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Abstract The equilibrium of aqueous D-glucaric acid was investigated via Nuclear Magnetic Resonance (NMR) spectroscopy. The NMR spectra of all four species (D-glucaric acid, D-glucaro-1,4-lactone, D-glucaro-6,3- lactone and D-glucaro-1,4;6,3-lactone) were assigned. A 1H NMR spectroscopy method was developed to investigate the kinetics of equilibration of the starting species (D-glucaro-1,4-lactone and D-glucaro-1,4;6,3-dilactone). The equilibration was investigated under neutral conditions as well as conditions with increasing acidity. Each experiment set contained 50-100 1HNMR spectroscopy experiments that were run on the same sample using a program that built in delays. Dimethyl sulfoxide was used as an internal standard, and its signal size was used as a scale to report the changes in relative concentration of the four species throughout the experiment sets. Under neutral conditions D-glucaro-1,4-lactone is relatively stable against equilibration, while D-glucaro-1,4;6,3-dilactone is not. Under acidic conditions both compounds equilibrate within approximately 30,000 seconds. After equilibration under acidic conditions D-glucaric acid is the dominant species, while the relative concentration of D-glucaro-1,4-lactone is slightly higher than that of D-glucaro-6,3-lactone. The relative equilibrium concentration of D-glucaro-1,4;6,3-dilactone is low. A mechanism for the equilibration of aqueous D-glucaric acid was proposed and equilibrium constants and estimates of rate constants were derived from the experimental data. These rate constants were used in MATLAB simulations that were compared to the experimental data. MATLAB simulations were used to alter the rate constants to improve the fits between experimental data and simulated data.

The ecological factors that maintain defensive chemical variation within and between plant species have intrigued ecologists for decades. While theory posits that polymorphisms may be maintained different forms of balancing selection, relatively few experimental studies have tested whether such balancing selection can maintain defensive chemical trait polymorphisms in nature. Further, evidence demonstrating balancing selection is rare for any trait. Here, I investigated a stereochemical trait polymorphism in Xanthium strumarium. This species is polymorphic with respect to the stereochemistry of the lactone ring junction of a prominent defensive compound class ? the sesquiterpene lactones. Individual plants typically produce only cis-fused or trans-fused lactones across their entire suite of compounds. Sesquiterpene lactones are known to influence feeding behavior and growth rates of various herbivores, but nothing is known about the ecological implications of variation in this stereochemical trait. I first examined whether sesquiterpene lactone stereochemical variation can influence folivore feeding behavior in the laboratory. Using pure sesquiterpene lactones in controlled feeding experiments, I found that laboratory-reared grasshoppers were less deterred by the cis-fused compounds than the trans-fused compounds. I then found that these patterns extended to the field: in common gardens, plants producing cis-fused lactones received more damage than plants producing trans-fused lactones. Additionally, folivore damage was negatively correlated with plant fitness. Taken together, these results indicate that herbivores can impose natural selection on this stereochemical trait polymorphism in nature. Finally, I found evidence that spatially variable selection leads to fitness patterns conducive to the maintenance of this polymorphism. Further, I found that the intensity of folivore damage across spatial scales predicted in which environments each morph outperformed the other, with plants producing cis-fused lactones achieving higher fitness than plants producing trans-fused lactones when herbivore pressure was low (and the reverse being true when herbivore pressure was high). This work demonstrates that relatively minor defensive chemical variation can have far-reaching impacts on the ecology and evolution of plant populations.

Contamination of seafood by marine toxins has been a consistent public health problem. Gymnodimine (GYM) is a member of a family of spirocyclic imine containing marine natural products which was shown to be highly toxic (LD50 96 mg/kg, intraperitoneal injection); thus ensuring public safety requires stringent monitoring of gymnodimine. Current detection methods for GYM and spirolides include the mouse bioassay and LC-MS-based detection techniques which, however, have significant limitations. Therefore, more efficient and convenient detection methods are required. Building on our recently completed total synthesis of (-)-gymnodimine, the synthesis of two haptens were targeted for eventual production of monoclonal antibodies (mAb) to be used in an eventual Enzyme-Linked Immunosorbent Assay (ELISA) for gymnodimine. As an extension of the intramolecular nucleophilic catalyzed aldol lactonization (NCAL) process from aldehyde acid to keto acid substrates, carbocyclic and nitrogen heterocyclic B-lactones were synthesized. Demonstration of the utility of the NCAL process for keto acids was applied to the synthesis of dihydroplakevulin A and the core of tussilagine. In addition, although initial attempts to develop guanidine catalysts for the asymmetric NCAL process were unsuccessful, homobenzotetramisole (HBTM) was found to be a suitable asymmetric catalyst for keto acid substrates. Finally, synthetic studies toward the proposed structure of thiolyngbyan are described. Thiolyngbyan was isolated from a blue-green algae and it exhibited antifungal activity.

Four new diterpenoids, cespitulactones A-B (1-2), C4B1 (7) and E3A (8), were isolated from the soft coral Cespitularia taeniata collected in Taiwan, at a depth of 25 m, together with five known compounds, cespitularin F (3), 6-O-acetylcespitularin F (4), cespitularin D (5), flaccidoxide-13-acetate (6) and florlide A (9). Compound 1 possesses a novel structure with a bond cleavage between C-10 and C-11, and having a 14-membered lactone ring junction between C-10 and C-12. Compounds 2 and 8 have the verticillane skeleton, and compound 9 possesses the cespitularane skeleton. Their structures were elucidated on the basis of extensive spectroscopic analysis and chemical derivatization. The isolated compounds were also evaluated for cytotoxicity toward human cancer cell lines. Compound 1 exhibited significant cytotoxicity against human cervical epitheloid carcinoma (HeLa) and colon adenocarcinoma (DLD-1) cancer cells with IC50 of 3.69 and 9.98 £gg/mL, respectively. Flaccidoxide-13-acetate (6) showed mild activity against human medulloblastoma (Daoy) and colon (WiDr) cancer cells at 16.9 and 13.8 £gg/mL, respectively.

Stuttgart, Univ., Diss., 2008.

Techn. Universiẗat, Diss., 2002--Braunschweig.

Paraoxonase 1 (PON1) is an enzyme synthesised in the liver that is associated with High Density Lipoprotein (HDL) and increasingly with a number of human diseases, including cardiovascular disease and cancer. PON1 is (i) a lactonase, hydrolysing aliphatic and aromatic lactones, (ii) a phosphotriesterase, acting on certain organophosphates and (iii) a peroxidase that combats lipid oxidation. The aim of this study was to investigate the extent to which PON1 might impact on the levels of lactone-induced and oxidative DNA damage. Initially, the plasmid DNA (pDNA) nicking assay was used to show that of the 12 lactones and homocysteine (HC) examined, only alpha angelica lactone (AAL), Furanone (FUR) and HC caused DNA single strand breaks (SSB) under physiological conditions. Further studies indicated that AP sites were not involved suggesting that DNA-phosphotriesters were responsible. AAL-reacted pDNA bound the damage sensing protein, Atl1, and AAL, HC and HC thiolactone (HCTL) -reacted calf thymus DNA inhibited the activity of the DNA repair protein, MGMT, both indicating the presence of O6-alkylguanines in DNA, although this could not be confirmed using MALDI-ToF MS analyses of tryptic digests of MGMT incubated with lactone-reacted DNA. The inhibition of rPON1 by these lactones and HC was determined using paraoxon as a substrate and two groups were identified comprising lactones that caused reductions in PON1 activity of (i) ˃15% (e.g. HCTL, and AAL) and (ii) ˂10% (e.g. FUR, and HC). The pDNA nicking assay then showed that only AAL and FUR induced DNA single strand breaks. PON1 itself nicked pDNA, and bound to group 1 lactone-reacted pDNA by an unknown mechanism, both effects not previously reported, but, with the possible exception of AAL, did not increase the extent of plasmid nicking. The MTT, cell viability assay, indicated that all of the lactones with the exception of γ-BL (IC50 >12 mM) were to some degree toxic in HepG2 cells with AAL being the most toxic (IC50 1.0 ±0.03 mM). It was not possible to quantify PON1 activity in HepG2 cells and agents that are reported to change the expression levels of PON1 had no detectable impact on the toxicity of AAL, γ-VL, FUR or MBL, so any possible effect of PON1 could not be determined. The neutral Comet assay showed that AAL and HCTL generated the highest levels of DNA double strand breaks and DNA fragmentation in HepG2 cells, and that this effect was greatly enhanced for most of the lactones by the addition of rPON1. The impact of PON1 on oxidative stress was investigated using serum samples collected as part of a previous case-control study of lung cancer. Initially, an in-house developed ELISA assay to quantify 8-hydroxydeoxyguanosine (8-OHdG) levels had insufficient sensitivity and poor reproducibility. Hence 8OHdG levels were measured using kits along with PON1 levels and other serum parameters including HDL-C, LDL-C, TG and apoAI in serum from 112 patients with lung cancer and 249 patients without. No correlation was found between serum level of PON1 activity and level of 8-OHdG in patients with lung cancer, however a negative non-significant correlation was found between PON1 and 8-OHdG in control. The level of 8-OHdG was significantly higher in lung cancer patients than in controls and in the controls, the OGG1 wild type genotype correlated with reduced levels of 8-OHdG in serum. These studies showed that certain lactones, are toxic and DNA damaging and this can be increased by PON1, suggesting that any association between PON1 and human disease will be substrate dependent and may be PON1 genotype-dependent.

Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Although five mAChR subtypes (M1-M5) share a high degree of homology, they display different physiological effects including controlling smooth muscle tone to neurotransmitter release in the CNS. Hence these receptor subtypes have been investigated as potential therapeutic targets for agents capable of treating Alzheimer's Disease, Parkinson's Disease, peptic ulcer disease, COPD, urinary incontinence, and muscle spasms. Our interest in the development of subtype selective muscarinic ligands led to previous reports detailing the identification of substituted lactones as lead muscarinic compounds. Later work involved molecular modifications of those leads that included the addition of aromatic groups with a variety of substitution patterns. These efforts led to an increase in receptor affinity and produced a lactone-based muscarinic ligand with an IC50 of 340nM. As a continuation of that work, additional novel ligands were designed based on the general pharmacophoric elements proposed for the lactone-based ligands. In that model, the lactone oxygens serve as H-bond acceptor moieties while different nitrogen containing heterocycles provide the requisite cationic group. These groups may be separated by linker groups of varying sizes. In order to synthesize the lactone-based ligands mentioned above, efficient synthetic routes are required for key precursors. These include but are not limited to: 1. A novel high yield synthesis of the hydroxyethyl-lactone precursor was designed using a carefully controlled Prins reaction. The method readily quenches a cationic intermediate and simultaneously protects hydroxyl groups in a single step. A mechanism for the new route to the precursor is proposed and its use in the preparation of the target compounds is presented 2. Microwave-assisted synthesis of various sterically hindered N-aryl piperazines has been developed allowing quick access to structurally diverse muscarinic ligands These synthesis along with other newly developed routes enabled ready access to 59 novel muscarinic ligands. The ligands were tested in a general muscarinic binding assay. The result was analyzed and SAR study was performed to direct ligand design. As a result of this work, ligand affinity was improved by over 100 folds compare to the lead molecules. Several promising compounds were selected and selectivity tested. / Pharmaceutical Sciences

Orientador: Ronaldo Aloise Pilli / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-18T11:54:19Z (GMT). No. of bitstreams: 1 Drekener_RobertaLopes_D.pdf: 4694535 bytes, checksum: 998a5a4160bc1a4929a466c8dcfe2462 (MD5) Previous issue date: 2011 / Resumo: Neste trabalho descrevemos os avanços sintéticos para obtenção das Criptomoscatonas D1 (41) e D2 (42), isoladas da Cryptocarya mandioccana, para as quais não se encontram sínteses descritas na literatura e suas estruturas tridimensionais ainda não foram elucidadas. As estratégias de síntese propostas para os isômeros 41 e 42 foram baseadas em utilizar como etapa chave a reação aldólica com indução remota 1,5-anti. A primeira proposta envolveu a síntese da metil cetona (R)-153 em 8 etapas e baixo rendimento global (4,2%). Uma nova proposta sintética foi formulada e resultou na síntese da metil cetona (+/-)-157 em 3 etapas e 54% de rendimento. Esta abordagem possibilitou a construção do esqueleto carbônico de 41 e 42, de maneira diastereosseletiva. A etapa aldol utilizando a metil cetona (+/-)-157 foi realizada com sucesso, levando a formação de um aduto diastereoisomérico preferencial (+/-)-162. Já o centro estereogênico em C2' foi formado via redução 1,3-sin empregando-se metodologia de Narasaka para a obtenção de (+/-)-165a e 1,3-anti com a utilização da metodologia de Evans para obtenção de (+/-)-165b. Após manipulações de grupos protetores e de grupos funcionais, os ésteres a,b-insaturados (+/-)-169a e b foram obtidos com a ligação dupla com geometria Z através de metodologia de Horner-Wadsworth-Emmons, com modificação de Still-Gennari. A etapa de remoção do grupo acetonídeo levando aos ésteres a,b-insaturados (+/-)-170a e b foi realizada em meio ácido brando, sendo a ciclização do anel lactônico destes produtos realizada na presença de óxido de dibutilestanho, com excelentes rendimentos em ambas as etapas. Esta abordagem permitiu a formação do composto (+/-)-171a em 6,7% de rendimento e do composto (+/-)-171b em 7,6% de rendimento, a partir do trans-cinamaldeído. A etapa de remoção do grupo protetor p-metoxibenzila, não foi alcançada utilizando-se DDQ ou ZrCl4. Desta forma, embora as sínteses dos compostos 41 e 42 não tenham sido finalizadas e, portanto, a estereoquímica dos produtos naturais da família das Criptomoscatonas D não tenha sido esclarecida, este trabalho permitiu mapear a síntese racêmica destes dois compostos de uma maneira eficiente, contribuindo para que estudos futuros possam definir as estruturas desses produtos naturais / Abstract: Natural compounds of the 5,6-dihydropyranone family, isolated from the genus Cryptocarya (Laureacae), have attracted scientific interest due to their biological activities. Among these compounds, we highlight Cryptomoscatone D1 (41) and D2 (42), isolated from C. mandiocanna, for which a definitive proof of structure is still lacking. In this work, we describe our synthetic efforts toward these compounds. The synthetic strategies proposed for the syntheses of isomers 41 and 42 were based on a key aldol reaction with 1,5-anti remote induction. The first approach involved the synthesis of methyl ketone (R)-153 in 8 steps, however in low overall yield (4.2%). The second synthetic approach led to the synthesis of methyl ketone (+/-)-157, in three steps and 54% overall yield. The aldol step involving methyl ketone (+/-)-157 successfully led to the formation of the diastereoisomer (+/-)-162. The stereogenic center at C2'in (+/-)-165a was established via Narasaka's 1,3-syn reduction while Evans 1,3-anti reduction afforded (+/-)-165b. After manipulation of the protecting and functional groups, a,b-unsaturated esters (+/-)-169a and b were obtained with the desired Z double bond via the Still-Gennari modification of the Horner-Wadsworth-Emmons olefination reaction. Cleavage of the acetonide leading to the a, b-unsaturated d-hydroxyesters (+/-)-170a and b was achieved under mild acidic conditions, and cyclization was performed in the presence of dibutyltin oxide, in excellent yields for both steps. This approach allowed the formation of compound (+/-)-171a in 6.7% yield and compound (+/-)-171b in 7.6% yield, from trans-cinnamaldehyde. Removal of p-methoxybenzyl ether failed using DDQ or ZrCl4 methodologies. Although our studies did not elucidate the structures of Cryptomoscatone D1 (41) and D2 (42), they are a valuable contribution for future efforts aimed to unambiguously establish the structure of these natural products / Doutorado / Quimica Organica / Doutor em Ciências