Ph.D.

The exciting and intriguing biological effects associated with amelanocyte stimulating hormone, α-MSH, have initiated a variety of studies to identify the biologically important conformations of the backbone and sidechains structures. To further investigate the topology of the critical "message" residues, several bicyclic (sidechain-to-sidechain) and x¹ restricted peptides have been synthesized and biologically examined, and selected analogues have been studied by NMR techniques and by theoretical molecular modeling studies. Bicyclic melanotropic peptides possessing the central sequence, Cys⁴-Asp⁵-His⁶-DPhe⁷-Arg⁸-Trp⁹-Cys¹⁰-Lys¹¹, exhibited nearly identical nanomolar biological potencies in the lizard skin bioassay. Molecular modeling studies suggested the hypothesis of incomplete receptor binding by either the His or Trp residue as being responsible for the decrease in potencies relative to a-MSH, and the parent monocyclic peptides from which these bicyclic peptides were designed. This hypothesis is further supported by twodimensional NMR studies of a representative bicyclic peptide, Ac-Cys⁴-Asp⁵-His⁶-DPhe⁷-Arg⁸-Trp⁹-Cys¹⁰-Lys¹¹-NH₂. Peptides synthesized to probe the topographical space of the tryptophan residue at position nine provided extraordinary results regarding the biological phenomena of prolongation. These peptides were based on the template, Ac-Nle⁴-Asp⁵-His⁶-DPhe⁷-Arg⁸-Xaa⁹-Lys¹⁰-NH₂, where Xaa consists of the four isomers of β-MeTrp, DTrp , and L- or D-TCA residues. Some of these peptides were selected for studies on the cloned hMC1 melanocortin receptor. These studies resulted in a partial hypothesis accounting for the prolonged biological activities observed in other physiological assay systems. Conformational analysis by solution 20 NMR techniques revealed similar peptide backbone secondary structure features with main differences of structure occurring in the sidechain x¹ space. The implications and results are discussed. Homology-based molecular modeling of the hMC1 melanocortin receptor was also undertaken and provided evidence for ligand-receptor interactions which are being tested by receptor mutagenesis studies. This three-dimensional computer model provides an opportunity to probe detail chemical ligand-receptor interactions and further study differences in biological activities and biological mechanisms.

The isomers of cyclo(Trp-Pro) (cyclo(L-Trp-L-Pro), cyclo(L-Trp-D-Pro), cyclo(D-Trp-LPro) and cyclo(D-Trp-D-Pro)) have been successfully synthesized and screened for biological activity. High percentage yields were obtained by using the three phase synthesis system, which involves the synthesis of the intermediate protected linear dipeptides, followed by the removal of the protecting Boc groups. This step is followed by cyclization and crystallization of the isomers. The diketopiperazines rings of cyclo(L-Trp-L-Pro) and cyclo(D-Trp-D-Pro) contain cisamide bonds, while cyclo(L-Trp-D-Pro) and cyclo(D-Trp-L-Pro) contain trans-amide bonds. These bonds govern the conformation of the diketopiperazines ring. The isomers have shown different degrees of biological activity, possibly as a result of the orientation of the side chain of tryptophan and this difference in conformation, leading to varying interactions between isomer and a range of receptors. Under experimental conditions, 10-3 M cyclo(L-Trp-D-Pro) and cyclo(D-Trp-L-Pro) showed effective anticancer activity against the cervical cancer cell line, HeLa, resulting in a <50% reduction in cell viability. Cytotoxicity screening with cyclo(D-Trp-L-Pro) indicated that it was hepatocyte-specific in its toxicity, whilst the other isomers were cytotoxic against the other cell types tested. At 1mg/ml, cyclo(L-Trp-L-Pro) proved to be an effective antimicrobial agent against Gram positive bacteria, while cyclo(L-Trp-DPro) effectively inhibited the growth of the Gram negative bacteria, Esherichia coli. Cyclo(D-Trp-L-Pro) proved to be effective against Streptococcus, while cyclo(D-Trp-DPro) effectively reduced viability of the yeast, Candida albicans. Cyclo(D-Trp-L-Pro) was the only isomer to show Ca2+-channel antagonism, whilst the other isomers resulted in opening of the Ca2+-channel. No effects were observed on K+-channel activity for all the isomers tested. The isomers also proved to be valuable antiarrhythmic agents by effectively reducing the time spent in ventricular tachycardia and arrhythmia, as well as decreasing the time for the heart rate to return to a normal sinus rhythm. Furthermore, cyclo(L-Trp-D-Pro) showed positive chronotropic activity, while cyclo(D-Trp-L-Pro) ii showed negative chronotropic activity. In addition, cyclo(L-Trp-D-Pro) and cyclo(D-Trp- L-Pro) also increased the coronary flow rate. 0.125 –1 mM Cyclo(L-Trp-D-Pro) decreased aggregation in washed platelets induced by thrombin. All isomers increased adhesion to an artificial surface when the platelets were stimulated by ADP, yet caused reduced adhesion when the platelets were stimulated by thrombin. These results prove the potential of these compounds as novel agents in a range of biological fields, indicating that a combination of L- and D- amino acids may prove more effective than an agent consisting solely of L-amino acids.

During the past few decades, analogs of purine nucleosides have been described that are modified either in the heterocyclic base, sugar moiety or both, and many of these modified nucleosides display antiviral and/or antineoplastic activity. The Class II mesoionic purinones are isosteric with their non~mesoionic purinone counterparts. It is conceivable that the mesoionic purinone nucleosides might constitute an entirely novel class of modified nucleosides with potential chemotherapeutic activity. That the mesoionic heterobases are bioisosteric as well as isosteric with non-mesoionic purinones was realized by demonstrating that certain mesoionic xanthine derivatives, such as Anhydro-8-ethylw-5-hydroxy-7-oxo-l,3,4-thiadiazolo[3,2-a]pyrimidinium hydroxide and Anhydro-6-p-chlorobenzyl-8-ethyl-5-hydroxy-7-oxo—l,3,4—thiadiazolo [3,2-a]pyrimidinium hydroxide were comparable in potency to test compound, theophylline as inhibitors of adenosine binding at the A1 site. Three different types of mesoionic nucleosides were subsequently designed and synthesized as potential antineoplastic agents. Mesoionic thiadiazolopyrimidine nucleosides, i.e. Anhydro-6-ethyl-8-(2’,3',5'-tri-O-acetyl-D-ribofuranosyl)MS-hydroxy-7-oxo-l,3,4~thiadiazolo{3,2-a]pyrimidinium hydroxide and Anhydro-8-(2',3'.5’-tri-O-acetyl-D-ribofuranosyl) -5-hydroxy-7-oxo-l,3,4-thiadiazolo[3,2-a]pyrimidinium hydroxide were designed to serve as potential pro-drugs of 2-amino-l,3,4~thiadiazole mononucleotide which has been reported to be a potent inhibitor of inosine monophosphate dehydrogenase. The O~acylated derivatives of the target compounds were prepared by the acid catalyzed condensation of D-ribose with 2-ATD followed by protection of the hydroxyl groups and subsequent cyclization to the mesoionic products; anomeric separation was achieved by column chromatography; All attempts to deprotect the hydroxyl groups of the mesoionic nucleosides resulted in hydrolytic ring-opening of the mesoionic heterobase. The 0-acetyl derivatives of the mesoionic thiadiazolopyrimidine nucleosides were evaluated for antineoplastic activity but were found to be inactive. The mesoionic thia-zolinopyrimidine nucleoside, i.e. Anhydro-6-ethyl-8-(D-2‘-deoxyribo-furanosyl-5-hydroxy-7-oxo-2,3-dihydrothiazolo[3,2-a]pyrimidinium hydrox- ide, prepared in a similar fashion to the mesoionic thiadiazolopyrimie- dine nucleosides, was designed as a potential inhibitor of the enzyme thymidylate synthetase. The mesoionic thiazolinopyrimidine nucleoside, was obtained as the a anomer and was not evaluated for antineoplastic activity. The mesoionic imidazothiazine nucleoside, i.e. Anhydro-l- (2',3',5'-tri-O-acetyl-D-ribofuranosyl)-5-hydroxy-7-oxoimidazo[2,l-b] thiazinium hydroxide was prepared as a potentially useful agent, due to its structural and isosteric similarity with purine nucleosides. The mesoionic imidazothiazine nucleoside was prepared by a cyclization reaction between the tri-0-acetyl-D-ribofuranosyl imidazole-2-thione and carbon suboxide. The mesoionic imidazothiazine nucleoside was not stable at room temperature or in aqueous Solution. While the results of this study on the chemotherapeutic utility of mesoionic nucleosides was rather discouraging, knowledge has been.gained that might be of value for the future design and synthesis of useful mesoionic nucleosides.

In part I of this thesis are described the structural determinations of sitsirikine, dihydrositsirikine and isositsirikine, three new alkaloids from Vinca rosea Linn. Sitsirikine, C₂₁H₂₆O₃N₂, and dihydrositsirikine, C₂₁H₂₈ O₃N₂, were isolated as an inseparable mixture, which was shown by hydrogenation studies to be comprised of an olefin and its dihydro derivative. The formation of formaldehyde upon ozonisation of the mixture, and of propionic acid in a modified Kuhn-Roth oxidation of dihydrositsirikine demonstrated that, sitsirikine possessed a vinyl group. Both sitsirikine and dihydrositsirikine gave mono-acetates, and the N.M.R. data indicated that primary hydroxyl groups were present in the original alkaloids. A methyl ester function suggested by spectral evidence was established by hydride reduction of dihydrositsirikine to a diol. Since the diol, yielded an acetonide, it was inferred that dihydrositsirikine possessed a β-hydroxy-ester unit. The U.V. spectrum of dihydrositsirikine was characteristic of an indole chromophore, which the mass spectrum showed to be part of a tetrahydro-β-carboline system. Dehydrogenatioh afforded a compound with a flavocoryline-type U.V. spectrum, and this suggested that sitsirikine was a relative of the tetracyclic corynantheine class of alkaloids. This was confirmed by conversion of dihydro-corynantheine into dihydrositsirikine. The structure of the related indole alkaloid isositsirikine, C₂₁H₂₆O₃N₂, was determined by a similar series of reactions. Ozonolysis yielded acetaldehyde, which authenticated the ethylidene group indicated by the N.M.R. spectrum. Acetylation afforded a mono-acetate, whose N.M.R. spectrum suggested that isositsirikine had a primary hydroxyl function. A methyl ester was established by hydride reduction to a diol, which formed an acetonide and hence showed the presence of a β-hydroxy-ester unit in the original alkaloid. Since dehydrogena-tion of dihydro-isositsirikine yielded flavocoryline, a tetracyclic structure very similar to that of sitsirikine could be postulated for isositsirikine. Part II is concerned with the chemistry of cleavamine, a scission product, of the Vinca alkaloid catharanthine. Treatment of catharanthine with aqueous acid in the presence of a reducing agent, led to the isolation of descar-bomethoxycatharanthine, cleavamine and two epimeric dihydro-cleavamines. A tentative mechanism for the reaction is proposed, which can account for the formation of these compounds. Reduction of catharanthine in glacial acetic acid provided carbomethoxy-dihydrocleavamine. Mercuric acetate oxidised this compound to a mixture of two immonium ions, both of which underwent transannular cyclisations. One of the ions gave the known Iboga alkaloids coronaridine and dihydrocathafanthine, whereas the other afforded pseudo-vincadifformine - a synthetic analogue of the known Vinca alkaloid vincadifformine. The structure of pseudo-vincadifformine was determined by conversion into compounds which had U.V., I.R., N.M.R. and mass spectra completely analogous to the corresponding derivatives of vincadifformine. Similar transannular cyclisations to the above are postulated in the scheme advanced by Wenkert for the biogenesis of Iboga and Aspidosperma alkaloids, and the significance of our results with regard to this theory is duscussed. The formation of coronaridine and dihydrocatharanthine in the reaction constituted partial syntheses of these alkaloids, and the potential use of transannular cyclisations in laboratory syntheses of Iboga and Aspidosperma alkaloids is also considered. / Science, Faculty of / Chemistry, Department of / Graduate

A major gap in sexual assault casework is demonstrated when DNA is not recovered. Oftentimes, if DNA evidence is not present on the collection swabs, the sexual assault kit (SAK) is not further analyzed. Due to the "CSI effect," DNA is commonly understood as highly identifiable evidence, potentially leading to increased condom usage to eliminate or reduce DNA transfer during a sexual assault. Therefore, the analysis of condoms and sexual lubricants is pertinent. The purpose of this research is to develop analytical protocols to potentially connect unknown substances recovered in a SAK to known lubricant reference samples. Sexual lubricants were analyzed using Fourier transform infrared spectroscopy, gas chromatography-mass spectrometry, and direct analysis in real time-high resolution mass spectrometry. Analytical protocols were developed using 162 sexual lubricants comprised of bottled lubricants, condoms, and personal hygiene products. A statistical model was developed from 112 of the samples using hierarchical cluster analysis (HCA), principal component analysis (PCA), and linear discriminant analysis (LDA) to determine appropriate sample groupings that resulted in at least 97% accurate classification for each instrument. Assigned truth classes for the remaining 50 samples were developed using Pearson correlation coefficients (PCCs) to predict classification accuracy for unknown samples. The FTIR data resulted in a 96% accurate prediction, 54% for GC-MS, and 42% for DART-HRMS, showing the need for expansion of the sample set in future analysis. Potential storage conditions of SAK swabs were evaluated using PCCs to identify optimal swab storage conditions, which was determined to be a humidity-controlled environment around 22 °C. Then, post-coital swab samples from volunteers using an unknown condom were analyzed using the developed protocols. The data was analyzed using PCC, PCA, and LDA to compare the classification to the "ground truth" of the sample to determine potential applications of this research in SAK analysis.

Magnetic materials are currently being developed in the areas of pharmacology and medicinal chemistry for use in applications such as drug delivery and magnetic resonance imaging. Magnetic fluids are being used in audio equipment and hard disk drives. Their suspension in a particular fluid is promoted by the adsorption or reaction of steric or electrostatic stabilizers, which are appropriate for the particular medium. Critical to the success of these magnetic fluids is the development of the steric stabilizers, which must prevent the coagulation of the metal particles. Polymeric materials are one of the most suitable nonmagnetic media to disperse the magnetic nanoparticles, forming polymeric nanocomposites in ferrofluids. We have developed strategies in molecular nanoscience to design polymeric systems for stabilization of magnetic nanoparticles. Ring opening metathesis polymerization (ROMP) was used to prepare a series of novel, well-defined diblock copolymers of bicyclo[2.2.1]hept-5-ene 2-carboxylic acid 2-cyanoethyl ester and bicyclo[2.2.1]hept-2-ene, consisting of both anchoring and steric stabilizing blocks. Both ester and cyano groups were incorporated into the polymers to chelate and stabilize the iron oxide magnetic nanoparticles. These polynorbornene-based copolymers were characterized by GPC, along with 1H NMR, FTIR, DSC, and TGA. Using diblock copolymers as stabilizers, nanostructured maghemite (γ-Fe2O3) magnetic ferrofluids were prepared in toluene or cyclohexanone via thermal decomposition of Fe(CO)5 and then the oxidation of iron nanoparticles. Transmission electron microscopic (TEM) images showed a highly crystalline structure of the γ-Fe2O3 nanoparticles, with average particle size varying from 5 to 7 nm. Polymer films containing iron oxide nanoclusters were also prepared from the diblock copolymers. For comparison, a commercial triblock copolymer (BASF PluronicR F127) surfactant was used to prepare stabilized ferrofluids. In addition to γ-Fe2O3 nanoparticles, other types of magnetic nanoparticles, such as FePt, were investigated using this triblock copolymer as a stabilizer. The results indicated that the norbornene diblock copolymers could also be used for the preparation of FePt stabilized magnetic ferrofluids in the future research work.

<p>Capillary electroseparation methods<b> </b>are exciting new techniques with very broad application areas and vast potential in pharmaceutical and biomedical analysis.</p><p> To improve the limit of detection (LOD) capillary zone electrophoresis (CZE) has been combined with isotachophoretic (ITP) preconcentration in a single capillary. Using the ITP-CZE combination the LOD can be improved at least 100-fold. Laser-induced fluorescence (LIF) detection is more sensitive and more selective than the most common detection technique, UV, and the intensity and focusing capability of LIF fits well with the small dimensions in CZE. The total sensitivity enhancement attained for a new acetylcholinesterase inhibitor, NXX-066, by using ITP-CZE-LIF was more than 5500-fold compared to CZE-UV.</p><p> Capillary electrochromatography (CEC) combines the high separation efficiency of CZE with the vast possibilities to improve selectivity of HPLC. We have examined different ways to solve the problem of extensively tailing peaks and studied the influence of the mobile phase composition on the electrochromatographic performance for a number of tricyclic antidepressants and related quaternary ammonium compounds. (1) Adding aliphatic amines to the mobile phase in reversed phase CEC. The effect on the chromatographic performance was coupled to the hydrophobicity of the additive and the amine of our choice was dimethyloctylamine. (2) Silica-based cation exchangers with different pore sizes. The large-pore materials promoted pore flow, but this had no positive influence on the performance. The small-pore (highest surface area) particles gave the best selectivity. (3) Designing special continuous beds. As the bed is covalently attached to the capillary wall, problems related to retaining frits are avoided. The stationary phase most suitable for our analytes had a molar ratio of 1:80 between the functional ligands, vinyl sulphonic acid and isopropyl groups, respectively. The LOD was lowered 26000-fold by dissolving the sample in a low-conducting medium.</p>

<p>Capillary electrophoresis-mass spectrometry has been used successfully for the analysis of a wide range of analytes such as chiral local anaesthetics, sulphonated reactive dyes and endogenous neurotransmitters and neuropeptides.</p><p>The partial filling technique was used in CE-MS for chiral separation of bupivacaine and ropivacaine using the non-volatile selector β-cyclodextrin. By only partially filling the capillary with selector and using capillaries coated with polyacrylamide to suppress the electroosmotic flow, introduction of the selector into the mass spectrometer was avoided. An impurity of 0.25% of the R-enantiomer of ropivacaine in the S-form could be detected.</p><p>The partial filling technique was developed further using CE employing two different selectors in separate plugs in the capillary. This enhanced the separation efficiency and offered greater flexibility in controlling the separation.</p><p>By using transient-isotachophoresis (tITP)-CE-MS it was possible to concentrate and detect classical neurotransmitters and neuropeptides with masses ranging from 104 Da to 1642 Da. γ -Aminopropyltriethoxysilane coated capillaries were used to minimize adsorption of the peptides onto to capillary surface. Endogenous dopamine, glutamate, γ-aminobutyric acid (GABA), acetylcholine, methionine-enkephalin and substance P 1-7 were detected in the striatum of marmoset monkey.</p><p>Sulphonated dyes obtained from single textile fibres were analysed using CE-MS. Capillary electrophoresis was found to be a good way of removing the excess amounts of glucose present in the sample that would otherwise interfere with the electrospray ionisation. </p><p>Automatic function switching, originally developed for use together with liquid chromatography, was found to be a great method for acquiring MS/MS data when doing infusion experiments saving both time and sample without decreasing the quality of the MS/MS data. It was also found to be a more time efficient way than using the precursor ion scanning mode on the Q-TOF to obtain precursor ion data.</p>

<p>The aim of this work was to explore the structural and functional diversity of polypeptides that are found in plants. Expanding knowledge of simililarities between plant use of these compound and animal use promises exceptional opportunities for finding, from plant research, new structures with biomedical and biotechnological potential.</p><p>A fractionation protocol was developed and applied to many plant species, providing fractions enriched in polypeptides, amenable to chemical and biological evaluation. From one species, the common field pansy (<i>Viola arvensis</i>), a 29-amino-acid residue polypeptide was isolated, named varv A, which revealed a remarkable macrocyclic structure (i.e., N- and C-termini are joined) stabilised by three knotted disulfides<i>. </i></p><p>Varv A, together with an increasing number of homologous peptides, form the currently known peptide family of cyclotides. Their stable structure makes them an attractive scaffold for protein engineering. In addition, they display a wide range of biological activities (e.g., antimicrobial, cytotoxic, and insecticidal). As a part of this work, the cytotoxic effects of varv A and two other isolated cyclotides were evaluated in a human cell-line panel: all were active in the low µM range. Most likely, these effects involve pore formation through cell membranes.</p><p>Cyclotides were found to be common in the plant family<i> Violaceae; </i>with eleven cyclotides isolated and sequenced from V. arvensis, V. cotyledon, and<i> Hybanthus parviflorus. </i>For six members of the genus <i>Viola</i>, cyclotide expression profiles were examined by liquid chromatography-mass spectrometry (LC-MS): all expressed notably complex mixtures, with single species containing more than 50 cyclotides. These profiles reflect the evolution of the genus<i>.</i></p><p>To assess these mixtures, a rational strategy for MS based amino acid sequencing of cyclotides was developed, circumventing inherent structural problems, such as low content of positively charged amino acids and the macrocyclic structure. This was achieved by aminoethylation of cysteines, which, following tryptic digestion, produced fragments of size and charge amenable to MS analysis. This method was also modified and used for mapping of disulfide bonds<i>. </i></p><p>Methods for isolation and characterisation developed in this work may prove useful not only for further studies on macrocyclic polypeptides from plants, but also for other plant peptides and disulfide-rich peptides from animals.</p>