Abstract not available.

Cancer is the second leading cause of the death in the US. Cancer patients who do not respond to surgery and radiation or others with hematological malignancies are usually treated with chemotherapy. The ability of cancer cells to develop multidrug resistance (MDR) and the undesirable side effects of chemotherapeutic drugs are the major obstacles to successful chemotherapy. The marine environment is an enormous resource of unique and highly bioactive compounds with significant anticancer, anti-inflammatory, analgesic, immunomodulatory, and anti-viral activities. The Red Sea sponge Callyspongia (＝Siphonochalina ) siphonella is a rich source of triterpenoids. The sponge was reported to contain nineteen polyepoxysqualene-derived triterpenoids, divided into four groups, according to their different skeletons, including the major group sipholanes. Interestingly, these triterpenoids were not reported for their biological activity. In a search for bioactive marine natural products to treat cancer, eleven new terpenoids were isolated from the sponge C. siphonella, along with known sipholenols A, G, and sipholenone A. The structures of new compounds were elucidated by 1D and 2D NMR spectroscopy, HRMS, and X-ray crystallography. Major sipolanes, sipholenol A and sipholenone A, were subjected to biocatalytic and semisynthetic transformations to generate structurally diverse analogs and optimize their biological activities. The isolated sipholanes and their derivatives were evaluated for their ability to reverse ABCB1/P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) in human epidermoid cancer cells and for their antiproliferative activity against the highly malignant ＋SA mouse mammary epithelial cells. Four triterpenoids, sipholenol A, sipholenone E, sipholenol L, and siphonellinol D were found to reverse P-gp-mediated resistance to colchicine at non-cytotoxic concentrations. A pharmacophore model consisting of three hydrophobic points and two hydrogen bond acceptors was generated for the active sipholane P-gp modulators. Several semisynthetic sipholane derivatives, particularly esters and ethers, showed remarkably enhanced antiproliferative activity in ＋SA cancer cells compared to their parent sipholanes. In conclusion, these findings strongly suggest that sipholane triterpenoids represent a novel class of anticancer marine natural products.

Triclosan, 5-chloro-2-2,4-dichlorophenoxy)phenol, is an active ingredient in many household disinfectants and has been extensively used in improving environmental hygiene. The chemical can be found as an antiseptic component in medical products such as hand disinfecting soaps, medical skin creams, dental products and many household cleansers. Triclosan has been found as a contaminant of rivers, lakes, costal water, breast milk and human urine and so on. In Europe, about 350 tonnes of triclosan are produced annually for commercial applications. Recent studies suggested that triclosan can be undergone cyclization to form 2,8-dichlorodibenzo-p-dioxin 2,8-DCDD) in aqueous solution under UV irradiation. In order to understand triclosan and its relative components of photo-transformation and metabolism in environmental water, mouth wash, toothpaste, breast milk, rat urine and plasma comprehensively, we have performed several projects and their main results are shown as below. In this thesis, triclosan in the waste, river and coastal water samples collected in Hong Kong was analyzed by GC-ITMS. 13C12 -triclosan was used as internal standard in the quantitative analysis. Water samples were extracted and cleaned-up with a C18 solid-phase extraction cartridge. The recoveries of triclosan in spiked coastal water at three different concentrations were 83% to 110%. The method detection limit was 0.25 ng/l for triclosan in one-liter water, and the relative standard deviation and relative error were less than 11.0% and 12.3%, respectively n ＝ 3). The method was successfully applied to analyze water samples collected from rivers, coastal water bodies and wastewater treatment plants at ng/l levels. The formation of dioxins including 2,7/2,8-dichlorodibenzo-p -dioxin 2,7/2,8-DCDD), 2,3,7-trichlorodibenzo-p-dioxin 2,3,7-TrCDD), 1,2,8-trichlorodibenzo-p-dioxin 1,2,8-TrCDD), 2,3,7,8-tetrachlorodibenzo-p-dioxin 2,3,7,8-TeCDD), 1,2,3,8-tetrachlorodibenzo- p-dioxin 1,2,3,8-TeCDD) and chlorinated triclosan were studied in the presence of low concentration of active chlorine in filtered seawater under UV irradiation 365nm) and dark condition. 2,3,7,8-TeCDD was not detected under dark condition when concentration of triclosan was 0.2 mg/ml and active chlorine was 2.0 mg/ml while 2,7/2,8-DCDD, 2,3,7-TrCDD, 1,2,8-TrCDD, 1,2,3,8-TeCDD and chlorinated trilosan were determined. Under the same concentration of triclosan and active chlorine, 2,3,7,8-TeCDD was detected after the UV irradiation from 120 to 960 min. Formation rate and levels of dioxins and chlorinated triclosan were compared for the experiments with different concentration of triclosan and active chlorine. A GC-MS method was developed for the analysis of 2,8-DCDD in toothpaste and mouthwash. After liquid-liquid extraction of aqueous phase with n-hexane, the extract was cleaned by a silica-bonded C18 solid-phase extraction SPE) cartridge and analyzed. The detection limit was 0.96 ng/g and 0.83 ng/g for 1.0 g toothpaste and mouthwash, respectively. The recovery of 2,8-DCDD ranged from 87.5% to 104.2%. The relative errors were less than 12.5%, and the intra-day as well as inter-day precisions represented by RSD were less than 11.2% and 10.6%, respectively. The highest concentration of 2,8-DCDD was 56.3 ＋/- 8.5 ng/g in toothpaste and 180.6 ＋/- 19.2 ng/g in mouthwash. Photo-degradation of spiked triclosan to 2,8-DCDD in toothpaste and mouthwash under UV irradiation was performed to validate the formation mechanism of 2,8-DCDD in toothpaste and mouthwash. A method involving liquid-liquid extraction, gel permeation chromatography clean up and GC-MS analysis was developed for the determination of triclosan in the breast milk. The calibration curve gave a linear dynamic range of 1.0 to 150 ng/g with correlation coefficient of 0.9996 and the recoveries ranged from 86.2% to 96.6% for the spiked milk samples containing triclosan at three different concentrations. The relative standard deviation and relative error were 0.75% to 9.0% and -7.2 ＋/- 3.4% to 9.7 ＋/- 0.9%, respectively. The limit of quantification of the method was 0.15 ng/g milk weight or 4.8 ng/g lipid. The method was successfully applied for the determination of triclosan in human breast milk. The results of triclosan concentration in 148 breast milk samples were ranged from not detected to 53.04 ng/g lipid based). In vitro metabolic study of triclosan was performed using Sprague-Dawley SD) rat liver S9 and microsome, while the oral metabolism was investigated on SD rat. Twelve metabolites were identified by using in-source fragmentation from HPLC-APCI/ITMS. Compared to ESI/MS and tandem mass spectrometry that gave little fragmentation for triclosan and its metabolites, the in-source fragmentation under APCI provided intensive fragmentations for structural identifications. The obtained results indicated that the glucuronidation and sulfonation was the major pathway of phase II metabolism and the hydroxylated products were the major phase I metabolites. Glucose, mercapturic acid and cysteine conjugates of triclosan were also observed in the urine samples of rat oral administrated with triclosan. The in vitro metabolic rates of triclosan and its major glucuronidation metabolite were determined. Plasma samples from the SD rats after oral administration of 5 mg/kg triclosan were analyzed by ultra performance liquid chromatography-triple quadrupole mass spectrometry methods for pharmacokinetic study of triclosan. The limit of quantification of the developed analytical method was 10.8 ng/ml. The recovery, accuracy, precision and repeatability were satisfactory. The half time of elimination was 48.5 ＋/- 10.5 hours. Triclosan and its metabolites, including two hydroxylated and sulfated triclosan, one glucuronidated triclosan and one sulfated triclosan were detected in rat plasma.

The recent growth of the market of chemically synthesized peptides as therapeutics exemplifies their many advantages over small molecules as drug candidates. Solid phase techniques have been shown to be ideal for manufacturing peptides, but large excesses of reagents and solvent, unpredictably difficult couplings, and time consuming analysis are currently inhibiting efficient automated syntheses. Nonetheless, the combination of low toxicity, higher specificity, and diversity available with peptide therapeutics has raised interest in overcoming the problems of their challenging and costly synthesis. A fluidized bed system unlimited scalability) was constructed and used for the synthesis of peptides on the solid phase and real-time monitoring of deprotection and coupling reactions was achieved. The fluidized bed has been characterized and shows promise of providing a more efficient means of producing large quantities of peptides. A survey of reported chemical libraries shows that the functional diversity present is limited when compared to naturally occurring peptides and proteins. This is the result of the fact that including monomers with reactive functional groups either leads to impractical protecting group schemes or unwanted reactivity with reaction conditions causing poor yields. As the synthesis of large libraries does not allow for the complete analysis of each synthetic step, researchers must compromise between adding diversity and ensuring acceptable yields. Often the more conservative choice is made based upon conjecture and fear of unwanted reactivity. A new methodology has been developed to quickly screen a diverse set of functional groups representing possible monomers) against the reaction conditions that are to be used for library synthesis. The results of the screen can be applied towards the design and synthesis of more diverse small molecule libraries. Furthermore, a more efficient method of reaction optimization is introduced using an analytical construct in tandem with electrospray ionization mass spectrometry.

Unraveling the nanoscale processes of biological pathways via the testing, replication, and visualization of the underlying mechanisms remains a persistent challenge in the study of these critical life-governing systems. Recent advances in the field of chemically induced dimerization have unlocked multiple tools for the exploration of these facets of biology, including the development of switchable signaling systems, assertion of control over protein localization in the cell, and regulation of gene expression. An additional revelation through protein complexation by chemical induction is the construction of multivalent protein-based nanostructures, capable of bearing multiple targeting agents. However, stochastic assembly of these proteins has proven unsatisfactory in generating homogeneous populations. Herein, we have taken the initial steps toward developing a protein-based biomolecular language for nanostructural assembly. Through gel filtration analysis, we have characterized the ability of interfacial point mutations to modulate the stability of a bis-methotrexate bis-MTX) induced E. coli dihydrofolate reductase DHFR) dimer over a dynamic range of 1.5 kcal/mol. Furthermore, we have employed single-molecule fluorescence assays to demonstrate the stabilization of a heterodimeric DHFR dimer, yielding 4-fold selectivity for the heterodimer over either corresponding homodimer. In addition to our experimental characterization of the chemically induced DHFR dimer, we have also taken steps toward the construction of a tripartite computational model of dimerization in an effort to predict the effects of further mutations. We have tested a number of molecular mechanics force fields against quantum mechanical benchmarks and discovered that the MMFF94, OPLS2005, and AMBER force fields yield the most accurate electrostatic and configurational treatment of the complex bis-MTX dimerizer. While initial attempts at calculating the binding free energy of the macromolecular complex have been unsuccessful, we have gleaned important insights into the complexities of modeling this three-body system. The advances described within the following work delineate important aspects of protein interface remodeling in a chemically induced system and provide an avenue toward the further development of both a computational model of protein interactions and the future directed assembly of protein based materials and therapeutic nanostructures.

DNA-directed chemotherapies continue to play an important role in modern oncology. The cytotoxic complex, [PtClen)ACRAMTU)]NO 3)2 en ＝ ethane-1,2-diamine； ACRAMTU ＝ 1-[2-acridine-9-ylamino)ethyl]-1,3-dimethylthiourea) PT-ACRAMTU, 31), is a dual platinating/intercalating DNA binder that, unlike clinical platinum agents, does not induce DNA cross-links. The research in this dissertation was concerned with establishing structure–activity relationships SAR) in this novel class of DNA-targeted antitumor agents with the ultimate goal of improving the biological activity of the prototypical agent, PT-ACRAMTU. Two ways were explored of tuning the reactivity and target interactions of platinum, which was considered critical in changing the pharmacological properties of this pharmacophore. The first approach involved a structurally minimally invasive modification of the prototype to enhance its chemical stability and reactivity with DNA. This was achieved by introducing a new inert nonleaving group imino group) in place of thiourea. The first attempt involved the synthesis of a guanidine analogue of ACRAMTU, 1-[2-acridine-9-ylamino)ethyl]-1,3-dimethylguanidine 38), by adding N-acridin-9-yl-N-methylethane-1,2-diamine 36) to a Boc-activated carbodiimide Me-N＝C＝N-Boc), obtained by desulfuration of N-methylthiourea 44) with HgCl2. While the study was able to delineate unusual pathways to novel cyclic and spirocyclic acridine derivatives, the ultimate goal of generating a guanidine analogue of PT-ACRAMTU was not reached due to the chelating properties of the guanidinato group verified by 195Pt NMR). Instead, platinum-mediated amidination chemistry afforded two amidine derivatives of PT-ACRAMTU 54 and 55) with greatly enhanced activity IC50 values of 26 nM and 28 nM, respectively) in H460 non-small-cell lung cancer NSCLC). The amidination reaction involved addition of the secondary amine in 36 across the activated CN bond of platinum-bound propionitrile EtCN). Complex 54 proved to be a more efficient DNA binder t 1/2 ＝ 65 min) than PT-ACRAMTU 31) t1/2 ＝ 234 min), and showed considerably reduced reactivity with N-acetylcysteine compared to PT-ACRAMTU, based on a mechanistic study using time-dependent 1H NMR and 2-D HMQC NMR spectroscopy, as well as in-line liquid chromatography–electro-spray mass spectrometry LC–ESMS). A cellular imaging study using confocal fluorescence microscopy was performed to study the subcellular distribution of complex 54. The results show higher cellular levels of 54 than of PT-ACRAMTU and suggest that the drug reaches the nucleus, although a significant amount of compound seems to be trapped in the lysosomes. A H460 mouse xenograft study showed complex 55 slows tumor growth by 40% when administered at a dose of 0.5 mg/kg, but the compound was quite toxic and caused weight loss in the animals. The concentrations of platinum detected using inductively-coupled plasma electrospray mass spectrometry ICP–MS) in various biological tissues recovered from the euthanized mice reveal that, while compound 55 reaches the tumors, most of the detected platinum accumulated in the kidneys, the major organ of excretion. The high level of platinum in the kidneys most likely contributes to the toxicity observed in the treated animals. Complex 55 is the first non-cross-linking platinum agent endowed with promising activity in NSCLC. In the second approach, novel thiourea- and guanidine-modified acridine-4-carboxamides and a corresponding platinum–intercalator conjugate have been synthesized and evaluated as cytotoxic agents. The point of attachment of the platinum-modified linker was changed from the 9-position to the 4-position of acridine to alter the DNA binding of the conjugate and its cytotoxicity. The IC50 value of 2.4 muM determined in H460 lung cancer cells for [PtClen) N-2-1,3-dimethylthioureido)ethyl)acridine-4-carboxamide)]NO 3)2 58) indicates that this strategy has no advantage over the 9-aminoacridine-based prototype.

Many of the drugs used in cancer chemotherapy target DNA to kill malignant cells. Some of them form DNA interstrand crosslinks ICLs), which are extremely cytotoxic lesions that block essential metabolic process such as replication, transcription and recombination by forming covalent bonds between opposite strands of DNA. Despite the importance of chemotherapeutic agents that rely on ICLs for their efficacy, the mechanisms by which these lesions are repaired remains poorly understood. A major impediment in studying ICLs repair has been the limited availability of well-defined substrates. This dissertation describes the development of a new strategy for the synthesis of defined site-specific ICLs in high yields and purity. This strategy relies on the incorporation of ICL precursors bearing reactive aldehyde functionalities on complementary strands of DNA, followed by ICL formation via double reductive amination. We were able to synthesize different crosslinks that are isosteric to the therapeutic nitrogen mustard NM) ICLs, introducing substitution of a few atoms to make them more stable and therefore more suitable for chemical, structural and biological studies. The synthetic substrates were validated through molecular dynamic studies, confirming that our mimic has all the essential structural features to its natural counterpart. Modeling data also demonstrate that both the natural and the synthetic ICL induce a bend in the DNA, which could play an important role in the way the lesion is repaired. Our synthetic approach furthermore allows for the synthesis of major groove ICLs with different degrees of distortion, providing unique and valuable tools for biochemical and cell biological studies of ICL repair.

Hydrolytic enzymes constitute one of the largest and most diverse protein classes in Nature and play key roles in nearly all physiological and pathological processes. The mammalian serine hydrolase superfamily contains a remarkable number of uncharacterized members, with at least 40-50% of these enzymes lacking experimentally verified endogenous substrates and products. Assignment of metabolic and cellular functions to these enzymes requires the development of pharmacological tools to selectively perturb their activity. We describe herein a functional proteomic strategy to systematically develop potent and selective inhibitors for uncharacterized serine hydrolases, and its application to create a highly potent and selective inhibitor of the brain enriched enzyme alpha/beta-hydrolase-6, monoacylglycerol lipase and alpha/beta-hydrolase-11. We also comprehensively profiled serine hydrolase activities in adipocyte cell lines, identified WWL113 as a selective and potent inhibitor for carboxylesterase3 in 10T1/2 cell line and demonstrated its potential application in metabolic syndrome. We anticipate that the methods described herein will facilitate the development of selective chemical probes to annotate the metabolic and (patho)physiological functions of many of the uncharacterized serine hydrolases that currently populate eukaryotic and prokaryotic proteomes.

Uric acid, a component in human kidney stones, crystallizes in an anhydrous (UA), a metastable dihydrate (UAD), a rare monohydrate, and an ionized monosodium urate form in vivo. In the body, these crystals exhibit a variety of morphologies and colors which differ from laboratory grown uric acids. These observations provided the motivation to study the effect of several molecular dyes and physiologically relevant metal cations on the crystallization of UA and UAD. All cationic and neutral dopants investigated were included in UA and UAD crystals, while anionic dyes were excluded. At low concentrations, the dyes were preferentially incorporated into the {001} and {201} growth sectors of UA. In UAD, variable inclusion behaviors were encountered. Inclusions occurred on the {011} growth sector/hillock, on the {102} growth sector, and non-specific inclusions were also observed. Most of the dyes induced morphological changes in UA and UAD crystals at higher concentrations. The amount of dye/metal cation included and absorption spectra of the dyes in the single crystals of both UA and UAD were determined.

For years, the pharmaceutical industry has relied heavily on crystalline active pharmaceutical ingredients APIs) that can be approved by the Federal Drug Administration FDA) as neutral compounds, salts, or solvates of said neutral compounds and salts. Yet, the solid crystalline form can have unexpected and unfavorable effects on properties such as solubility, bioavailability, efficacy, etc., due to different polymorphic forms of the API. A drug can be present in multiple forms and interconvert between forms during isolation, manufacturing, storage, and transport of the end product. These unwelcome problems could be alleviated or even eliminated by the formation of a liquid drug, which possesses no crystal structure. Unfortunately, research in this area has been limited to solubilization of solid drugs into various drug delivery vehicles such as emulsions, suspensions, and liposomes. However, it is possible for a drug to crystallize from these vehicles during the manufacturing, storage, and transportation. Thus, a new method to liquefy pharmaceuticals, thereby reducing problems associated with the solid-state, is needed. A potential solution is the use of ionic liquids IL), defined as salts that melt below 100&deg；C. Since ILs are salts it is possible to combine a pharmaceutical ion with any desired counter ion, thereby, providing a level of tunablity that is not possible with current techniques. This IL modular strategy was the basis for the research discussed here, in which APIs with known problems were combined with GRAS generally regarded as safe) compounds or FDA-approved APIs, which resulted in ILs displaying dual biological functionality. This strategy was successful in producing a wide range of ILs, all containing at least one pharmaceutically active ion. The physical property set for these synthesized ILs was varied, as it is difficult to predict how two ionic organic compounds will interact. However, common trends regarding melting point depression, thermal stability, and solubility were determined. The most exciting results were exhibited during the biological testing, as several of the synthesized ILs demonstrated improved biological activity over the precursor ions. Additionally, the drug mechanism, at a cellular level, was found to be modified when contained within an IL. This indicates that ILs behavior differently in the body than simple halide containing salts. Overall, the obtained results signify that ILs can serve as pharmaceuticals, in which these liquid salts eliminate problems associated with the solid-state and displayed to synergistic physical and biological properties.