TcTS and a Pd2,6ST. These compounds were used for the detection of neuraminidase activity in a number of biological samples and led to the discovery of neuraminidase activity from Bacillus pumilus and Arthrobacter aurescens, two different bacteria in which the presence of neuraminidases had never been described. Secondly, TcTS was used to study lipid glycosylations. Glycans in biological systems can be associated to complex lipidic microdomains and the presence of these microdomains can affect the activity of some enzymes. In case of Trypanosoma cruzi trans-sialidase, a decreased activity was detected when the acceptor substrate was part of the aggregated lipid rafts compared to activity observed when the reaction was performed using fully dispersed substrate. Thirdly, the sialylation of glycoarrays using Pd2,6ST was studied. For the first time, sialylated glycans with alpha2,6- glycosidic linkages were successfully incorporated into a gold glycoarray platform, which had been previously developed for the label-free detection of carbohydrate-protein interactions. Successful enzymatic incorporation of sialic acids onto the arrays was confirmed with commercial available lectins. Finally, by using the gold glycoarray platform containing both 2,3 and 2,6 linked sialic acids as well as other common glycans, the carbohydrate-binding properties of the surface proteins of the bacterium Lactobacillus reuteri was studied using MALDI-ToF MS techniques. For first time, strong interactions were observed between a mucus binding protein and Neu5Ac alpha2,6-linked glycans, with much weaker binding to 2,3-linked analogues. Such glycan structures have been identified in abundant manner in colon mucins and this study contributes to the understanding of complex interactions between mucins and probiotic organisms as well as pathogenic bacteria. These studies show that glycan arrays can contribute both to the understanding of probiotics as well as to the identification of glycan binding proteins as targets for new drugs.

Tuberculosis (TB) is the disease arising from infection by Mycobacterial tuberculosis and kills millions of people every year. Difficulties in the treatment of TB and the emergence of multiple-drug resistant and extreme-drug resistant M. tuberculosis strains have increased interest in finding new antimycobacterial agents. The cell wall of mycobacteria is essential for the survival of these bacteria and enzymes involved in its assembly are key targets for anti-mycobacterial chemotherapy. One of the largest components of the cell wall is the arabinogalactan, which is composed of arabinofuranose (Araf) and galactofuranose (Galf) residues. These monosaccharides are incorporated into the polysaccharide by arabinosyltransferases and galactosyltransferases that employ decaprenolphophoarabinose (DPA) and uridine diphospho-galactofuranose (UDP-Galf) as the donor substrate, respectively. The synthesis of analogues of DPA and UDP-Galf as potential inhibitors of mycobacterial glycosyltransferases is presented in the thesis. Carbohydrate mimics of Araf and Galf that have a bicyclo[3.1.0]hexane at the core were prepared. Key steps involved the formation of bicyclo[3.1.0]hexane system via an intramolecular displacement reaction followed by a separation by converting a mixture of enantiomers into diastereomers. The absolute configuration of these species was confirmed by X-ray analysis of a crystalline derivative of the Araf analogue. The bicyclo[3.1.0]hexane based mimics were then alkylated with various aldehydes through reductive amination to form the DPA and UDP-Galf analogues. The synthesis of the sulfonium ion analogs of Galf was also carried out. The precursor of these compounds, a cyclic sulfide, was synthesized in nine steps from D-arabinitol. The key step is a conversion of an olefin into hydroxymethyl group thus establishing a stereogenic centre that is essential in forming a molecule that is a mimic of the galactofuranose ring. This sulfide was then coupled with alkyl halides to form sulfonium ion compounds in good yields. All of the DPA and UDP-Galf analogues were tested for their ability to inhibit GlfT2, a key galactofuranosyltransferase involved in the synthesis of the galactan portion of the mycobacterial arabinogalactan. Most of the compounds showed weak inhibition of the enzyme; however, a few were moderately active and are the mode of inhibition of these analogues is currently being studied.

No description available.

Natural products occupy a central role as the majority of currently used antibiotic and anticancer agents. Among these are type-II polyketide synthase (PKS)-derived molecules, or polyketides, which are produced by many representatives of the genus Streptomyces. Some type-II polyketides, such as the tetracyclines and the anthracycline doxorubicin, are currently employed as therapeutics. However, several polyketide molecules exhibit promising biological activity, but due to toxic side effects or solubility concerns, remain undeveloped as drugs. Gilvocarcin V (GV) (topoisomerase II inhibitor) has a novel mechanism of action: [2+2] cycloaddition to thymine residues by the 8-vinyl side chain and cross-linking of histone H. Mithramycin blocks transcription of proto-oncogenes c-myc and c-src by forming an Mg2+-coordinated homodimer in the GC-rich minor groove of DNA. The purpose of this research was to investigate the biosynthesis of several type II polyketide compounds (e.g. chrysomycin, elloramycin, and mithramycin) with the goal of improving the bioactivities of these drugs through combinatorial biosynthesis. Alteration of the glycosylation pattern of these molecules is one promising way to improve or alter the bioactivities of these molecules. To this end, an understanding of the glycosyltransferases and post-polyketide tailoring enzymatic steps involved in these biosynthetic pathways must be established. Four specific aims were established to meet these goals. In specific aim 1, the biosynthetic locus of chrysomycin A was successfully cloned and elucidated, which afforded novel biosynthetic tools. Chrysomycin monooxygenases were found to catalyze identical roles to their gilvocarcin counterparts. Cloning of deoxysugar constructs (plasmids) which could direct biosynthesis of ketosugars, NDP-D-virenose, and NDP-D-fucofuranose in foreign pathways was undertaken in specific aim 2. Finally, these “sugar” plasmids were introduced into producer organisms of elloramycin and mithramycin pathways in specific aims 3 and 4 to interrogate the endogenous glycosyltransferases in order to alter their glycosylation patterns. These experiments resulted in the successful generation of a newly glycosylated tetracenomycin, as well as premithramycin, and mithramycin analogues. In specific aim 4, a new mithramycin analogue with an altered sugar pattern rationally designed and improved structural features was generated and structurally elucidated.

The abundant cell-surface lipophosphoglycan (LPG) of Leishmania parasites plays a central role throughout the eukaryote’s life cycle. A number of LPG-defective mutants and their complementing genes have been isolated and have proven invaluable in assessing the importance of LPG and related glycoconjugates in parasite virulence. While ricin agglutination selection protocols frequently result in lpg- mutants, one L. donovani variant we isolated, named JABBA, was found to be lpg+. Procyclic (logarithmic) JABBA expresses significant amounts of a large-sized LPG, larger than observed from procyclic wild-type but similar in size to LPG from wild-type from metacyclic (stationary) phase. Structural analysis of the LPG from logarithmically-grown JABBA by capillary electrophoresis protocols revealed that it averaged 30 repeat units composed of the unsubstituted Gal(β1,4)Man(α1)-PO4 typical of wild-type L. donovani. Analysis of JABBA LPG caps indicated that 20% are the disaccharide Glc(β1,2)Man, trisaccharide Gal(β1,4)[Glc(β1,2)]Man, and tetrasaccharide Gal(β1,4)[Glc(β1,2)Man(α1,2)]Man in addition to wild-type Gal(β1,4)Man and Man(α1,2)Man and Gal(β1,4)[Man(α1,2)]Man terminating caps, These glucose containing isoforms were absent in stationary parasites. Consistent with these structural observations, analyses of the relevant glycosyltransferases in JABBA microsomes involved in LPG biosynthesis showed a two-fold increase in elongating mannosylphosphoryltransferase activity and up-regulation of a β-glucosyltransferase activity. The β-glucosyltransferase in both JABBA and wild-type in vitro produced a β-glucosidase sensitive and β-galactosidase sensitive trisaccharide, indicative of the mannose of repeating units and caps being used in substrate, novel in comparison to other Leishmania species. Furthermore, the caps of JABBA LPG are cryptic in presentation as shown by the loss of binding by the lectins ricin, peanut agglutinin and concanavalin A and reduced accessibility of the terminal galactose residues to oxidation by galactose oxidase. These results indicate that LPG from JABBA is intriguingly similar to the larger LPG in wild-type parasites that arises following the differentiation of the non-infectious procyclic promastigotes to infectious, metacyclic forms, and has a unique β-glucosyltransferase not active in vivo in wild-type parasites.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Thesis (M.S.)--Ohio University, March, 2010. / Title from PDF t.p. Includes bibliographical references.