Dissertationen zum Thema „Carbohydrate“

Carbohydrate-Carbohydrate interactions (CCIs) operate in a number of biologically significant environments. These carbohydrate based interactions on cell surfaces, which have been shown to be highly specific and polyvalent, are believed to mediate cell recognition and adhesion within biological processes. Two synthetic glycosylated scaffolds have been synthesised as model systems and the scaffolds chosen were designed to orientate interacting carbohydrates close in space to one another thereby allowing the study of these important CCIs.

Carbohydrate-carbohydrate interactions (CCIs) have been shown to be an important interaction in molecular recognition. These interactions present characteristics such as the synergistic effect with other interactions e.g. protein-protein interactions, specificity, polyvalency and in some cases requirement of divalent cations. CCIs remain insufficiently documented due to the weakness of such an interaction, which is difficult to probe by classical techniques and study at the molecular level in a monovalent system has not been performed. In order to study the CCI, a peptide based on alanine and lysine has been designed. Carbohydrates have been ligated to this peptide and changes in a-helix and random coil conformations are examined using CD spectroscopy. This system was shown to function as a reporter for CCls through changes in the conformations of the peptide. A second system that will be employed to study eCls is utilising the thiol-thioester exchange reaction. This involves a reaction between a carbohydrate with a thioester linkage and a carbohydrate linked to a thiol moiety. The resulting equilibrium is to be probed using HPLC. Synthetic routes have been developed in order to obtain the desired thiols and thioester. First studies showed that a CHO-π interaction can be quantified using this system. The carbohydrates to be attached are: Lex, sLex, LeY. The synthesis of Lex has been improved within the Gallagher group. A new synthesis of LeY was performed using an armed/disarmed strategy. Meanwhile, studies towards the synthesis of sLex were carried out.

Protein-carbohydrate recognition is an important target for inhibitor development. Improved inhibitor design requires a fundamental molecular basis of these interactions. This thesis describes the preliminary structural studies on three carbohydrate processing enzymes, UDP-galactopyranose mutase, alpha-D-glucose-1-phosphate thymidylyltransferase and TDP-glucose 4,6-dehydratase. These enzymes are found in important human pathogens such as Mycobacterium tuberculosis and Salmonella typhimurium. The major focus of the thesis has been on UDP-galactopyranose mutase, the enzyme responsible for catalysing synthesis of the thermodynamically unfavourable 5 membered ring form of galactose, UDP-galactofuranose from the thermodynamically favoured 6 membered ring form, UDP-galactopyranose. UDP-galactofuranose plays a key role in mycobacterial cell walls. This thesis also describes work with concanavalin A. This legume lectin is an invaluable model for the study of protein-carbohydrate interactions. Two concanavalin A complexes are discussed. Both structures clear up misunderstandings in the literature and provide an insight into designing enzyme inhibitors.

Carbohydrates are ubiquitous in nature and have become the focus of much scientific investigation. The proteins which recognise carbohydrates have become widely used in the areas of cell and molecular biology. Protein - carbohydrate interactions have been probed by theoretical, structural and thermodynamic techniques. The lectins are a class of carbohydrate binding proteins which bind carbohydrates through non covalent interactions such as hydrogen bonds and van der Waals interactions. In addition to these interactions, other factors play an important role in determining affinity such as carbohydrate conformation, solvent reorganisation and changes in the protein binding site. The legume lectin concanavalin A specifically recognises mannose and glucose terminal residues. The thermodynamics of concanavalin A binding to carbohydrates has been well documented. Concanavalin A binds the core trimannoside and pentasaccharide of the biantennary glycan found on mammalian cell surfaces with a high affinity. This thesis describes the structural basis of carbohydrate binding by con A, through the interpretation of crystal structures of concanavalin A bound with α1-2 mannobiose, methyl α1-2 mannobioside, the core pentasaccharide of the biantennary glycan and fructose. The structural information obtained from these structures is related to thermodynamic information available and unravels the importance of the role played by carbohydrate conformation, solvent reorganisation and statistical population of ligand in determining affinity. This work helps to develop an understanding of the physical basis of carbohydrate recognition.

In this thesis the synthesis of several hydrolytically stable carbohydrate mimics with the potential to function as glycosidase or lectin inhibitors are described. This work is presented in Chapters 2-5. Chapters 2 and 3 describe synthetic efforts for producing carbasugars, and include the first synthesis of 1,2-bis-epi-valienamine and the preparation of two previously known aminocarbasugars. All three compounds were synthesised starting from D-mannose, using ring-closing metathesis as the key step. 1,2-Bis-epi-valienamine was found to inhibit Cellulomonas fimi β-mannosidase with a Ki value of 140 mM. Also included is the development of a novel synthetic route from cheap D-fructose to three mannose-mimicking carbasugars using a ring-closing metathesis strategy. Two of the compounds are potential inhibitors of the FimH adhesin. In Chapters 4 and 5 the synthesis of a number of pseudodisaccharides are presented; valienamine- and epi-valienamine-containing pseudodisaccharides and a small library of S-linked pseudodisaccharides were prepared. Various synthetic strategies were explored, including an alkylation strategy, Mitsunobu couplings, and sulfonate displacements. This is the first report on the synthesis of a valienamine pseudodisaccharide with β-lyxo-configuration. Two of the S-linked pseudodisaccharides were found to bind to Concanavalin A with high affinity. The final chapter (Chapter 6) of this thesis focuses on the development of a carbohydrate epimerisation method using transition metal catalysis. Two equilibrium constants involving gluco/manno- and gluco/allo-alcohols were determined via this method.
At the time od doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 3: Manuscript. Paper 5: Manuscript.

It has been widely recognized that aging will cause a profound decrease in glucose tolerance end increase insulin resistance. These changes have been found to occur at a relatively early age. Narimiya [54], has documented these changes in younger rots end has shown alterations in glycogen metabolism to occur prior to nine months of age. Exercise in the form of running has been shown to attenuate these changes. In Vivo, glucose, insulin end muscle glycogen have interrelated functions. The role of muscle glycogen is to provide energy for the muscle's contractile process. Insulin is needed at rest to allow glucose to enter the muscle and be stored as glycogen. The purpose of this study is to pinpoint when changes in glycogen metabolism occur while looking at the influence of exercise end weight restriction on the process. METHODS: Male Sprague Dawley rats ages 1.5-4.0 months of age were divided into three groups control (CN), pairfed (PF), and exercise trained (ET). The ET cages were equipped with voluntary running wheels attached to an automatic revolution counter. At 1.5 months, a group of controls were sacrificed and treated as the 4 mo. animals described below. Following training the hindlimbs of CN, ET, end PF were surgically isolated and glucose uptake examined by perfusing them with a bovine blood preparation, which contained insulin and glucose. Pre and post samples of the soleus, plantaris, and red and white vastus were removed and assayed for glycogen. RESULTS: The 1.5 mo. CN had significantly greater glucose uptake then any other group for both the insulin and non-insulin infused groups. The ET had significantly greater uptake than the other 4 mo. groups at 90 and 120 min in the insulin infused group. In the 1.5 mo. CN when insulin is not present all muscles will break down glycogen. When insulin is present, glycogen is used in all but the white vastus. In the 4 mo. CN all muscles except the soleus break down glycogen. In the ET the glycogen breakdown pattern is similar regardless of whether insulin is present or not. In the PF, glycogen breakdown is depressed and decidely different from the other treatment groups. CONCLUSIONS: There is an age dependent decrease in glucose tolerance and insulin resistance at or before 4 mo. of age. Training prevented some of this loss but did not stop the decline. Weight restriction had a nominal, if any, benefit in reducing insulin resistance and raising glucose tolerance with aging.

Glycoproteins have diverse and essential roles within biological systems. They are formed by enzymatic addition of saccharides to proteins during, or shortly after, translation. However, saccharides can also react with proteins non-enzymatically, a process termed glycation, which can cause impaired function and improper folding. Glycated proteins further react to form advanced glycation end-products, which have been implicated in the pathogenesis and progress of many diseases. Due to this pathological effect, glycation has been studied as a potential biomarker of these diseases. Photoaffinity labelling is a technique that is used to investigate the structure, and presence, of biological molecules; a precedent exists for its use in the study of carbohydrates in biological systems. Chapter 1 outlines the background of this thesis exploring previous studies of glycation, its effects, and methods used in recognition and photoaffinity labelling. Chapter 2 details the design and synthesis of a novel photoaffinity probe, and the optimisation of this synthesis. The target molecule was successfully produced and simpler alternatives to the initial synthetic route with similar yields are discussed. In Chapter 3 the use of the photoaffinity probe is studied. Labelling trials were performed on three proteins: human serum albumin (HSA), macrophage migration inhibitory factor (MIF), and casein. Mass spectrometry showed that the experiments with both HSA and MIF were successful, while the procedure appeared to lead to degradation of casein. Additionally, our work into developing techniques for identifying labelled samples is detailed. A diol-doped electrophoresis gel was not successful created, however, staining protein samples in polyacrylamide gel electrophoresis with curcumin showed promise. Chapter 4 explores the electrochemistry of the photoaffinity probe and details the use of the probe in functionalising a fluorine doped tin oxide (FTO) glass electrode. Cyclic voltammograms of Alizarin Red S (ARS), obtained using a treated electrode, suggest that surface functionalisation was successful.

Thesis (MTech (Food Technology))--Cape Peninsula University of Technology, 2013.
Acha starch was isolated and purified from clean and milled acha grain. Functional, thermal and physicochemical properties of acha starch were analysed using appropriate methods. Wheat starch was used as the reference standard. Acha bread from acha grain was baked and the consumer sensory acceptability was evaluated and white wheat bread was used as the reference standard. The effect of baking, boiling, steaming and microwaving on the starch and glycemic properties of the acha starch was evaluated. With regard to thermal properties, gelatinisation temperature of acha and iburu starches typifies that of waxy starch. Acha starch has similar retrogradation temperature profiles as that of wheat. There were however significant differences in some of the functional properties (pasting and turbidity) and physico-chemical properties (in vitro starch digestibility), but no significant difference in the texture profile analysis (TPA) and water binding capacity (WBC). WBC of both acha varieties was higher than that for wheat starch. Due to its high break down viscosity, white acha starch can be included in foods that are subjected to high temperature processing. This indicates that both acha starch varieties can be used for hot and cold desserts as well as for soft jelly like sweets and confectionery toppings. A prescreening exercise using carboxymethyl cellulose (CMC), Xanthan gum, yeast and acha starch as the variables was successful in concluding a recipe which rendered acha bread with the optimum specific loaf volume for both white and black acha bread. The optimum recipe consisted 8.0 % acha starch, 2.0 % xanthan gum, 2.0 % CMC and 1.0 % yeast. The majority of the consumer panellists found the crust colour, taste and aroma to be moderately desirable. This implies that most consumers find acha bread to have the potential to be marketed as wheat free bread. The different processing methods baking, boiling, microwaving and steaming, affected the black and white acha starch hydrolysis. The amount of starch hydrolysed for the different processing methods was in the following order: baking > boiling > microwaving > steaming. It can thus be concluded that different processing methods affects the micro structure and physical properties of the acha and wheat samples which thus influence their starch hydrolysis. The equilibrium percentage of starch hydrolysed after 180 min incubation was affected differently for the various starches, black acha, white acha and wheat starch by the different processing methods and times. In the case of baking black acha starch and wheat bread were affected similarly. However, this was not the case for microwaving, steaming and boiling, where both acha starch varieties and wheat starch were affected in the same way. The rate of starch hydrolysis for both acha varieties and wheat grain for the different processing methods, steaming, boiling, microwaving and baking was affected to the same degree respectively.

Thesis (Ph. D.)--Ohio State University, 2005.
Title from second page of PDF file. Document formatted into pages; contains xix, 347 p.; also includes graphics. Includes bibliographical references (p. 183-192). Available online via OhioLINK's ETD Center

La Tesis que se presenta trata sobre el desarrollo de nuevos métodos selectivos de síntesis de carbohidratos. En la primera parte de la tesis se estudió la epoxidación de glicales utilizando catalizadores de Mo y ácido meta-cloroperbenzoico (MCPBA), en relación con la obtención de mano-oligosacáridos. En todos los casos estudiados, el epóxido formado se abría in situ para dar lugar al correspondiente diol o glicósido. Se observó un efecto director por parte de los hidroxilos libres, lo que permitía obtener derivados de manosa a partir de glucosa. En el caso del Mo, los resultados figuran entre los mejores descritos para epoxidación de glicales con catalizadores. Los resultados con MCPBA fueron excelentes, aplicándose este último procedimiento a la síntesis ortogonal de dadores de glicosilo. En la segunda parte, relacionado con la síntesis del cardiotónico digitoxina, se estudiaron dos metodologías sintéticas para obtener 2-desoxi-glicósidos mediante reacción de olefinación-ciclación-glicosilación y a través de síntesis asimétrica.
This thesis deals with two topics connected with carbohydrate chemistry. The first part presents epoxidation and dihydroxylation reactions of glycals using Mo-catalysts and m-chloroperbenzoic acid (MCPBA) towards the synthesis of manno oligosaccharides. The oxidation of glycals afforded in all cases the corresponding diols or glycosides, as a consequence of the in situ opening of the epoxides initially formed. Free hydroxyl groups were observed to direct the stereoselectivity of the epoxidation, and then manno derivatives were obtained from unprotected or partially protected glucals. The results using Mo-catalysts are among the best described for the catalytic epoxidation of glycals. The results using MCPBA were excellent, and this methodology was applied to the synthesis of orthogonally protected glycosyl donors. The second part is related to the synthesis of the cardiotonic digitoxin. Two different strategies were studied in order to obtain 2-deoxy-glycosides through olefination-cyclization-glycosylation reaction and using asymmetric synthesis.

Nanoporous carbon materials are widely used in industry as adsorbents or catalyst supports, whilst becoming increasingly critical to the developing fields of energy storage / generation or separation technologies. In this thesis, the combined use of carbohydrate hydrothermal carbonisation (HTC) and templating strategies is demonstrated as an efficient route to nanostructured carbonaceous materials. HTC is an aqueous-phase, low-temperature (e.g. 130 – 200 °C) carbonisation, which proceeds via dehydration / poly-condensation of carbon precursors (e.g. carbohydrates and their derivatives), allowing facile access to highly functional carbonaceous materials. Whilst possessing utile, modifiable surface functional groups (e.g. -OH and -C=O-containing moieties), materials synthesised via HTC typically present limited accessible surface area or pore volume. Therefore, this thesis focuses on the development of fabrication routes to HTC materials which present enhanced textural properties and well-defined porosity. In the first discussed synthesis, a combined hard templating / HTC route was investigated using a range of sacrificial inorganic templates (e.g. mesoporous silica beads and macroporous alumina membranes (AAO)). Via pore impregnation of mesoporous silica beads with a biomass-derived carbon source (e.g. 2-furaldehyde) and subsequent HTC at 180 oC, an inorganic / carbonaceous hybrid material was produced. Removal of the template component by acid etching revealed the replication of the silica into mesoporous carbonaceous spheres (particle size ~ 5 μm), representing the inverse morphological structure of the original inorganic body. Surface analysis (e.g. FTIR) indicated a material decorated with hydrophilic (oxygenated) functional groups. Further thermal treatment at increasingly elevated temperatures (e.g. at 350, 550, 750 oC) under inert atmosphere allowed manipulation of functionalities from polar hydrophilic to increasingly non-polar / hydrophobic structural motifs (e.g. extension of the aromatic / pseudo-graphitic nature), thus demonstrating a process capable of simultaneous control of nanostructure and surface / bulk chemistry. As an extension of this approach, carbonaceous tubular nanostructures with controlled surface functionality were synthesised by the nanocasting of uniform, linear macropores of an AAO template (~ 200 nm). In this example, material porosity could be controlled, showing increasingly microporous tube wall features as post carbonisation temperature increased. Additionally, by taking advantage of modifiable surface groups, the introduction of useful polymeric moieties (i.e. grafting of thermoresponsive poly(N-isopropylacrylamide)) was also demonstrated, potentially enabling application of these interesting tubular structures in the fields of biotechnology (e.g. enzyme immobilization) and medicine (e.g. as drug micro-containers). Complimentary to these hard templating routes, a combined HTC / soft templating route for the direct synthesis of ordered porous carbonaceous materials was also developed. After selection of structural directing agents and optimisation of synthesis composition, the F127 triblock copolymer (i.e. ethylene oxide (EO)106 propylene oxide (PO)70 ethylene oxide (EO)106) / D-Fructose system was extensively studied. D-Fructose was found to be a useful carbon precursor as the HTC process could be performed at 130 oC, thus allowing access to stable micellular phase. Thermolytic template removal from the synthesised ordered copolymer / carbon composite yielded functional cuboctahedron single crystalline-like particles (~ 5 μm) with well ordered pore structure of a near perfect cubic Im3m symmetry. N2 sorption analysis revealed a predominantly microporous carbonaceous material (i.e. Type I isotherm, SBET = 257 m2g-1, 79 % microporosity) possessing a pore size of ca. 0.9 nm. The addition of a simple pore swelling additive (e.g. trimethylbenzene (TMB)) to this system was found to direct pore size into the mesopore size domain (i.e. Type IV isotherm, SBET = 116 m2g-1, 60 % mesoporosity) generating pore size of ca. 4 nm. It is proposed that in both cases as HTC proceeds to generate a polyfuran-like network, the organised block copolymer micellular phase is essentially “templated”, either via hydrogen bonding between hydrophilic poly(EO) moiety and the carbohydrate or via hydrophobic interaction between hydrophobic poly(PO) moiety and forming polyfuran-like network, whilst the additive TMB presumably interact with poly(PO) moieties, thus swelling the hydrophobic region expanding the micelle template size further into the mesopore range.
Nanoporöse kohlenstoffbasierte Materialien sind in der Industrie als Adsorbentien und Katalysatorträger weit verbreitet und gewinnen im aufstrebenden Bereich der Energiespeicherung/erzeugung und für Trennverfahren an wachsender Bedeutung. In der vorliegenden Arbeit wird gezeigt, dass die Kombination aus hydrothermaler Karbonisierung von Zuckern (HTC) mit Templatierungsstrategien einen effizienten Weg zu nanostrukturierten kohlenstoffbasierten Materialien darstellt. HTC ist ein in Wasser und bei niedrigen Temperaturen (130 - 200 °C) durchgeführter Karbonisierungsprozess, bei dem Zucker und deren Derivate einen einfachen Zugang zu hochfunktionalisierten Materialien erlauben. Obwohl diese sauerstoffhaltige Funktionalitäten auf der Oberfläche besitzen, an welche andere chemische Gruppen gebunden werden könnten, was die Verwendung für Trennverfahren und in der verzögerten Wirkstofffreisetzung ermöglichen sollte, ist die mittels HTC hergestellte Kohle für solche Anwendungen nicht porös genug. Das Ziel dieser Arbeit ist es daher, Methoden zu entwickeln, um wohldefinierte Poren in solchen Materialien zu erzeugen. Hierbei führte unter anderem der Einsatz von anorganischen formgebenden mesoporösen Silikapartikeln und makroporösen Aluminiumoxid-Membranen zum Erfolg. Durch Zugabe einer Kohlenstoffquelle (z. B. 2-Furfural), HTC und anschließender Entfernung des Templats konnten poröse kohlenstoffbasierte Partikel und röhrenförmige Nanostrukturen hergestellt werden. Gleichzeitig konnte durch eine zusätzliche Nachbehandlung bei hoher Temperatur (350-750 °C) auch noch die Oberflächenfunktionalität hin zu aromatischen Systemen verschoben werden. Analog zur Formgebung durch anorganische Template konnte mit sog. Soft-Templaten, z. B. PEO-PPO-PEO Blockcopolymeren, eine funktionelle poröse Struktur induziert werden. Hierbei machte man sich die Ausbildung geordneter Mizellen mit der Kohlenstoffquelle D-Fructose zu Nutze. Das erhaltene Material wies hochgeordnete Mikroporen mit einem Durchmesser von ca. 0,9 nm auf. Dieser konnte desweiteren durch Zugabe von Quell-Additiven (z. B. Trimethylbenzol) auf 4 nm in den mesoporösen Bereich vergrößert werden. Zusammenfassend lässt sich sagen, dass beide untersuchten Synthesewege nanostrukturierte kohlenstoffbasierte Materialien mit vielfältiger Oberflächenchemie liefern, und das mittels einer bei relativ niedriger Temperatur in Wasser ablaufenden Reaktion und einer billigen, nachhaltigen Kohlenstoffquelle. Die so hergestellten Produkte eröffnen vielseitige Anwendungsmöglichkeiten, z. B. zur Molekültrennung in der Flüssigchromatographie, in der Energiespeicherung als Anodenmaterial in Li-Ionen Akkus oder Superkondensatoren, oder als Trägermaterial für die gezielte Pharmakotherapie.

CHARACTERIZATION OF CARBOHYDRATE BASED VACCINES Variables influencing the immunogenicity and physicochemical properties of glycoconjugate vaccinesMany aspects can influence the immunogenicity of conjugate vaccines and the main variables investigated so far are the size of the saccharide moiety, the saccharide:protein ratio in the purified conjugate, the conjugation strategy, the nature of the spacer and the protein carrier. The size of the saccharide moiety and saccharide/protein ratio were investigated in different works such as Seppälä and Mäkelä in one of the first studies on the effect of size and chemistry on the immunogenicity of dextrans-protein conjugates found that dextrans of low molecular weight conjugated to chicken serum albumin, induced strong anti-dextran responses in mice, while increasing the dextrans' size resulted in reduced immunogenicity.47 Peeters et al. showed that a synthetic tetramer of Hib capsular polysaccharide repeating unit, conjugated to a protein carrier, induced in adult mice and non-human primates antibody levels comparable to a commercial Hib conjugate and higher than those induced by a trimer, indicating that for Hib a minimum of eight sugars is needed for a proper immunological response.48 Laferriere et al. found little influence of the carbohydrate chain length on the immunogenicity of pneumococcal conjugate vaccines in mice.49 Pozsgay et al. studied the immunogenicity in mice of synthetic Shigella dysenteriae type 1 LPS oligosaccharides conjugated to human serum albumin (HSA). The authors found that octa-, dodeca-, and hexadecasaccharide fragments induced high levels of lipopolysaccharide binding IgG antibodies in mice after three injections and were superior to a tetrasaccharide conjugate. The influence of the carbohydrate/protein ratio was different for the three conjugates. The octasaccharide-HSA conjugate with the highest density evoked a good immune response, while in the case of dodeca- and hexadecasaccharide conjugates, the median density was optimal.50 These studies suggest that oligosaccharide chain length and hapten loading might be interconnected in determining the immunogenicity of glycoconjugate vaccines. The spacer is a short linear molecule that is generally linked to the polysaccharide chain or to the protein or to both moieties, depending on the chemistry, used to facilitate the coupling between the protein and sugar. There are evidences in the literature which suggest that rigid, constrained spacers like cyclohexyl maleimide, elicit a significant amount of undesirable antibodies, with the risk of driving the immune response away from the targeted epitope on the hapten.51 52 The use of a flexible alkyl type maleimido spacer has been reported as a way to overcome the previous observed immunogenicity of cyclic maleimide linkers.53 A number of protein carriers have been used so far in preclinical and clinical evaluation of conjugate vaccines. 54 55 56 57 58 59 60Proteins such as diphtheria and tetanus toxoids, which derive from the respective toxins after chemical detoxification with formaldehyde, were initially selected as carrier because of the safety track record accumulated with tetanus and diphtheria vaccination. CRM197, a non-toxic mutant of diphtheria toxin61 which instead does not need chemical detoxification, has been extensively used as carrier for licensed Hib, pneumococcal, meningococcal conjugate vaccines and for other vaccines being developed. An outer membrane protein complex of serogroup B meningococcus has been used by Merck as carrier for their Hib conjugate vaccine.62 GSK in their multivalent pneumococcal conjugate vaccine introduced the use of the Hib-related protein D as carrier for most of the polysaccharides included into the vaccine.63 64 The team of John Robbins made extensive use of the recombinant non toxic form of Pseudomonas aeruginosa exo-toxin as carrier for Staphylococcus aureus type 5 and 8 as well as for Salmonella

Chapter 1 describes the synthesis of a 1,4-dicarbonyl compound 1 which was constructed by a sequence involving opening of a protected glucose epoxide with allyl magnesium chloride, alkylation and Wacker oxidation; the 1,4-dicarbonyl compound 1 readily underwent cyclisation under basic conditions to produce the cyclopentaannulated sugar derivative 2. Treatment of the cyclopentaannulated sugar derivative with N-bromosuccinimide and subsequent treatment with activated zinc completed the fragmentation of 2 to furnish the cyclopentanes 3. Chapter 2 describes the reduction of 2 which furnished a mixture of allylic alcohols 4 in a ratio of 8:1 in favour of either isomer depending on the conditions and reagents employed. The application of a Stork silyl methylene radical cyclisation of the a-cyclopentaannulated derivative led to a 2:1 mixture of trans and cis fused tricyclic ring systems. Treatment of the allylic cyclopentaannulated derivative, however, led to a single cis-fused product 5. This is in contrast to previous examples which show a mixture of cis and trans-fused 6,5-ring systems. Chapter 3 describes the continuation of work in the Jenkins group directed towards the synthesis of taxanes from glucose. A model study was undertaken to show the viability of a stepwise diene synthesis utilising selenium chemistry. This succesful model study showed that an enone to diene conversion was possible. The application of this methodology to the C-ring synthon 6 produced 7, the most advanced intermediate to date, as a single isomer.

A series of O-diphenylphosphinyl monosaccharide derivatives, e.g. 1,2:5,6-di-O-cyclohexylidene-3-O-diphenyl phosphinyl--D-glucofuranose [60], has been synthesised by reaction of a free hydroxy group in a sugar with Ph_2PCl. C-Diphenylphosphinyl and diphenylarsino derivatives, e.g. methyl 4,6-O-benzylidene-3-deoxy-3-C-diphenylarsino--D-altropyranoside [89], were synthesised via reaction of Ph2PLi or Ph2AsLi with p-tosyl, mesyl, epoxy or carbonyl substituted sugar reagents. Steric factors play a large part in determining the reactivity of the precursor sugars towards the phosphorous or arsenic nucleophilic reagents. Conformations of the arsenic and phosphorus derivatives were assigned from the 13C, 1H and 31P NMR spectra. Analogous phosphinyl and arsino derivatives have the same conformations in solution. Generally, the pyranosides prefer the 4C1 conformation in solution while the furanoses and furanosides prefer a symmetrical twist. Ribofuranosides [52], [67] and [83], prefer the 2T3 conformation as to the mannofuranosides [56], [57], [58], [63] and [64], whereas xylofuranose derivatives [53] and [86] prefer the 3T2. The solid state NMR of [65] and [89] were also obtained and compared with the solution NMR. No major conformational differences were evident. The following alicyclic carbohydrate derivatives were also synthesised - 1,4:3,6-dianhydro-2,5-dideoxy-2,5-bis-C-diphenylarsino-L-iditol [99], 1,4:3,6-dianhydro-5-deoxy-5-C-diphenylarsino-2-O-p-tosyl-L-gulitol [100], 1,4:3,6-dianhydro-2-deoxy-2-C-diphenylarsino-L-iditol [97] and the 2-O- p -tosyl derivative [98] and 1,4:3,6-dianhydro-5-C-diphenyl-phosphinyl-2-O- p -tosyl-L-iditol [81]. Reactions of the carbohydrate derivatives with transition metal hexacarbonyls were also carried out. Methyl 4,6-O-benzylidene-2-deoxy-2-C-diphenylarsino--D-altropyranoside [88] gave a tetracarbonyl complex [114] on reaction with Cr(CO)_6 and two products from W(CO)_6, viz W_2(CO)_8(AsPh_2)_2 [111] and the dimer [W_2(CO)_9(H)L]. - [112]. The bridged dimer W_2(CO)_10(AsPh_2)_2 [113] was obtained from the reactions of the bis-diphenylarsino derivatives of L-iditol [99] and diisopropylidene-D-galactose [82]. The complex [Rh(1,5-cyclooctadiene)L]. &'43 BF_4. - [116] was synthesised on reaction of the chlororhodium dimer [Rh(COD)Cl]_2 with the bis-diphenylarsino-L-iditol derivative [L&'61 99].

Studies were performed to determine the concentration of soluble carbohydrates and starch in chrysanthemum plants at various stages of vegetative and floral development. Leaves of pot chrysanthemum (Chrysanthemum morifolium 'Charm' or 'Favor') grown under full irradiance had more soluble carbohydrates and starch than those grown with 65% irradiance reduction. Both showed clear diurnal variation in carbohydrate concentrations. Inflorescences exhibited no diurnal fluctuations in total soluble carbohydrate (TSC). Sucrose was the only translocated carbohydrate in chrysanthemums in quantities detectable by HPLC. In a postproduction environment, leaf and stem TSC remained relatively unchanged while inflorescence TSC decreased significantly. Reducing sugars (glucose + fructose) accounted for up to 84% of the inflorescence TSC. Relative levels of starch and fructans over time suggests an alternate use of fructans and starch as pools of available reserve carbohydrate during floral development. Fructans were shown to decrease in polymerization in both petals and inflorescences as petals expanded.

In this study, we investigated the potential applications of carbohydrates in the development of new antibiotics. To tackle the problem of multidrug-resistant variants of M. tuberculosis (MDR-TB), we investigated the biosynthesis pathways of trehalose, which has contributed to significant drug resistance. Some new methods were developed for the synthesis of potential inhibitors (6-azido-trehalose and 6,6'-diazido-trehalose) that have been designed to imitate the intermediate molecule (trehalose 6-phosphate, TPP) of OtsA-OtsB pathway. At the same time, some new antibacterial agents based on trehalose have been synthesized. Members of the 1,2,3-triazole family have shown interesting biological properties. Steroid derivatives have been developed in antibiotics area. Therefore click reaction was utilized to build a 1,2,3-triazole ring and combine cholesterol with different carbohydrate moieties. A series of new compounds has been synthesized and their bioactivities have been tested.

The work embodied in this thesis pertains to an attempt to understand better, the molecular basis of protein-carbohydrate recognition. For this purpose a systematic study was undertaken, not only of the energetics of lectin-sugar interactions, which serve as molecular recognition prototype of protein-carbohydrate interactions, but also of the complex effects of solvent water molecules surrounding both the species in solution state. The systems chosen for investigation include the specific recognition of sugars by lectins from diverse families, leguminosae and moraceae. The following salient aspects of the molecular recognition process constitute the focus of this thesis: • Effect of site specifically modified, deoxy-, fluorodeoxy-, or methoxy- substituted D-galactopyranoside binding to lectins. Isothermal titration calorimetric (ITC) investigations of the binding of these sugars to a model lectin permitted the correct prediction of the architecture of the primary binding site in the absence of x-ray crystal or NMR structure of the combining site (Ref. 7). The study provided the only unambiguous means of a site specific mapping of the hydrogen-bond donor- acceptor relationship of the monosaccharide within the primary combining site of the lectin. • Novel features of lectin-sugar recognition. Molecular interactions and forces contributing to the stabilization of the saccharides in the primary combining site of lectins. Binding of site specifically modified fluoro- substituted D- galactopyranosides to WBA I led to the demonstration of the involvement of C- F««»H-0 hydrogen bonds in stabilizing the saccharide within the combining site of lectin (Ref. 7). Implication of the novel C-H«**O hydrogen bonds at the specificity determining C-4 position in enabling the methoxy- substituted D- galactopyranoside to be stabilized within the primary binding site of galactose specific lectins WBA I and jacalin. • Development of a novel coupled osmotic-thermodynamic approach for investigating the role of water molecules in determining the specificity of lectin- sugar interactions. The results obtained led to the first direct demonstration of a differential uptake of water molecules accompanying the specific process of recognition of sugars by lectins (Ref 2) • On the origin of enthalpy-entropy compensation, a ubiquitous phenomenon accompanying the thermodynamics of several ligand binding reactions in aqueous solutions in general and the molecular recognition involving all known lectin-sugar interactions, in particular. The results provide the first unequivocal solution state proof of water reorganization as the source of enthalpy-entropy compensation (Ref 3). A new diagnostic test of a true osmotic effect in molecular recognition phenomena was proposed (Ref. 2) and validated (Ref. 3). As an introduction, Chapter 1 is a comprehensive review of literature that touches upon the diverse properties of lectins and our present understanding of their multifarious roles and applications, which has led to their christening, perhaps appropriately, as molecules that mediate the 'social' functions of cells and tissues. Although a challenge it is still, to decipher the "glycocode", it is apparent that the fundamental basis of the recognition function of lectin-sugar interactions is the initial specific binding of the saccharide molecule by the globular proteinaceous lectin molecule. It is imperative, therefore, that an incisive investigation of the origin of specificity of the binding reaction as well as the solvent effects influencing both the interacting species be undertaken for a better understanding of the complete molecular recognition process. Towards this end is introduced in Chapter 1 our present understanding of the results on lectin-sugar interactions from two complementary approaches viz structural, including X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, as well as thermodvnamic ones, which have provided important information on the architecture of the combining sites, the dynamic modes of saccharide recognition and forces involved therein. Despite a detailed knowledge available from such methods, a structure-energetics correlation has persisted as a current challenge of the field. Towards achieving this goal, studies on the energetics of the recognition of sugars by lectins were undertaken, with an aim to better understand the origin of specificity of lectin-sugar interactions. This thesis attempts to provide new insights on some of the possible lacunae precluding structure-energetics correlation and suggests ways to overcome them. Chapter 2 deals with ITC investigation of the effect of deoxy-, fluorodeoxy-, and methoxy- substitutions on the binding of monosaccharides to the primary combining site of the lectin WBA I isolated from the mature seeds of the leguminosae family member Psophocarpus tetragonolobus as well as the moraceae lectin jacalin. These studies provide valuable information on the hydrogen-bond donor-acceptor relationships within the combining site of the lectins wherein the sugar molecule is liganded with the amino-acid residues of the lectin. This study is relevant for understanding the origin of specificity of monosaccharide binding within the primary combining site of the lectins. It has recently become apparent that there is a predisposition in three-dimensional space, of the donor-acceptor pairs within the sugar binding site of the lectins. Hence there appears to be a stereochemical basis of distinguishing the recognition of the donor group vis-a-vis that of the acceptor group and that their spatial disposition determines the specificity of the saccharide recognition. Unambiguous assignment of which of the groups within the hydrogen bonded pairs is a donor and which one is the acceptor assumes greater importance. The ITC measurements of the binding of deoxy-, flurodeoxy-and methoxy-derivatives of D-galactopyranoside (oc-D-Gal) to the basic lectin from winged bean Psophocarpus tetragonolobus, WBA I revealed that each of the ligands bind to WBA I with the same stoichiometry of one per subunit (29 kDa) of WBA I. The binding enthalpies for various derivatives were essentially independent of temperature and showed complementary changes with respect to binding entropies. Replacement of the hydroxyl group by fluorine or hydrogen on C3 and C4 of the galactopyranoside eliminated their binding to the lectin, consistent with C3-OH and C4-OH acting as hydrogen bond donors. The affinity for C2 derivatives of galactose decreased in the order: GalNAc>2MeOGal>2FGal=Gal>2HGal which suggests that both polar and non-polar residues surround the C2 locus of galactose, consistent with the observed high affinity of WBA I towards GalNAc, where the acetamido group at C2 position is probably stabilized by both non-polar interactions with the methyl-group and polar interactions with the carbonyl group. The binding of C6 derivatives followed the order: Gal>6FGal>D-Fuc»6MeOGal=L-Ara indicating the presence of favourable polar interactions with a hydrogen bond donor in the vicinity. Based on these results the hydrogen bond donor-acceptor relationship of the complexation of methyl-a-D-galactopyranoside with the primary combining site of WBA I was proposed (Ref. /), which was subsequently validated by the crystal structure of methyl-a-D-galactopyranoside complexed with WBA I. This chapter also describes the results from ITC studies on the binding of monosaccharides and disaccharides to the lectin jacalin isolated from the mature seeds of the moraceae family member Artocarpus integrifolia. The novel observation about the existence of C-F*«*H-0 and C-H**»O hydrogen bonds in lectin-sugar interactions is also discussed in this chapter. Chapter 3 is a description of the detailed investigation on the role of water molecules in influencing the energetics of lectin-sugar recognition. A novel coupled osmotic-thermodynamic approach was developed to dissect the role of water molecules in determining the recognition of the sugars by lectins. For this purpose, the model system of mannotriose-concanavalin A was used because atomic level structural information on these complexes were available. The work described in this chapter, is the first solution state evidence for the role of water molecules in the specific interaction of carbohydrates with a legume lectin, concanavalin A (Con A) (Ref. 2). Sugar binding to Con A was accompanied by linear changes in the logarithm of binding constants as a function of neutral osmolyte strength, and were described by well defined negative slopes characteristic for each sugar. As these changes were independent of the chemical nature of the osmolyte used, the results were rationalized in terms of a true osmotic effect. It was demonstrated that the specific recognition of the branched trimannoside (3,6-di-0-(a-D-mannopyranosyl)~a-D-mannopyranoside), the individual dimannosidic arms (3-<9-(a-D-mannopyranosyl)-a-D-mannopyranoside, and 6-0-(a-D-marmopyranosyl)-a-D-mannopyranoside) and the monomeric unit D-mannopyranoside by Con A was accompanied by differential uptake of water molecules; 1,3 and 5 respectively. We also observed a conservation of the compensatory behaviour of binding enthalpies and entropies in the presence as well as absence of osmolytes. This provided the first definitive evidence that water-reorganization plays a direct role in effecting the phenomenon of enthalpy-entropy compensation in protein-ligand interactions in general and lectin-sugar interactions in particular, and that the specificity of lectin-sugar recognition is characterized by a differential uptake of water molecules. Chapter 3 also describes the first experimental identification of the origin of enthalpy-entropy compensation (EEC), a ubiquitous phenomenon accompanying the thermodynamics of multifarious biomolecular recognition processes. By coupling direct microcalorimetry with osmotic stress technique, an experimental handle was devised to test the hypothesis that solvent reorganization could be the source of EEC. The results provided an unequivocal demonstration that an osmotic change in water activity alone, at the same temperature and pH, is sufficient to result in the conservation of EEC during the molecular recognition of specific ligands by macromolecules belonging to thermodynamically diverse and unrelated systems, a compelling evidence that the primary source of EEC in aqueous solutions is attributable to reorganization of solvent water molecules, thus validating the test for the role of water reorganization as a source of EEC (Ref. 3). This provides the first definitive evidence for the notion that there is a direct involvement of water molecules in originating the EEC effect. Despite the generality of the results it is urged that several systems be subjected to a vigorous application of the coupled osmotic-thermodynamic approach proposed herein before constituting it as a proof. Suffice to say, it is perhaps heartening that at last one has a handle to test the role of water molecules in effecting EEC in the solution state and appreciate the diverse roles played by water molecules in mediating molecular recognition reactions. The proposal presented in Ref 2, that the strong isoequilibrium relationship of enthalpy with entropy during the recognition of saccharides by Con A studied under osmotic stress, be considered as diagnostic of a true osmotic effect was subsequently validated in a thermodynamically diverse and unrelated system of peptide recognition by monoclonal antibody, the results from which are discussed in an Appendix (A) to this thesis (Ref 4). That the stabilities of these lectins are not hampered in the presence of osmolytes was demonstrated using differential scanning calorimetry (DSC) (Ref 2). During the course of these DSC studies, we discovered an unusual feature in an animal galectin. Despite possessing the legume lectin fold, the 14-kDa S- type lectin exhibits multiple oligomeric states that are influenced profoundly by complementary ligands and surprisingly do not dissociate at the denaturation temperature. These results are discussed in an Appendix (B) to this thesis (Ref. 5). The general discussion and conclusions drawn from this work are summarized in chapter 4. Briefly, the following salient conclusions can be drawn from the work presented in this thesis: 1. Unambiguous assignment of hydrogen-bond donor-acceptor relationship at each of the hydroxyl group of the monosaccharide bound to the lectin belonging to different families has been demonstrated (Refs. 1,6). 2. First report of novel hydrogen bonds in lectin-sugar interactions such as C- F«MH-0 (Ref 1) and C-H^*O hydrogen bonds (Ref 6). 3. Unusual structural stabilities in a galectin with a fold similar to that in legume lectins but with starkly different thermodynamic stabilities (Ref 5). 4. We have demonstrated for the first time in solution state, that water molecules are involved in the specific recognition of sugars by concanavalin A (Ref 2). It appears that lectin-sugar recognition reactions are, in general, mediated by a net uptake of water molecules during the binding process (Ref 7). 5. We have provided the first experimental demonstration that reorganization of water molecules is the source of enthalpy-entropy compensation in molecular recognition processes (Ref 3). 6. We provide evidence for another facet in the recognition of antigens by antibodies, viz water release accompanying the binding reaction (Ref 4). The studies reported in this thesis provide the foundation for embarking on a systematic study not only of the origin of specificity of lectin-sugar recognition but also of the complex roles that water molecules play in mediating these molecular recognition processes. These specific binding reactions wherein non-linear thermodynamics predominates and precludes a direct structure-energetics correlation emphasize the need to account for the effect of solvent water molecules in lectin-sugar interactions in particular and, without any overemphasis, in molecular recognition processes in general.

The work embodied in this thesis pertains to an attempt to understand better, the molecular basis of protein-carbohydrate recognition. For this purpose a systematic study was undertaken, not only of the energetics of lectin-sugar interactions, which serve as molecular recognition prototype of protein-carbohydrate interactions, but also of the complex effects of solvent water molecules surrounding both the species in solution state. The systems chosen for investigation include the specific recognition of sugars by lectins from diverse families, leguminosae and moraceae. The following salient aspects of the molecular recognition process constitute the focus of this thesis: • Effect of site specifically modified, deoxy-, fluorodeoxy-, or methoxy- substituted D-galactopyranoside binding to lectins. Isothermal titration calorimetric (ITC) investigations of the binding of these sugars to a model lectin permitted the correct prediction of the architecture of the primary binding site in the absence of x-ray crystal or NMR structure of the combining site (Ref. 7). The study provided the only unambiguous means of a site specific mapping of the hydrogen-bond donor- acceptor relationship of the monosaccharide within the primary combining site of the lectin. • Novel features of lectin-sugar recognition. Molecular interactions and forces contributing to the stabilization of the saccharides in the primary combining site of lectins. Binding of site specifically modified fluoro- substituted D- galactopyranosides to WBA I led to the demonstration of the involvement of C- F««»H-0 hydrogen bonds in stabilizing the saccharide within the combining site of lectin (Ref. 7). Implication of the novel C-H«**O hydrogen bonds at the specificity determining C-4 position in enabling the methoxy- substituted D- galactopyranoside to be stabilized within the primary binding site of galactose specific lectins WBA I and jacalin. • Development of a novel coupled osmotic-thermodynamic approach for investigating the role of water molecules in determining the specificity of lectin- sugar interactions. The results obtained led to the first direct demonstration of a differential uptake of water molecules accompanying the specific process of recognition of sugars by lectins (Ref 2) • On the origin of enthalpy-entropy compensation, a ubiquitous phenomenon accompanying the thermodynamics of several ligand binding reactions in aqueous solutions in general and the molecular recognition involving all known lectin-sugar interactions, in particular. The results provide the first unequivocal solution state proof of water reorganization as the source of enthalpy-entropy compensation (Ref 3). A new diagnostic test of a true osmotic effect in molecular recognition phenomena was proposed (Ref. 2) and validated (Ref. 3). As an introduction, Chapter 1 is a comprehensive review of literature that touches upon the diverse properties of lectins and our present understanding of their multifarious roles and applications, which has led to their christening, perhaps appropriately, as molecules that mediate the 'social' functions of cells and tissues. Although a challenge it is still, to decipher the "glycocode", it is apparent that the fundamental basis of the recognition function of lectin-sugar interactions is the initial specific binding of the saccharide molecule by the globular proteinaceous lectin molecule. It is imperative, therefore, that an incisive investigation of the origin of specificity of the binding reaction as well as the solvent effects influencing both the interacting species be undertaken for a better understanding of the complete molecular recognition process. Towards this end is introduced in Chapter 1 our present understanding of the results on lectin-sugar interactions from two complementary approaches viz structural, including X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, as well as thermodvnamic ones, which have provided important information on the architecture of the combining sites, the dynamic modes of saccharide recognition and forces involved therein. Despite a detailed knowledge available from such methods, a structure-energetics correlation has persisted as a current challenge of the field. Towards achieving this goal, studies on the energetics of the recognition of sugars by lectins were undertaken, with an aim to better understand the origin of specificity of lectin-sugar interactions. This thesis attempts to provide new insights on some of the possible lacunae precluding structure-energetics correlation and suggests ways to overcome them. Chapter 2 deals with ITC investigation of the effect of deoxy-, fluorodeoxy-, and methoxy- substitutions on the binding of monosaccharides to the primary combining site of the lectin WBA I isolated from the mature seeds of the leguminosae family member Psophocarpus tetragonolobus as well as the moraceae lectin jacalin. These studies provide valuable information on the hydrogen-bond donor-acceptor relationships within the combining site of the lectins wherein the sugar molecule is liganded with the amino-acid residues of the lectin. This study is relevant for understanding the origin of specificity of monosaccharide binding within the primary combining site of the lectins. It has recently become apparent that there is a predisposition in three-dimensional space, of the donor-acceptor pairs within the sugar binding site of the lectins. Hence there appears to be a stereochemical basis of distinguishing the recognition of the donor group vis-a-vis that of the acceptor group and that their spatial disposition determines the specificity of the saccharide recognition. Unambiguous assignment of which of the groups within the hydrogen bonded pairs is a donor and which one is the acceptor assumes greater importance. The ITC measurements of the binding of deoxy-, flurodeoxy-and methoxy-derivatives of D-galactopyranoside (oc-D-Gal) to the basic lectin from winged bean Psophocarpus tetragonolobus, WBA I revealed that each of the ligands bind to WBA I with the same stoichiometry of one per subunit (29 kDa) of WBA I. The binding enthalpies for various derivatives were essentially independent of temperature and showed complementary changes with respect to binding entropies. Replacement of the hydroxyl group by fluorine or hydrogen on C3 and C4 of the galactopyranoside eliminated their binding to the lectin, consistent with C3-OH and C4-OH acting as hydrogen bond donors. The affinity for C2 derivatives of galactose decreased in the order: GalNAc>2MeOGal>2FGal=Gal>2HGal which suggests that both polar and non-polar residues surround the C2 locus of galactose, consistent with the observed high affinity of WBA I towards GalNAc, where the acetamido group at C2 position is probably stabilized by both non-polar interactions with the methyl-group and polar interactions with the carbonyl group. The binding of C6 derivatives followed the order: Gal>6FGal>D-Fuc»6MeOGal=L-Ara indicating the presence of favourable polar interactions with a hydrogen bond donor in the vicinity. Based on these results the hydrogen bond donor-acceptor relationship of the complexation of methyl-a-D-galactopyranoside with the primary combining site of WBA I was proposed (Ref. /), which was subsequently validated by the crystal structure of methyl-a-D-galactopyranoside complexed with WBA I. This chapter also describes the results from ITC studies on the binding of monosaccharides and disaccharides to the lectin jacalin isolated from the mature seeds of the moraceae family member Artocarpus integrifolia. The novel observation about the existence of C-F*«*H-0 and C-H**»O hydrogen bonds in lectin-sugar interactions is also discussed in this chapter. Chapter 3 is a description of the detailed investigation on the role of water molecules in influencing the energetics of lectin-sugar recognition. A novel coupled osmotic-thermodynamic approach was developed to dissect the role of water molecules in determining the recognition of the sugars by lectins. For this purpose, the model system of mannotriose-concanavalin A was used because atomic level structural information on these complexes were available. The work described in this chapter, is the first solution state evidence for the role of water molecules in the specific interaction of carbohydrates with a legume lectin, concanavalin A (Con A) (Ref. 2). Sugar binding to Con A was accompanied by linear changes in the logarithm of binding constants as a function of neutral osmolyte strength, and were described by well defined negative slopes characteristic for each sugar. As these changes were independent of the chemical nature of the osmolyte used, the results were rationalized in terms of a true osmotic effect. It was demonstrated that the specific recognition of the branched trimannoside (3,6-di-0-(a-D-mannopyranosyl)~a-D-mannopyranoside), the individual dimannosidic arms (3-<9-(a-D-mannopyranosyl)-a-D-mannopyranoside, and 6-0-(a-D-marmopyranosyl)-a-D-mannopyranoside) and the monomeric unit D-mannopyranoside by Con A was accompanied by differential uptake of water molecules; 1,3 and 5 respectively. We also observed a conservation of the compensatory behaviour of binding enthalpies and entropies in the presence as well as absence of osmolytes. This provided the first definitive evidence that water-reorganization plays a direct role in effecting the phenomenon of enthalpy-entropy compensation in protein-ligand interactions in general and lectin-sugar interactions in particular, and that the specificity of lectin-sugar recognition is characterized by a differential uptake of water molecules. Chapter 3 also describes the first experimental identification of the origin of enthalpy-entropy compensation (EEC), a ubiquitous phenomenon accompanying the thermodynamics of multifarious biomolecular recognition processes. By coupling direct microcalorimetry with osmotic stress technique, an experimental handle was devised to test the hypothesis that solvent reorganization could be the source of EEC. The results provided an unequivocal demonstration that an osmotic change in water activity alone, at the same temperature and pH, is sufficient to result in the conservation of EEC during the molecular recognition of specific ligands by macromolecules belonging to thermodynamically diverse and unrelated systems, a compelling evidence that the primary source of EEC in aqueous solutions is attributable to reorganization of solvent water molecules, thus validating the test for the role of water reorganization as a source of EEC (Ref. 3). This provides the first definitive evidence for the notion that there is a direct involvement of water molecules in originating the EEC effect. Despite the generality of the results it is urged that several systems be subjected to a vigorous application of the coupled osmotic-thermodynamic approach proposed herein before constituting it as a proof. Suffice to say, it is perhaps heartening that at last one has a handle to test the role of water molecules in effecting EEC in the solution state and appreciate the diverse roles played by water molecules in mediating molecular recognition reactions. The proposal presented in Ref 2, that the strong isoequilibrium relationship of enthalpy with entropy during the recognition of saccharides by Con A studied under osmotic stress, be considered as diagnostic of a true osmotic effect was subsequently validated in a thermodynamically diverse and unrelated system of peptide recognition by monoclonal antibody, the results from which are discussed in an Appendix (A) to this thesis (Ref 4). That the stabilities of these lectins are not hampered in the presence of osmolytes was demonstrated using differential scanning calorimetry (DSC) (Ref 2). During the course of these DSC studies, we discovered an unusual feature in an animal galectin. Despite possessing the legume lectin fold, the 14-kDa S- type lectin exhibits multiple oligomeric states that are influenced profoundly by complementary ligands and surprisingly do not dissociate at the denaturation temperature. These results are discussed in an Appendix (B) to this thesis (Ref. 5). The general discussion and conclusions drawn from this work are summarized in chapter 4. Briefly, the following salient conclusions can be drawn from the work presented in this thesis: 1. Unambiguous assignment of hydrogen-bond donor-acceptor relationship at each of the hydroxyl group of the monosaccharide bound to the lectin belonging to different families has been demonstrated (Refs. 1,6). 2. First report of novel hydrogen bonds in lectin-sugar interactions such as C- F«MH-0 (Ref 1) and C-H^*O hydrogen bonds (Ref 6). 3. Unusual structural stabilities in a galectin with a fold similar to that in legume lectins but with starkly different thermodynamic stabilities (Ref 5). 4. We have demonstrated for the first time in solution state, that water molecules are involved in the specific recognition of sugars by concanavalin A (Ref 2). It appears that lectin-sugar recognition reactions are, in general, mediated by a net uptake of water molecules during the binding process (Ref 7). 5. We have provided the first experimental demonstration that reorganization of water molecules is the source of enthalpy-entropy compensation in molecular recognition processes (Ref 3). 6. We provide evidence for another facet in the recognition of antigens by antibodies, viz water release accompanying the binding reaction (Ref 4). The studies reported in this thesis provide the foundation for embarking on a systematic study not only of the origin of specificity of lectin-sugar recognition but also of the complex roles that water molecules play in mediating these molecular recognition processes. These specific binding reactions wherein non-linear thermodynamics predominates and precludes a direct structure-energetics correlation emphasize the need to account for the effect of solvent water molecules in lectin-sugar interactions in particular and, without any overemphasis, in molecular recognition processes in general.

Purpose: To investigate the differential effects of solutions providing varying concentrations of carbohydrate and/or protein ingested between 200-meter sprints on sprint time. Subjects: Recruitment was from the Georgia State University track and field team. Methods: The study protocol was approved by the Georgia State University IRB. Ten subjects, 18 to 21 years of age, consented to be included in the study. Nine subjects (7 females; 2 males) completed trial 1, six subjects (5 females; 1 male) completed trial 2, and three subjects (2 females; 1 male) completed the final trial. Each trial consisted of a 200-meter sprint followed by the immediate ingestion of a post-exercise recovery beverage within the first fifteen minutes of a one-hour recovery period. Following the one-hour of recovery, subjects sprinted a second 200-meter sprint. Beverage solutions were formulated to contain 1.2 g of protein (PRO), 1.2 g carbohydrate (CHO), or 1.2 g carbohydrate with protein (CHO/PRO) per kg of subject body weight. Using a single blind, non-randomized design, subjects received the same recovery beverage in each trial. Each trial consisted of either PRO (trial 1), CHO (trial 2), or CHO/PRO (trial 3), with one week separating trials. Sprint times were recorded in seconds and ten hundredths of a second using a manual, digital stopwatch. Results: During PRO, two subjects sprinted faster (x= -.25 sec), three subjects saw no change in sprint time, and four subjects sprinted slower (x= +.98 sec). During CHO, two female subjects sprinted faster between sprints (x= -.85 sec); and all other subjects (n=4) sprinted slower (x= +.73 sec). During CHO/PRO, no subjects sprinted faster from sprint 1 to sprint 2 (x= +.33 sec) Conclusions: Post-exercise nutritional supplementation effects varied among subjects, with some subjects performing better following PRO, while others experiencing improvements with CHO. In general, subjects performed better following consumption of the CHO beverage. Of those who ran faster between sprints, the CHO beverage resulted in an average improvement of -.85 sec, while the PRO beverage resulted in an average improvement of -.25 sec. On average, CHO resulted in faster 2nd sprints (x= +.20 sec) than the PRO beverage (x= +.47 sec) or the CHO/PRO beverage (x= +.33 sec). Continued research in this population is necessary for elucidation of study results. This investigation may serve as the foundation for future, related studies.

It has been well established that both carbohydrate-loading before and carbohydrate ingestion during exercise can enhance endurance performance by supplying carbohydrate for oxidation. However, the precise mechanism(s) underlying the proposed ergogenic effects of these procedures remain to be established. The studies in this thesis were therefore designed to examine the effects of carbohydrate-loading and carbohydrate ingestion on fuel substrate kinetics.

Inhibiting the formation of ice is an essential process commercially, industrially, and medically. Compounds that work to stop the formation of ice have historically possessed drawbacks such as toxicity or prohibitively high active concentrations. One class of molecules, ice recrystallization inhibitors, work to reduce the damage caused by the combination of small ice crystals into larger ones. Recent advances made by the Ben lab have identified small molecule carbohydrate analogues that are highly active in the field of ice recrystallization and have potential in the cryopreservation of living tissue. A similar class of molecules, kinetic hydrate inhibitors, work to prevent the formation of another type of ice – gas hydrate. Gas hydrates are formed by the encapsulation of a molecule of a hydrocarbon inside a growing ice crystal. These compounds become problematic in high pressure and low temperature areas where methane is present - such as an oil pipeline. A recent study has highlighted the effects of antifreeze glycoprotein, a biological ice recrystallization inhibitor, in the inhibition of methane clathrates. Connecting these two fields through the synthesis and testing of small molecule ice recrystallization inhibitors in the inhibition of methane hydrates is unprecedented and may lead to a novel class of compounds.

L’objectiu d’estudi de la tesi és el desenvolupament de nous mètodes sintètics per a la l’obtenció d’oligosacàrids i tio-anàlegs mitjançant processos polimèrics d’obertura d’anell. Aquestes estructures es poden trobar en una gran varietat de productes naturals on formen part de substàncies biològicament actives. D’aquesta manera, s’han desenvolupen rutes sintètiques per l’obtenció de carbohidrats epóxidats, episulfurats, carbonatats i tiocarbonatats. Els monòmers sintetitzats han estat sotmesos a diferents reaccions d’oligomerització. Curiosament, la cerca de nous mètodes de síntesi d’epitio-carbohidrats va donar lloc al descobriment de sucres 1,3-oxazolin-2-tioderivats. Aquests nous compostos han esdevingut inhibidors altament específics de β-glucosidases amb un gran potencial contra la malaltia de Gauche. Els diferents estudis d’inhibició es troben recollits en la memòria.
The final goal of this thesis is the development of strategic methods fro the synthesis of well-defined 1,2-linked oligosaccharides and S-linked thio-analogues through different polymerization techniques. These structures form a complex group of biomolecules with an unsurpassed structural diversity, performing a variety of biological functions. In this context, the present work aimed to develop new procedures in carbohydrate chemistry, focusing in the oligomerization reactions. Thus, efficient syntheses have been studied for accessing the suitable carbohydrate based monomers (including epoxides, episulfides, carbonates and tiocarbonates). Unexpectedly, the synthesis of epithiocarbohydrates afforded carbohydrate-derived 1,3-oxazolidine-2-thiones. These compounds have proved to be quite attractive as new enzyme inhibitors for Gaucher disease. All enzyme inhibition studies can be found in the manuscript.

It has long been observed that some organo-fluorine compounds exhibit enhanced biological activity over their non-fluorinated precursors, however reasons for these unusual properties still remain poorly understood. An explanation which has been widely used relates to the ability of the C-F fragment of the analog to participate in hydrogen-bonding interactions with its protein receptor. For this reason, fluorinated carbohydrates have been used as hydrogen-bonding probes with a number of proteins. Thus there exists a need for a systematic investigation into the hydrogen-bonding ability of the C-F fragment, and the enzyme glycogen phosphorylase provides an excellent subject for such a study. The glucopyranose binding site in the inactive (T-state) conformation of the enzyme has been well characterised and high resolution crystallographic data is available. Thus by comparison of kinetic and crystallographic data for the natural effectors and the fluorinated substrate analogs considerable insight into the hydrogen bonding ability of the C-F fragment and the nature of carbohydrate-protein interactions should be gained. Little is known about the active (R-state) conformation of the enzyme and about the T-state to R-state transition. Use of fluorinated analogs of the enzymes natural substrate, glucose-l-phosphate, could also shed light on these questions. With these aims in mind, all of the isomeric mono-fluorinated derivatives of glucose and glucose-l-phosphate have been synthesised. Some deoxy and difluorinated analogs of glucose and mannose have also been prepared. Kinetic results obtained using the analogs of glucose indicate that the 3 and 6 positions of the sugar participate in strong hydrogen-bonding interactions with the protein while the other positions are only involved in relatively weak interactions. These results agree well with recent X-ray crystallographic data. None of the analogs of glucose-l-phosphate exhibited any substrate activity. The 2-deoxyfluoro analog had a similar affinity to glucose-1-phosphate and therefore probably binds in the same mode. The lack of substrate activity in this case can be explained by the destabi1isation of the putative oxo-carbonium ion intermediate at C(l), by the adjacent fluorine substituent. The other analogs of glucose-l-phosphate showed lower affinity for the enzyme. The similar inhibition constants obtained for these compounds suggested a binding mode in which the glucopyranose ring contributes little to the overall binding energy. This has led to the proposal of a molecular mechanism for the T-state to R-state transition.
Science, Faculty of
Chemistry, Department of
Graduate

The rapid detection of pathogenic organisms to ensure appropriate administration of treatment remains a global healthcare challenge. This is becoming increasingly difficult, as identification of the organism alone is no longer enough, with the rise of drug resistance amongst many pathogens it is becoming increasingly important that both the pathogen and drug resistance are identified. Currently, rapid identification can be achieved through a variety of techniques. However, many of these techniques are expensive, require extensive sample preparation, or highly trained personnel to run with results often not rapidly available. This leaves health care professionals to make point-of-care treatment decisions based on symptoms without any indication of drug resistance. The use of carbohydrate microarrays for pathogen detection has been identified as both a method for detection but also as a basis for identifying new drug targets. This exploits the initial protein-carbohydrate interaction that many pathogens utilise in the initial stages of infection. However, the use of microarrays is also challenging, as highly sensitive identification of pathogens often requires expensive or synthetically challenging oligosaccharides or coupling with a highly sensitive detection method thus limiting its point of care application. Herein we describe the coupling of a facile surface chemistry for glycan addition with a powerful statistical algorithm to improve the sensitivity of a cheap monosaccharide functionalised surface without using expensive detection methodologies. This technique was then applied to the detection and identification of toxic lectins, bacterial samples and finally the life-stage specific detection of Plasmodium falciparum (one of the parasites responsible for human malaria). In this last case, drug resistance related to carbohydrate binding profile was also observed.

The macrotricyclic system as a receptor for carbohydrates in organic as well as aqueous media has been extensively studied within the Davis group. The hydrogen bonding and CH-π interactions are the two main non covalent interactions operating for the recognition of carbohydrates in our system. In principle higher affinities could be achieved by enhancing either of these types of interactions. The possibility of enhancing H-bonding interactions with host-guest was explored by the introduction of heterocyclic linkers in the 'temple' receptors. Macrocycle with pyrrole linker 132 and pyridine linker 131 (Figure i) were synthesised and studied for their affinities with different carbohydrate substrates. Pyrrole macrotricycle 132 bound D- Glucose selectively over other substrates with K; = 18 M-1. However pyridine macrotricycle 131 did not show selectivity and higher affinities with any substrates. Figure i. Macrotricycle with pyridine linker 131 and pyrrole linker 132. In collaboration with Roelens group in Firenze, Italy, a series of water soluble C3-symmetric receptors were synthesized and studied for their affinity for carbohydrates. Unfortunately, these series of receptors did not bind the substrates strong enough to be detected by lH NMR titration studies.