Since 1893, acetate has been regarded as a very important precursor of naturally occurring phenolic compounds. Although intensive work has been directed at the complete elucidation of this biogenetic pathway, a satisfactory method has yet to be devised for the synthesis of the polyketide chains proposed as intermediates in the acetate scheme. Labelling studies with such compounds would provide information regarding their intermediate role in the acetate route to aromatic compounds. Efforts to date to synthesize these extended poly-β-ketone chains have been unsuccessful. Polypyrones, containing potential polyketide chains, appeared to offer a feasible solution. Synthesis of these compounds by fusing successive C₃ units to triacetic lactone proved successful, and by this method, bis-, tris-, and tetrapyrone systems were constructed. Basic hydrolysis of these fused pyrone systems, followed by intramolecular aldol condensation, demonstrated that naturally occurring phenolic compounds could be synthesized by the cyclization of a polyketide intermediate. / Science, Faculty of / Chemistry, Department of / Graduate

Thesis (Ph. D.)--University of Wisconsin--Madison, 1958. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 92-97).

Thesis (M.Sc.)--The Chinese University of Hong Kong. / Bibliography: leaves 90-96.

Aspergillus niger souche G131 est un champignon qui produit en quantité des métabolites secondaires appartenant à la famille des naphtho-gamma-pyrones (NγPs). Ces NγPs sont des pigments qui présentent des intérêts industriels de par leurs importants potentiels antiradicalaires. L’objectif de ce doctorat est la production à l’échelle pilote et en continu de NγPs à travers la culture du champignon sur milieu solide. Le choix de la fermentation en milieu solide (FMS) comme processus de culture repose sur des aspects d’ordre qualitatif et quantitatif de production, ainsi que sur des raisons économiques et éthiques, relatives à la protection de l’environnement avec notamment la possibilité de valoriser des coproduits agricoles comme milieu de culture pour le champignon. Dans un premier temps, ce travail s’intéresse à la caractérisation de la composition et des potentialités associées aux molécules produites par la souche. Ces potentialités incluent les activités anti-radicalaires et les mesures de cytotoxicité. La thèse porte également sur la caractérisation de la physiologie de croissance de la souche en FMS et sur l’optimisation des conditions de culture par la méthodologie des plans d’expériences pour l’augmentation de la production de NγPs. Une stratégie originale d’optimisation adaptée aux contraintes posées par la FMS est d’ailleurs proposée. Finalement, un transfert d’échelle de production est réalisé au moyen d’un bioréacteur prototype innovant permettant la production à l’échelle pilote de milieu fermenté en continu. Dans son dernier chapitre, ce travail s’intéresse donc à la mise au point des paramètres opératifs qui entourent la production continue de NγPs par FMS. / Aspergillus niger strain G131 is a non-ochratoxigenic filamentous fungus producing high quantities of secondary metabolites known as naphtha-gamma-pyrones (NγPs). NγPs are pigments of industrial interest in reason of their high antioxidant properties. The aim of this dissertation is the continuous, pilote-scaled production of these NγPs through the cultivation of the fungus on solid medium. The choice of solid state fermentation (SSF) as cultivation method is not only driven by quantitative and qualitative considerations, but also by economical and ethical concerns related to environmental protection. SSF allows, in fact, a direct valorization of agricultural byproducts as the solid medium for the fungal growth. First, this work deals with the characterization of the composition and potentialities associated with the molecules produced by the strain, which include antioxidant and cytotoxic activities. Second, the dissertation focuses on the characterization of the fungal growth’s physiology on solid medium and on the optimization of the culture conditions using experimental methodology in order to increase NγPs production. For this purpose, an original optimization strategy is proposed to overcome specific constraints connected to SSF. Finally, a scale transfer of the production is advanced by means of an innovative prototype bioreactor continuously producing fermented material. The final chapter of this work addresses the development of parameters regarding the continuous NγPs production using SSF.

Nous avons mis au point une réaction tandem couplage/cyclisation entre des dérivés acides carboxyliques ß-iodés et des alcynes vrais en présence sels de cuivre. Cette transformation conduit régio et stéréosélectivement à la formation de ?-alkylidènebuténolides. Les différents essais effectués ont montré que la méthodologie tolère un grand nombre de substrats et de fonctions. L’extension de la réactivité sur des acides carboxyliques a,ß-dihalogénés nous a permis d’accéder régio et stéréosélectivement à la formation d’a-halogéno-?-alkylidènebuténolides. La conservation d’un halogène sur le buténolide obtenu dénote la flexibilité de la transformation et permet ensuite d’étendre la structure du buténolide par les réactions de couplage croisés. Cette flexibilité nous a permis d’accéder à la synthèse de rétinoïdes et de précurseurs de nostoclides I et II. Une étude annexe sur des substrats de type aromatiques et hétéroaromatiques a montré l’invertion de la régiosélectivité au profit de la formation d’a-pyranone. Ces essais ont conduit à la synthétiser de nombreux indoles, isocoumarines et thiophènes originaux. / We have developed a tandem coupling/cyclization reaction between ß-iodided acrylic acid derivatives and true alkynes in the presence of copper salts. This transformation led regio and stereoselectively to the formation of ?-alkylidenebutenolides. Different tests have shown that the method tolerates many substrates and functions. The extension of the reactivity on a,ß-dihalogenated acrylic acids derivatives allowed us to access regio and stereoselectively to the formation of a-halo-?-alkylidenebutenolides. The conservation of a halogen on the butenolides obtained indicates the flexibility of the process and allows then the extention of the butenolide structure by cross-coupling reactions. This flexibility has allowed us to access to the synthesis of retinoids and nostoclides I and II precursors. An annex study on other substrates like aromatic and heteroaromatic systems showed the inversion of the regioselectivity of our methodology in favor of the formation of a-pyranone. These tests led to the synthesis of many original indoles, isocoumarines and thiophenes moieties

Modular polyketide synthases (PKSs) are the largest enzymes known to man and are responsible for synthesizing some of the most important human medicines. Their ability to construct stereochemically-rich carbon chains containing diverse substituents has inspired the biosynthetic community to engineer these factories for the in vitro synthesis of a small library of polyketide compounds. New complex polyketides are discovered every year, yet the lack of compound prohibits characterization and testing of these new compounds for medicinal properties. Smaller polyketide compounds generated in vitro could be organically manipulated to generate larger, more complex polyketide natural products and natural product analogs. Chemoenzymatic approaches like this would be extremely beneficial to the scientific community; however, there are still obstacles that must be overcome before the use of PKS for the preparative synthesis of an in vitro generated polyketide library would prove fruitful: purchasing substrates such as methylmalonyl-CoA is cost-prohibitive, PKSs are often difficult to express and purify, and the products generated are typically nonchromophoric. The use of a malonyl-CoA ligase from Streptomyces coelicolor (MatB) was investigated for the enzymatic synthesis of polyketide extender units such as methylmalonyl-CoA (Chapter 2). MatB synthesized a total of 5 CoA-linked extender units in vitro: malonyl-, methylmalonyl-, ethylmalonyl-, hydroxymalonyl- and methoxymalonyl-CoA. Two ternary complex structures of MatB with bound product and leaving group were also solved to sub-2Å resolution. MatB generated extender units were employed in the module-catalyzed synthesis of a triketide pyrone. The selectivity of a PKS module to incorporate a variety of side chains into triketide pyrones was also investigated (Chapter 3). A total of 10 triketide pyrone compounds were synthesized, 5 produced via modular "stuttering" and one possessing a terminal alkyne chemical handle. Lastly, nonchromphoric polyketide products were made visible upon copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) with fluorescent sulforhodamine B azide revealing insights into in vitro reactivites of a PKS module (Chapter 4). The work described in this dissertation has helped advance the scientific community towards procuring an in vitro synthesized polyketide library for future synthetic applications. / text

Doctor of Philosophy / Department of Chemistry / Duy H. Hua / Four research projects were carried out and they are described in this dissertation. Glycogen synthase kinase-3 beta (GSK3β) plays a pivotal and central role in the pathogenesis of Alzheimer's disease (AD) and protein kinase C (PKC) controls the function of other proteins via phosphorylation and involves in tumor promotion. In pursuit of identifying novel GSK3β and/or PKC inhibitors, substituted quinoline molecules were designed and synthesized based on the structure-activity-relationship studies. Synthesized molecules were evaluated for their neural protective activities and selected molecules were further tested for inhibitory activities on GSK3β and PKC enzymes. Among these compounds, compound 2 was found to have better GSK3β enzyme inhibitory and MC65 cell protection activities at low nanomolar concentrations and poor PKC inhibitory activity whereas compound 3 shows better PKC inhibitory activity. This demonstrates the potential for uses of quinoline scaffold in designing novel compounds for AD and cancer. Pharmacokinetics and distribution profiles of two anti-Alzheimer molecules, CP2 and TP70, discovered in our laboratory were assessed using HPLC/MS. Plasma samples of mice and rats fed with TP70 via different routes over various times were analyzed to quantify the amounts of TP70 in plasma of both species. Distribution profiles of TP70 in various tissues of mice were studied and results show that TP70 penetrated the blood brain barrier and accumulated in the brain tissue in significant amounts. Similarly, the amount of CP2 in plasma of mice was analyzed. The HPLC analysis revealed that both compounds have good PK profiles and bioavailability, which would make them suitable candidates for further in vivo efficacy studies. Nanodelivery of specific dsRNA for suppressing the western corn rootworm (WCR, Diabrotica virgifera virgifera) genes was studied using modified chitosan or modified polyvinylpyrrolidinone (PVP) as nanocarriers. Computational simulation studies of dsRNA with these polymers revealed that nanoparticles can be formed between dsRNA and modified chitosan and PVP polymers. Nanocarriers of hydroxylated PVP (HO-PVP) and chitosan conjugated with polyethylene glycol (PEG) were synthesized, and analyzed using IR spectroscopy. Particle sizes and morphology were evaluated using AFM and encapsulation was studied using UV spectroscopy. However, the formation of stable nanoparticles with dsRNA could not be achieved with either of the polymers, and further efforts are ongoing to discover a better nanocarrier for nanodelivery of siRNA by using chitosan-galactose nanocarrier. In our efforts to discover a novel class of tripeptidyl anti-norovirus compounds that can strongly inhibit NV3CLpro, a set of tripeptidyl molecules were synthesized by modifying the P1 - P3 of the substrate peptide including a warhead. It was found that the replacement of P1 glutamine surrogate with triazole functionality does not improve the inhibitory activities of the compounds. In addition, the synthesis of a known dipeptidyl compound (GC376) was carried out for evaluating its efficacy on feline infectious peritonitis (FIP) in cats.

Doctor of Philosophy / Department of Chemistry / Duy H. Hua / Three research projects were carried out and they are described below. The synthesis of substituted phenolic compounds including halogenated di- and trihydroxybenzenes, aminophenols, and substituted di-tert-butylphenols are described. Redox potentials of the synthesized molecules along with various known laccase substrates were measured, and an inverse relationship between the oxidation potential and the efficiency of oxidation by laccase of halogenated hydroxybenzenes and aminophenols is demonstrated. The synthesized substituted phenols were found to be substrates but not inhibitors of laccase. We discovered a new class of di-tert-butylphenols compounds that inhibits the growth of mosquito larvae at low concentrations. Compound 17, 2,4-di-tert-butyl-6-(3-methyl-2-butenyl) phenol caused greater than 98% mortality of third-instar larvae of Anopheles gambiae in the concentration of 0.18 µM. These compounds do not inhibit laccases. It appears that they affect a new target of the mosquito that is different from those of currently existing pesticides. Two anti-Alzheimer molecules, CP2 and TP70, discovered in our laboratory were studied for their pharmacokinetics and distribution. The distribution of CP2 and TP70 in mouse brain region and various tissues of mice were examined. HPLC analysis revealed that CP2 treatment in primary neurons accumulates in mitochondria fraction. Similarly, the amount of CP2 in the brain tissue from wild type and APP/PS1 mice treated with 25 mg/kg/daily for 2 months also have the highest concentration in the mitochondria fractions in the hippocampus. The results show that CP2 and TP70 can penetrate the blood brain barrier and accumulate in the tissue in significant amounts. Pharmacokinetics and bioavailability of compound TP70 were determined. Area under the curve and bioavailability value F were calculated, and data show that TP70 has a good PK profile and bioavailability. For the preparation of a novel tripeptidyl norovirus 3C-like protease (3CL[superscript]pro) inhibitor, the P3 unnatural amino acid, (S)-3-hydroxyphenylalanine was synthesized. The P3 is designed to increase the polarity with the addition of the alcohol group. After combining the P3 unnatural amino acid with the P1 and P2 to form the novel tripeptidyl compound, a study comparing the relations between the structure and its activity (SAR) will confirm whether prediction is correct in our pursuit for an antiviral therapeutic drug in the form of a protease inhibitor.

Parkash Amarender. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 158-172). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Table of contents --- p.ii / Abstract --- p.xi / 撮要 --- p.xiv / List of Abbreviations --- p.xvi / List of Tables --- p.xvii / List of Figures --- p.xix / Chapter CHAPTER 1. --- INTRODUCTION / Chapter 1.1 --- Ribosome-inactivating proteins (RIPs) --- p.3 / Chapter 1.2 --- General Properties of RIPs --- p.5 / Chapter 1.2.1 --- Structure --- p.5 / Chapter 1.2.1.1 --- Type I and Type II RIPs --- p.5 / Chapter 1.2.1.2 --- Small RIPs --- p.10 / Chapter 1.2.2 --- Distribution --- p.12 / Chapter 1.2.3 --- Physicochemical properties --- p.15 / Chapter 1.3 --- Enzymatic activities of RIPs --- p.17 / Chapter 1.3.1 --- N-glycosidase activity --- p.17 / Chapter 1.3.2 --- Polynucleotide:adenosine glycosidase activity --- p.21 / Chapter 1.3.3 --- Ribonuclease (RNase) activity --- p.24 / Chapter 1.3.4 --- Deoxyribonucleolytic (DNase) activity --- p.25 / Chapter 1.3.5 --- Multiple depurination --- p.26 / Chapter 1.3.6 --- Inhibition of protein synthesis --- p.27 / Chapter 1.4 --- Biological activities of RIPs --- p.29 / Chapter 1.4.1 --- Interaction of ribosome-inactivating proteins with cells --- p.29 / Chapter 1.4.1.1 --- Internalization of type 1 ribosome-inactivating proteins --- p.29 / Chapter 1.4.1.2 --- Internalization of type 2 ribosome-inactivating proteins --- p.32 / Chapter 1.4.2 --- Effects on laboratory animals --- p.33 / Chapter 1.4.3 --- Immunosuppressive activity --- p.33 / Chapter 1.4.4 --- Abortifacient activity --- p.34 / Chapter 1.4.5 --- Antiviral activity --- p.35 / Chapter 1.5 --- Physiological roles of RIPs --- p.37 / Chapter 1.6 --- Applications of RIPs --- p.39 / Chapter 1.6.1 --- Possible uses in experimental and clinical medicine --- p.39 / Chapter 1.6.1.1 --- Anti-tumor therapy --- p.40 / Chapter 1.6.1.2 --- Immune disorders --- p.42 / Chapter 1.6.1.3 --- Neuroscience research --- p.43 / Chapter 1.6.2 --- Applications in agriculture --- p.44 / Chapter 1.7 --- Arginine/Glutamate Rich Polypeptides (AGRPs) --- p.46 / Chapter 1.8 --- Objectives of the present study --- p.48 / Chapter 1.8.1 --- Rationale of the study --- p.48 / Chapter 1.8.2 --- Outline of the thesis --- p.50 / Chapter Chapter 2 --- Materials and methods / Chapter 2.1 --- Introduction --- p.52 / Chapter 2.2 --- Materials and methods --- p.54 / Chapter 2.2.1 --- Materials --- p.54 / Chapter 2.2.2 --- Preparation of crude extract --- p.55 / Chapter 2.2.3 --- Purification of proteins --- p.55 / Chapter 2.2.4 --- Molecular weight determination with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.61 / Chapter 2.2.5 --- Protein determination --- p.64 / Chapter 2.2.6 --- N-terminal amino acid sequence --- p.64 / Chapter 2.2.7 --- Preparation of rabbit reticulocyte lysate --- p.65 / Chapter 2.2.8 --- Assay for cell-free protein synthesis- inhibiting activity --- p.65 / Chapter 2.2.9 --- Assay for N-glycosidase activity --- p.66 / Chapter 2.2.10 --- Assay for ribonuclease activity --- p.70 / Chapter 2.2.11 --- Assay for antifungal activity --- p.71 / Chapter 2.2.12 --- Assay for dehydrogenase activity --- p.71 / Chapter Chapter 3 --- Purification and characterization of proteins from their respective sources. / Chapter 3.1. --- Purification and Characterization of Hispidin from Hairy melon (Benincasa hispida var. chieh-qua) / Chapter 3.1.1. --- Introduction --- p.73 / Chapter 3.1.2. --- Results --- p.76 / Chapter 3.1.2.1. --- Purification --- p.78 / Chapter 3.1.2.2. --- Molecular weight determination --- p.84 / Chapter 3.1.2.3. --- N-terminal amino acid sequence --- p.85 / Chapter 3.1.2.4. --- Assay for cell-free protein synthesis-inhibiting activity --- p.86 / Chapter 3.1.2.5. --- Assay for N-glycosidase activity --- p.87 / Chapter 3.1.2.6. --- Assay for ribonuclease activity --- p.88 / Chapter 3.1.2.7. --- Assay for dihydrodiol dehydrogenase activity --- p.88 / Chapter 3.1.2.8. --- Assay for antifungal activity --- p.89 / Chapter 3.1.2.9. --- "Assessment of purity, yield and activity" --- p.91 / Chapter 3.1.3. --- Discussion --- p.92 / Chapter 3.2. --- Purification and Characterization of Momorchin from Dried Bitter Gourd (Momordica charantia) Seeds / Chapter 3.2.1. --- Introduction --- p.95 / Chapter 3.2.2. --- Results --- p.99 / Chapter 3.2.2.1. --- Purification --- p.100 / Chapter 3.2.2.2. --- Molecular weight determination --- p.103 / Chapter 3.2.2.3. --- N-terminal amino acid sequence --- p.104 / Chapter 3.2.2.4. --- Assay for cell-free protein synthesis- inhibiting activity --- p.105 / Chapter 3.2.2.5. --- Assay for ribonuclease activity --- p.105 / Chapter 3.2.2.6. --- Assay for N-glycosidase activity --- p.106 / Chapter 3.2.2.7. --- "Assessment of purity, yield and activity" --- p.107 / Chapter 3.2.3. --- Discussion --- p.108 / Chapter 3.3.3. --- Purification and Characterization of Luffacylin from Sponge Gourd (Luffa cylindrica) / Chapter 3.3.1. --- Introduction --- p.110 / Chapter 3.3.2. --- Results --- p.113 / Chapter 3.3.2.1. --- Purification --- p.115 / Chapter 3.3.2.2. --- Molecular weight determination --- p.119 / Chapter 3.3.2.3. --- N-terminal amino acid sequencing --- p.120 / Chapter 3.3.2.4. --- Assay for cell-free protein synthesis- inhibiting activity --- p.121 / Chapter 3.3.2.5. --- Assay for ribonuclease activity --- p.121 / Chapter 3.3.2.6. --- Assay for N-glycosidase activity --- p.122 / Chapter 3.3.2.7. --- Assay for antifungal activity --- p.123 / Chapter 3.3.2.8. --- "Assessment of purity, activity and yield" --- p.124 / Chapter 3.3.3. --- Discussion --- p.125 / Chapter 3.4. --- Purification and Characterization of α and β Benincasin from fresh Winter Melon {Benincasa hispida var. dong-gua) Seeds / Chapter 3.4.1. --- Introduction --- p.127 / Chapter 3.4.2. --- Results --- p.129 / Chapter 3.4.2.1. --- Purification --- p.130 / Chapter 3.4.2.2. --- Molecular weight determination --- p.135 / Chapter 3.4.2.3. --- N-terminal amino acid sequence --- p.136 / Chapter 3.4.2.4. --- Assay for cell-free protein synthesis- inhibiting activity --- p.137 / Chapter 3.4.2.5. --- Assay for ribonuclease activity --- p.137 / Chapter 3.4.2.6. --- Assay for antifungal activity --- p.138 / Chapter 3.4.2.7. --- "Assessment of purity, activity and yield" --- p.140 / Chapter 3.4.3. --- Discussion --- p.141 / Chapter 3.5. --- Purification and characterization of Moschins from Pumpkin (Cucurbita moschata) Seeds / Chapter 3.5.1. --- Introduction --- p.143 / Chapter 3.5.2. --- Results --- p.145 / Chapter 3.5.2.1. --- Purification --- p.146 / Chapter 3.5.2.2. --- Molecular weight determination --- p.149 / Chapter 3.5.2.3. --- N-terminal amino acid sequence --- p.150 / Chapter 3.5.2.4. --- Assay for cell-free protein synthesis- inhibiting activity --- p.151 / Chapter 3.5.2.5. --- Assay for ribonuclease activity --- p.151 / Chapter 3.5.2.6. --- "Assessment of purity, activity and yield" --- p.152 / Chapter 3.5.3. --- Discussion --- p.153 / Chapter Chapter 4 --- General Discussion and Conclusion --- p.154 / References --- p.158