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Thiopeptides are posttranslationally-processed macrocyclic peptide metabolites, characterized by extensive backbone and side chain modifications that include a six-membered nitrogenous ring, thioazol(in)e/oxazol(in)e rings, and dehydrated amino acid residues. Thiostrepton A, produced by Streptomyces laurentii ATCC 31255, is one of the more structurally complex thiopeptides, containing a second macrocycle bearing a quinaldic acid. Thiostrepton A and other thiopeptides are of great interest due to their potent activities against emerging antibiotic-resistant Gram-positive pathogens, in addition to their antimalarial and anticancer properties. The ribosomal origins for thiopeptides have been established, however, few details are known concerning the posttranslational modification steps. Alteration to the primary amino acid sequence of the precursor peptide provides an avenue to probe the substrate specificity of the thiostrepton A posttranslational machinery. The information gathered from current studies can also be used to refine thiostrepton’s structure-activity relationship, providing insight into the key features of its scaffold that impart specificity toward each biological target. A fosmid-dependent biosynthetic engineering platform for thiostrepton A was developed and a series of thiostrepton analogs were successfully produced adapting this method. The seventh residue of thiostrepton A is predicted to be critical for the metabolite’s antibacterial activity. Our results were consistent this hypothesis and demonstrated that substitution of Thr7 in the thiostrepton A precursor peptide disrupts both biological activity and successful biosynthesis of the analogs. The thiostrepton biosynthetic machinery’s tolerances toward structural variation at the second and fourth positions of the TsrA core peptide were probed by the saturation mutagenesis of Ala2 and Ala4, respectively. Eight thiostrepton Ala2 variants were isolated with two analogs truncated at the N-terminus by one amino acid, bearing a shortened quinaldic acid-containing macrocycle. Our results suggested that the identity of the core peptide second residue influences the biosynthesis of a thiostrepton analog, however, not essential for the antibacterial and proteasome inhibitory activities of the full-length variants. Additionally, the quinaldic acid loop size affects thiostrepton’s antibacterial potency, but is not critical for the proteasome inhibitory activity. Sixteen thiostrepton analogs were isolated from Ala4 mutagenesis studies. We demonstrated that the identity of the amino acid residue at the fourth position in the thiostrepton scaffold is not critical to inhibit either the ribosome or the proteasome in vitro.

Master of Science / Department of Chemistry / Ping Li / Biodegradable polyhydroxybutyrates (PHBs) produced by a wide range of bacteria have been considered as an ideal alternative to petroleum-based plastics. Two types of mechanistic probes have been synthesized in order to understand the mechanism of PHB synthases (PhaCs). The first type is oxo analogs in which the sulfur in the coenzyme A (CoA) thioester has been replaced with an oxygen atom. A series of 3-R-hydroxybutyryl oxo CoA analogs, (HB)[subscript]nOCoA (n = 1, 2 and 3), were synthesized chemoenzymatically in good yields. Two models involving covalent catalysis with Cys have been proposed for the chain elongation catalyzed by PhaCs. The first involves an active site composed of two monomers in which the growing hydroxybutyrate (HB) chain alternates between Cys on each monomer. The second involves noncovalent intermediates (HB)[subscript]nCoA (n ≥ 2). Here the substrate analog HBOCoA was successfully employed to trap the noncovalent intermediates in the reactions catalyzed by class III PhaC from Allochromatium Vinosum, which supports our preferred second mechanistic model. Furthermore, it is also the first time that a wild-type (wt) synthase was used to investigate the chain elongation models. The other type of mechanistic probes is 3-R-hydroxyalkyl CoA that was used to investigate the substrate specificity of PhaCs from different classes. Substrate availability has been a challenge to study PHB synthases in vitro. Starting with commercially available dimethyl S-malate, the intermediate S-ethyl 2-(oxiran-2-yl) acetate 23 was synthesized via a ring-opening reaction involving lactone 21 and trimethylsilyl iodide followed by an oxidation reaction involving silver oxide. The regiospecific ring-opening reaction of epoxide 23 with different organometallic reagents afforded a straightforward access to ethyl 3-R-hydroxybutanoates attached with a variety of side chains. The final CoA compounds were obtained through the thiotransesterification reaction between corresponding benzenethioesters and the thiol group in CoA. This synthetic approach provides a new avenue to modifications of alkyl groups in 3-R-hydroxyalkyl CoA in an efficient manner.

This thesis describes the synthesis and preliminary enzymatic study of glutaryl-S-(8-aminooctyl)-L-cysteinylglycine and glutaryl-S-(10-aminodecyl)-L-cysteinylglycine as inhibitors of glyoxalase I. These analogs of glutathione were prepared as potential ligands for affinity chromatography purification of glyoxalase I. The compounds were synthesized by a seven-step procedure in overall yields of 24% for the octyl analog and 33% for the decyl analog. Both compounds exhibited mixed type inhibition of the enzyme, with the decyl derivative being more inhibitory than the octyl derivative. The inhibition was nonlinear (parabolic) for both compounds. Although less inhibitory than the corresponding S-substituted glutathione derivatives, these analogs are promising candidates for affinity chromatography ligands. Such compounds may also be useful in studying the mechanism of glyoxalase I.

Thesis advisor: Udayan Mohanty / Computational research can play a crucial component in the discovery of unique biochemical phenomena, from answering fundamental questions about molecular function and structure to the modeling of designed pharmaceuticals to cure many debilitating illnesses. Here computational methods are employed to examine the exquisite role that magnesium ions play in stabilizing ribosomal subunits responsible for protein translation and to analyze the potential of a proposed anticancer drug for a pathway that is impaired in the majority of human cancer cases. / Thesis (BS) — Boston College, 2011. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: College Honors Program. / Discipline: Chemistry.

Lo Ho Yin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. 142-149). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Wang Xin Ning. / "May 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 201-236). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Lee Chi-man. / "July 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Chen Yu. / Thesis submitted in: November 2001. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 98-112). / Abstracts in English and Chinese. / Acknowledgements --- p.I / List of publications --- p.II / Abstract --- p.III / Abstract (Chinese) --- p.IV / Abbreviations --- p.V / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Tea --- p.1 / Chapter 1.2 --- Green tea --- p.3 / Chapter a) --- Chemical composition of green tea --- p.3 / Chapter b) --- Pharmacological activities of green tea polyphenols --- p.6 / Chapter c) --- Pharmacokinetics of green tea polyphenols --- p.10 / Chapter 1.3 --- Objective --- p.14 / Chapter Chapter 2 --- Validation of analysis method for tea catechins --- p.15 / Chapter 2.1 --- Materials and methods --- p.16 / Chapter a) --- Preparation of a catechin-mixture from tea --- p.16 / Chapter b) --- Preparation of stock solutions --- p.18 / Chapter c) --- Preparation of biofluid samples --- p.18 / Chapter d) --- HPLC analysis of tea catechins --- p.19 / Chapter 2.2 --- Results --- p.21 / Chapter a) --- Catechin-mixture (tea extracts) --- p.21 / Chapter b) --- "Extraction from plasma, urine and feces" --- p.21 / Chapter c) --- HPLC analysis of biofluid samples --- p.23 / Chapter 2.4 --- Discussion --- p.26 / Chapter Chapter 3 --- Pharmacokinetics of tea catechins following administration of different doses of the catechin-mixture --- p.32 / Chapter 3.1 --- Materials and methods --- p.33 / Chapter a) --- Surgery and animal maintenance --- p.33 / Chapter b) --- Dosing and sample collection --- p.33 / Chapter c) --- Pharmacokinetics analysis of tea catechins --- p.35 / Chapter 3.2 --- Results --- p.36 / Chapter 3.3 --- Discussion --- p.50 / Chapter Chapter 4 --- Pharmacokinetics of tea catechins following administration of different doses of individual catechins --- p.52 / Chapter 4.1 --- Materials and methods --- p.53 / Chapter a) --- Chemicals and reagents --- p.53 / Chapter b) --- Pharmacokinetic study of tea catechins and the HPLC analysis --- p.53 / Chapter c) --- Pharmacokinetic analysis of tea catechins --- p.54 / Chapter 4.2 --- Results --- p.55 / Chapter 4.3 --- Discussion --- p.67 / Chapter Chapter 5 --- Plasma protein binding of tea catechins --- p.69 / Chapter 5.1 --- Introduction --- p.69 / Chapter 5.2 --- Materials and methods --- p.72 / Chapter a) --- Ultrafiltration --- p.72 / Chapter b) --- Preparation of stock solution --- p.73 / Chapter c) --- Determination of nonspecific binding of the catechins --- p.73 / Chapter d) --- Determination of ultrafiltration conditions --- p.74 / Chapter e) --- In vitro plasma protein binding assay --- p.74 / Chapter f) --- Statistical analysis --- p.75 / Chapter 5.3 --- Results --- p.76 / Chapter a) --- Nonspecific binding in ultrafiltration --- p.76 / Chapter b) --- Protein binding of the catechins --- p.76 / Chapter c) --- Statistical analysis --- p.76 / Chapter 5.4 --- Discussion --- p.80 / Chapter Chapter 6 --- Partition of tea catechins in red blood cell --- p.82 / Chapter 6.1 --- Materials and methods --- p.83 / Chapter a) --- Prepare of stock solution --- p.83 / Chapter b) --- In-vitro erythrocyte partition --- p.83 / Chapter c) --- Data Analysis --- p.83 / Chapter 6.2 --- Results --- p.85 / Chapter a) --- Selection of experiment conditions --- p.85 / Chapter b) --- Partition of catechins to RBC --- p.85 / Chapter 6.3 --- Discussion --- p.87 / Chapter Chapter 7 --- Comparison of pharmacokinetics of tea catechins in mixture form versus pure compound --- p.89 / Chapter Chapter 8 --- Conclusion --- p.95 / References --- p.98

Lau Kin Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 97-103). / Abstracts in English and Chinese. / Contents --- p.1 / Chapter Chapter 1 --- Introduction --- p.5 / Chapter 1.1 --- Calcium Signaling in Endothelial Cells --- p.5 / Chapter 1.1.1 --- Calcium and its functions --- p.5 / Chapter 1.1.2 --- "Second Messengers: Inositol-1,4,5-Triphosphate and Diacylglycerol" --- p.6 / Chapter 1.1.3 --- Propagation of Ca2+ Signals --- p.8 / Chapter 1.1.4 --- Ca2+-ATPases --- p.9 / Chapter 1.1.5 --- Regulation of Sarcoplasmic Reticulum --- p.10 / Chapter 1.1.6 --- Agonist-induced Ca2+ Entry --- p.11 / Chapter 1.2 --- Mechanism of Store-Operated Ca2+ Entry --- p.14 / Chapter 1.2.1 --- Signaling Mechanisms of SOC --- p.14 / Chapter 1.2.1.1 --- A Diffusible Messenger --- p.14 / Chapter 1.2.1.2 --- Conformational Coupling --- p.15 / Chapter 1.2.1.3 --- Vesicle Secretion --- p.16 / Chapter 1.3 --- Regulation of Ca2+ Entry by cGMP --- p.20 / Chapter 1.4 --- Molecular Structres of Store-operated Channels --- p.22 / Chapter 1.4.1 --- Drosophila Transient Receptor Potential (trp) Gene --- p.22 / Chapter 1.4.2 --- Trpl Gene --- p.23 / Chapter Chapter 2 --- Methods and Materials --- p.27 / Chapter 2.1 --- Materials --- p.27 / Chapter 2.1.1 --- Phosphate-buffered saline --- p.27 / Chapter 2.1.2 --- Culture Media and Materials --- p.27 / Chapter 2.2 --- Preparations and Culture of Cells --- p.28 / Chapter 2.2.1 --- Culture of Rat Aortic Endothelial Cells --- p.28 / Chapter 2.2.2 --- Culture of Human Bladder Epithelial Cell Line --- p.29 / Chapter 2.2.3 --- Culture of Human Embryonic Kidney Epithelial Cell Line --- p.29 / Chapter 2.3 --- Cell. Subculture and Marvest --- p.29 / Chapter 2.4 --- Intracellular Free Calcium Ions ([Ca2+]i) measurment --- p.30 / Chapter 2.4.1 --- Chemicals --- p.30 / Chapter 2.4.2 --- Bathing solutions --- p.31 / Chapter 2.4.3 --- Preparations of Cells for [Ca2+]i Measurement --- p.31 / Chapter 2.4.3.1 --- Plating cells on Glass Cover Slips for [Ca2+]i Measurement with PTI RatioMaster Fluorescence System --- p.31 / Chapter 2.4.3.2 --- Plating cells on Glass Cover Slips for [Ca2+]i Measurement with Confocal Imaging System and Confocal Laser Scanning Microscopy --- p.32 / Chapter 2.4.4 --- PTI RatioMaster Fluorescence System --- p.35 / Chapter 2.4.4.1 --- Experimental Setup --- p.35 / Chapter 2.4.4.2 --- Fura-2/AM Dye loading --- p.35 / Chapter 2.4.4.3 --- Background Fluorescence and [Ca ]i Measurement --- p.37 / Chapter 2.4.5 --- Confocal Imaging System --- p.37 / Chapter 2.4.5.1 --- Experimental Setup --- p.37 / Chapter 2.4.5.2 --- Fluo-3/AM Dye Loading --- p.39 / Chapter 2.4.5.3 --- [Ca2+]i Measurement --- p.39 / Chapter 2.4.6 --- Confocal Laser Scanning Microscopy --- p.40 / Chapter 2.4.6.1 --- Principles --- p.40 / Chapter 2.5 --- Cloning and expression of Trpl in HEK293 cell line --- p.43 / Chapter 2.5.1 --- Cloning of Htrpl Gene into pcDNA3 Vector --- p.43 / Chapter 2.5.1.1 --- Enzyme Digestion --- p.43 / Chapter 2.5.1.2 --- Gel electrophoresis and Isolation of Htrpl by GeneCIean II Kit --- p.44 / Chapter 2.5.1.3 --- Ligation of Trpl and pcDNA3 Vector --- p.44 / Chapter 2.5.1.4 --- Transformation --- p.47 / Chapter 2.5.1.5 --- Purification of cloned Trpl-pcDNA3 by QIAprep Spin Miniprep Kit --- p.47 / Chapter 2.5.2 --- Transfection of HEK293 Cells with Htrpl and pEGFP-Nl Vector --- p.48 / Chapter 2.5.2.1 --- Cell Preparation for Transfection --- p.48 / Chapter 2.5.2.2 --- Transfection --- p.48 / Chapter 2.5.3 --- Fluorescence Labeling of Expressed Htrpl Channel in HEK293 Cells --- p.49 / Chapter 2.5.3.1 --- Immunostaining with Anti-TRPCl Antibody --- p.49 / Chapter 2.5.3.2 --- Labeling with FITC2° Antibody --- p.50 / Chapter Chapter 3 --- Results --- p.51 / Chapter 3.1 --- Propagation of Ca2+ Signaling --- p.51 / Chapter 3.2. --- Effect of cGMP on SERCA --- p.55 / Chapter 3.2.1 --- ATP stimulated Ca2+ release from internal stores --- p.55 / Chapter 3.2.2 --- Effect of cGMP on the falling phase of [Ca2+]i --- p.55 / Chapter 3.2.3 --- Effect of CPA on the falling phase of [Ca2+]i --- p.58 / Chapter 3.2.4 --- Effect of KT5823 on cGMP --- p.63 / Chapter 3.3. --- Effect of cGMP on bradykinin-activated capacitative Ca2+ entry --- p.65 / Chapter 3.3.1 --- Bradykinin induced capacitative Ca2+ entry --- p.65 / Chapter 3.3.2 --- Effect of cGMP on Ca2+ entry activated by bradykinin --- p.67 / Chapter 3.3.3 --- Effect of KT5823 on the inhibitory effect of cGMP on Ca2+ entry activated by bradykinin --- p.67 / Chapter 3.3.4. --- Effect of cGMP and KT5823 on capacitative Ca2+ entry activated by a combination of different agonists. --- p.71 / Chapter 3.4 --- Cloning and expression of htrpl in HEK 293 cell line --- p.75 / Chapter 3.4.1 --- Optimizing transfection conditions using pEGFP-Nl --- p.78 / Chapter 3.4.2 --- Transient transfection of htrpl channel in HEK293 cells --- p.81 / Chapter 3.4.3 --- Channel properties of expressed htrpl channel --- p.84 / Chapter Chapter 4 --- Discussion --- p.88 / Chapter 4.1 --- Ptopagation of Ca2+ Signaling --- p.88 / Chapter 4.2 --- Effect of cGMP on[Ca2+]i of Vascular Endothelial Cells --- p.89 / Chapter 4.2.1 --- Effect of cGMP on SERCA --- p.89 / Chapter 4.2.2 --- Effect of cGMP on Regulation of Agonist-Activated Capacitative Ca2+ Entry --- p.92 / Chapter 4.2.3 --- Physiological Property of Expressed Htrpl in HEK293 cells --- p.95 / References --- p.97