Endoproteinase Glu-C (EPGIu-C, EC 3.4.21.19), an enzyme isolated from the bacteria Staphylococcus aureus, has been found to cleave specifically at the carboxyl-terminal side of glutamyl and aspartyl peptide bonds. EPGIu-C has been reported to be stable and active in the presence of common denaturants such as guanidinium chloride, urea and sodium dodecyl sulfate (Drapeau, G.R. (1977) Methods in Enzymology, 47:189-191). In order assess the denaturant stability and activity of EPGIu-C, the effect of three common protein denaturants, guanidinium chloride, urea, and sodium dodecyl sulfate (SDS) on the proteolytic activity of EPGIu-C was studied.The kinetics of the hydrolyis reaction catalyzed by EPGIu-C was determined using the chromophoric substrate N-tBOC-L-glutamic acid-a-phenyl ester (BGPE).To compare theurea is significantly greater at the higher concentrations of urea. These results suggest that a complete cleavage of proteins substrate by EPGlu-C might occur more rapidly in 8.0 M urea than in 6.0 M GuCl, since EPGlu-C will be operating at a significantly higher catalytic efficiency in the urea solution. / Department of Chemistry

Yes / Current drugs against human African trypanosomiasis (HAT) suffer from several serious drawbacks. The search for novel, effective, brain permeable, safe, and inexpensive antitrypanosomal compounds is therefore an urgent need. We have recently reported that the 4-aminoquinoline derivative huprine Y, developed in our group as an anticholinesterasic agent, exhibits a submicromolar potency against Trypanosoma brucei and that its homo- and hetero-dimerization can result in to up to three-fold increased potency and selectivity. As an alternative strategy towards more potent smaller molecule anti-HAT agents, we have explored the introduction of ω-cyanoalkyl, ω-aminoalkyl, or ω-guanidinoalkyl chains at the primary amino group of huprine or the simplified 4-aminoquinoline analogue tacrine. Here, we describe the evaluation of a small in-house library and a second generation of newly synthesized derivatives, which has led to the identification of 13 side chain modified 4-aminoquinoline derivatives with submicromolar potencies against T. brucei. Among these compounds, the guanidinononyltacrine analogue 15e exhibits a 5-fold increased antitrypanosomal potency, 10-fold increased selectivity, and 100-fold decreased anticholinesterasic activity relative to the parent huprine Y. Its biological profile, lower molecular weight relative to dimeric compounds, reduced lipophilicity, and ease of synthesis, make it an interesting anti-HAT lead, amenable to further optimization to eliminate its remaining anticholinesterasic activity. / Wellcome Trust

The design of new, improved NO-donor drugs is an important pharmacological objective due to the biological importance of nitric oxide. N-Hydroxyguanidines represent a useful class of NO donors where the mechanism of action is based on the biosynthetic pathway for NO. Thirty new N-arylalkyl-N’-hydroxyguanidines were synthesized and their vasodilatation activity examined by myography in rat aortic rings. The observed relaxations were reversed by ODQ, which is an inhibitor of the guanylate cyclase, implying that this was an NO dependent vasodilatation. The most active compounds were also tested in the isolated perfused kidney (IPK) giving the vasodilatation properties. Preliminary results indicated that N-phenyl-N’- hydroxyguanidine showed the best pharmacological profile with EC₅₀= 19.9 μM and ca. 100% reversibility with ODQ. A series of N-phenylalkyl-N’-hydroxyguanidines were synthesised. NO donor activity was found to be fairly constant up to three methylene groups, and then decreased. Substitutions in the benzene ring of N-phenylethyl-N’-hydroxyguanidine demonstrated that various electron-withdrawing and electron-donating groups in the para position did not significantly affect the NO donor activity of this series of analogues. The nitro and trifluoromethyl substituted compounds gave the best biological profiles. Additionally, a novel heterocyclic, N–furfuryl-N’–hydroxyguanidine possessed very promising vasodilatation properties. In general, almost all the N-arylalkyl-N’-hydroxyguanidines behaved as potent NO donors in the rat aorta assay. In order to establish the influence of the free NH₂ group in the hydroxyguanidine functionality on the vasodilatation properties, N,N-dimethyl and N-methyl-N’- hydroxyguanidines were successfully synthesised. Unfortunately, they have not been tested yet in the biological assay. However, their NMR spectra showed some unusual features and their detailed analysis and X-ray data are presented herein. In addition a series of hydroxamic acids was synthesised and the NO donor activity investigated using the same biological methodology. It was found that the 3-phenylpropionohydroxamic acid was the most potent compound with EC₅₀ = 6 μM and ODQ = 96%. However, behavior in the IPK indicated that hydroxamic acids did not undergo the same biological pathway as in the rat aorta. Two different types of enzyme-activated pro-drugs were designed using N-hydroxyguanidines as the NO donating molecule. Synthetic studies towards these targets were carried out using various synthetic approaches. The desired molecules have not yet been synthesised but the chemistry explored so far has indicated potentially more successful approaches that could be attempted.

Resorcinarenes can be functionalized at their upper and lower rims. In this work, the upper rim of a resorcinarene was functionalized with glutamic acids and the lower rim was functionalized with either methyl or undecyl alkyl groups. The cavitands were characterized by nuclear magnetic resonance (NMR), mass spectrometry (MS), UV-vis spectroscopy, dynamic light scattering (DLS) and electron microscopy. The binding of resorcinarene with amine guests was studied in DMSO by UV-vis titration. The obtained binding constants (K values) were in the range of 12,000-136000 M-1. The resorcinarenes were shown to form aggregates in a variety of solvents. The aggregates were spherical as confirmed by DLS, SEM and TEM experiments. Dynamic light scattering (DLS) experiments revealed the size of the aggregates could be controlled by cavitand concentration, pH, and temperature. The resorcinarene with undecyl alkyl group were adsorbed onto 55% cross-linked styrene-divinylbenzene resin to prepare a new stationary phases for ion chromatography (IC) columns. The new column packing material was applied in determination of uremic toxins and water contaminants. The new IC column afforded separation of the five uremic toxins : guanidinoacetic acid, guanidine, methylguanidine, creatinine, and guanidinobenzoic acid in 30 minutes. Detection and quantification of uremic toxins helps diagnose kidney problems and start patient care. Gradient elutions at ambient temperature with methanesulfonic acid (MSA) as eluent resulted in detection levels in water from 10 to 47 ppb and in synthetic urine from 28 to 180 ppb. Trace levels of creatinine (1 ppt) were detected in the urine of a healthy individual using the columns. The new IC stationary phase separated cationic pharmaceuticals including a group of guanidine compounds in surface water. Detection limits in the range of 5 - 32 µg L-1 were achieved using integrated pulsed amperometric detection (IPAD) for guanidine (G), methylguanidine (MG), 1,1-dimethylbiguanidine (DMG), agmatine (AGM), guanidinobenzoic acid (GBA) and cimetidine (CIM). Suppressed conductivity (CD) and UV-vis detection resulted in limits of detection similar to IPAD, in the range of 1.7 - 66 µg L-1, but were not able to detect all of the analytes. Three water sources, river, lake, and marsh, were analyzed and despite matrix effects, sensitivity for guanidine compounds was in the 100 µg L-1 range and apparent recoveries were 80-96 %. The peak area precision was 0.01 - 2.89% for IPAD, CD and UV-vis detection.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 134-148).

Diss. (sammanfattning) Linköping : Univ., 2002. / Härtill 4 uppsatser.

Cartilage Tissue Engineering. Joint pain and functional impairment due to cartilage damage from osteoarthritis and other means is a major source of disability for adults the world over. Cartilage is an avascular tissue with a very limited capacity for self repair. Current medical and surgical approaches to cartilage repair also have limited efficacy, and in all cases fail to completely restore a normal, healthy cartilage phenotype. Tissue engineering is a relatively new approach to cartilage repair that seeks to fabricate a replacement tissue, indistinguishable from healthy, native tissue. The basic idea of the tissue engineering approach is to seed tissue synthesizing cells into a shapeable, biocompatible/bioabsorbable scaffold that serves as a temporary extracellular matrix with a localized source of bioactive molecules to direct the development of new tissue. The challenge of tissue engineering is to identify cells, scaffolds, and growth conditions that will be optimal for tissue regeneration. The goal of the current studies was to evaluate one aspect of all three of the major components of cartilage tissue engineering: cell source, scaffolding material and preparation, and controlled growth factor delivery. We evaluated the chondrogenic potential of human nasal chondrocytes grown in calcium alginate in an in vivo culture system, the potential of computer-aided design and injection molding with calcium alginate to reliably reproduce complex geometries with high dimensional tolerances, and the potential for the controlled release of TGF-β1 from calcium alginate modified by the covalent addition of a recently discovered TGF-β binding peptide. We found that adult human nasal chondrocytes show significant chondrogenic potential when grown within an alginate scaffold. We also found that alginate is readily amenable to an injection molding process that utilizes precision made molds from computer-aided design and solid free form fabrication, allowing for the fabrication of tissue engineered constructs with very precise shape fidelity. Additionally, we found that calcium alginate could be reliably modified by the covalent addition of peptides, and that the addition of a newly discovered TGF-β binding peptide delayed the release of pre-loaded TGF-β1. Together these results show some of the encouraging prospects for cartilage tissue engineering. `Menière’s Syndrome.Menière’s syndrome is an inner ear disorder characterized by idiopathic endolymphatic hydrops with associated periodic tinnitus, vertigo, and progressive sensorineural hearing loss. It affects approximately 0.2% of the population, for whom it can be quite devastating. In addition to progressive hearing loss people with Menière’s syndrome are prone to sudden attacks of vertigo and tinnitus that are severe enough that they can lead to falls and potentially serious injury. People subject to frequent attacks are unable to drive, with obvious consequences on standard of living. In the current studies we evaluated the standard animal model of Menière’s syndrome by comparing cochlear turn specific hearing thresholds and the degree of hydrops in that turn. A positive correlation between these had previously been established in the study of human temporal bones from people with Menière’s syndrome, but had not been reported in the animal model. We also evaluated the potential of aminoguanidine, a relatively specific inhibitor of the inducible isoform of nitric oxide synthase, as a neuroprotective therapeutic agent for preservation of hearing in animals with surgically induced endolymphatic hydrops. We found, for the first time, a partial correlation between cochlear turn specific hydrops and hearing thresholds in the most commonly used animal model of Menière’s syndrome, helping to validate the utility of this animal model for future studies. We also found that aminoguanidine did indeed partially preserve hearing in animals with surgically induced Menière’s syndrome. This encouraging result appears to be the first report of a medical intervention protective against hearing loss in an animal model of Menière’s syndrome, and may help us to understand the etiology pathology seen in Menière’s syndrome.

Nitroguanidine (NQ) is a nitramine used in many propellant formulations. The purpose of this study was to develop a method for treating nitroguanidine process wastewaters. The major wastewater contaminants were found to be nitroguanidine and guanidine nitrate (GN) (an intermediate product). Pilot tests were conducted for the removal of nitroguanidine using granulated carbon adsorption (GAC) and ion exchange for the removal of GN. The nitroguanidine concentration in demonstration plant wastewater was reduced from 15.5 mg/L to less than 1 mg/L. The carbon capacity was found to be 0.045 g NQ/g carbon. Strong acid resin was found to reduce guanidinium ion Gu⁺ concentration from 14.5 mg/L to 1 mg/L with a resin capacity of 0.13 eq Gu⁺ /L resin. No significant reduction in resin capacity for Gu⁺ was experienced after five regenerant cycles. A unique two-step regeneration was used. This included a 5% calcium nitrate solution to produce a spent regenerant that could be recycled to the production process. This was followed by regeneration with a 15% sodium chloride solution to place the resin in the sodium form. Anion exchange was used for the removal of nitrate ions. / Master of Science

Cette thèse est constituée de trois parties : 1) Le contexte bibliographique, 2) le développement de réactions domino cupro-catalysées et 3) une approche vers la synthèse totale des raputindoles.La première partie introduit d’abord le concept de réactions domino ainsi que leurs applications, puis les réactions catalysées par du cuivre permettant de former des liaisons C-N sont passées en revue en incluant les couplages de Ullmann, Goldberg et de Chan-Lam, les séquences d’activation oxydante de liaisons C-H/formation de liaison C-N, l’insertion de nitrènes et l’hydroamination de liaisons C-C multiples. En se basant sur ces réactions élémentaires permettant de former une liaison C-N unique, les développements récents de réactions domino sont ensuite détaillés.La deuxième partie peut être subdivisée en 3 sections : 1) la synthèse de benzimidazoles, 2) la synthèse d’imidazoles and 3) la synthèse de guanidines. Un rappel des méthodes existantes pour la synthèse de ces motifs est proposé dans chaque section. Notre travail, basé sur la formation de liaisons C-N multiples selon une séquence cupro-catalysée domino, est ensuite détaillé. Celui-ci nous a permis d’aboutir au développement de voies d’accès aux benzimidazoles, en utilisant une réaction séquentielle catalysée par du cuivre en présence d’oxygène à partir d’acides boroniques et d’amidines, à la synthèse d’imidazoles par une réaction de di-amination d’alcynes vrai par des amidines et à l’obtention de guanidines et de 2-aminobenzimidines par une réaction à 3 composant. Ces réactions domino montrent une bonne efficacité et permettent d’assembler des hétérocycles à partir de précurseurs aisément accessibles.La dernière partie est consacrée à la synthèse des raputindoles. La structure, les activités et les réactions clé pour la construction de ces alcaloïdes sont discuté d’abord, nous amenant à proposer une rétrosynthèse pour accéder à ces molécules. Les réactions qui ont retenues notre attention pour construire ces molécules sont une annelation [3+2] irido-catalysée d’acides o-formylarylboronique et de 1,3-diènes, la synthèse de Leimgruber-Batcho pour obtenir des indoles et une séquence d’alkylboration-protodéboration. A partir de cela 3 stratégies ont été évaluées, montrant que l’accès à ce type de composé naturel est envisageable en combinant ces étapes. / This thesis consists in three parts: bibliographic background, copper-catalyzed reactions for synthesis of N-containing compounds, approach to the synthesis of raputindoles.The first part introduces the domino reactions and their applications, then, copper-mediated reactions for construction of C-N bond formation are reviewed including Ullmann, Goldberg and Chan-Lam coupling, oxidative C-H activation/C-N formation, insertion of nitrenes and carbenoids, and hydroamination of multi-C-C bonds. This can be used as guides to design domino reaction. Following these copper-mediated single C-N bond formation reactions, recent developments of copper-catalyzed domino reactions for synthesis of heterocycles are described.The second part can be divided into three sections: 1) synthesis of benzimidazoles, 2) synthesis of imidazoles and 3) synthesis of guanidines. Each section summarizes the existing methods used for their synthesis. Following it, our synthetic work involving copper-catalyzed C-N bond formation domino reactions is discussed in detail. Our objectives include the synthesis of benzimidazoles through copper-catalyzed sequential reaction of benzamidines and boronic acids, synthesis of imidazoles via copper-catalyzed domino reaction of benzamidines and acetylenes, and synthesis of guanidines and 2-aminobenzimidazoles by Cu-catalyzed three-component reaction of cyanamides, boronic acids and amines. These copper-catalyzed domino reactions show high efficiencies from readily available and simple starting materials.The last part is about the total synthesis of raputindoles. The structure and bioactivities of raputindoles and key reactions for the total synthesis of raputindoles are introduced first, the synthetic strategies are then proposed on basis of relative synthetic methods. The key reactions we use for the synthesis of raputindoles are iridium catalyzed [3+2] annulation of o-formylarylboronic acids and 1,3-dienes, Leimgruber-Batcho indole synthesis, transition-metal catalyzed SN2 substitution and alkylborylation-protondeborylation. According to the three strategies we proposed, lots of relative reactions were investigated. The results show that it is possible to synthesize the raputindole molecules based on the iridium catalyzed [3+2] annulation of 2-formylarylboronic acids and 1,3-dienes.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2014-08-11T14:46:08Z No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertacao River Souza Magalhaes.pdf: 1856967 bytes, checksum: 6da8a950fc3e088ddd4b5b2558c23691 (MD5) / Made available in DSpace on 2014-08-11T14:46:08Z (GMT). No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertacao River Souza Magalhaes.pdf: 1856967 bytes, checksum: 6da8a950fc3e088ddd4b5b2558c23691 (MD5) Previous issue date: 2011-02-25 / This work presents the synthesis of N-[-(4-methylphenyl)]-thiourea, the ligand N-[N'-(2-hydroxyethyll)-(4-methylphenyl)]-guanidine and the copper II complex. The compounds were characterized by infrared spectroscopy,1H NMR and X-ray diffraction. The results of X-ray shows the mode of ligand coordination to the metal ion, in addition to square planar structure, a connection is observed for the trans chromophore CuN2O2. Conformational studies of the molecule the ligand N-[N'-(2-hydroxyethyl)-(4-methylphenyl)]-guanidine and were performed to compare experimental data with calculated data. the computation of ligand molecule N-[N'-(2-hydroxyethyl)-(4-methylphenyl)]-guanidine shows the small energy difference between the threes tautomers, it is not possible to state firmly the most stable. The 1H NMR data show that the solution is more abundant in the tautomer II and III. Calculations of energy transition states for the reaction path. In addition to the calculations of imaginary for the characterization of transition states. / Este trabalho apresenta a síntese da N-[N'-(2-hidroxietil)-(4-metilfenil)]-tioureia, do ligante N-[N'-(2-hidroxietil)-(4-metilfenil)]-guanidina e do complexo de cobre II [Cu(oib)2]. Os compostos foram caracterizados por espectroscopia de infravermelho, RMN de 1H e difração de raios-X. O resultado de DRX mostra o modo de coordenação do ligante ao íon metálico, além da estrutura quadrado planar, é observado uma ligação trans para o cromóforo CuN2O2. Estudos conformacionais da molécula do ligante N-[N'-(2-hidroxietil)-(4-metilfenil)]-guanidina que foram realizados para confrontar dados experimentais com dados calculados. Os cálculos computacionais da molécula do ligante N-[N'-(2-hidroxietil)-(4-metilfenil)]-guanidina mostra uma pequena diferença de energia entre os três tautômeros, não sendo possível afirmar com firmeza o mais estável. Os dados de RMN 1H mostram que o mais abundante em solução é o tautômero II e o III. Cálculos de energia dos estados de transição para o caminho reacional. Além do cálculo das frequências imaginárias para caracterização dos estados de transição.