[Truncated abstract] The development of several carbohydrate-based pharmaceuticals has stimulated an increased interest in the field of carbohydrate chemistry. The discovery of Acarbose and invention of Miglitol, treatments for type II diabetes, as well as the influenza treatments, Relenza and Tamiflu, have been largely responsible for this increased interest. These treatments operate by the inhibition of glycoside hydrolases, a group of enzymes important in a variety of biological processes. This thesis involves the study of a group of glycoside hydrolase inhibitors known as azasugars, which are nitrogen-containing sugar mimics . . . The final chapter, Chapter 4, focuses on the testing of these disaccharides as a possible alternative carbohydrate source for pre-term infants. Initially, commercially available glycoside hydrolases were used to detect any hydrolysis of the four disaccharides, with (206) exhibiting the most promising results (to provide D-glucose and D-galactose). Detailed kinetic studies were then conducted using homogenates obtained from pig intestinal mucosa. Unfortunately, the results indicated that (206) was unsuitable as an alternative carbohydrate source for pre-term infants.

No / Aqueous solutions of hen egg lysozyme (3% w/v) were dispersed and precipitated by a homogenous mixture of supercritical carbon dioxide-ethanol using the Solution Enhanced Dispersion by Supercritical fluid (SEDS) process. The effects of different working conditions, such as temperature, pressure and the flow rates of the solution and ethanol, on the particle-formation process were studied The morphology, particle size and size distribution and biological activity of the protein were determined The precipitates were examined with high-sensitivity differential scanning calorimetry (HSDSC) and high-performance cation-exchange chromatography Particle size measurements showed the precipitates to be aggregates with primary particles of size 1-5 ¿m. The similarity of HSDSC data for unprocessed and processed samples indicated that the different physical forces that stabilise the native form of lysozyme are unchanged after SEDS processing. From FT-Raman spectroscopic studies secondary structural changes were observed in certain SEDS-produced lysozyme, with most processed samples displaying a slightly more disordered secondary structure than the unprocessed sample However, SEDS samples produced at 200 bar and 40 C exhibited negligible disturbance Thus the SEDS process utilising aqueous solution was able to bring about size reduction of lysozyme with minimal loss of biological activity.

Fung Pik Ha. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 116-121). / Acknowledgments --- p.i / Table of Contents --- p.ii / List of Figures --- p.v / List of Tables --- p.x / Abstract --- p.xi / Chapter Chapter I --- Introduction --- p.1 / Chapter Chapter II --- Literature Reviews / Chapter II.l --- Glycosidase --- p.3 / Chapter II.2 --- Biosynthesis of N-linked Glycoprotein --- p.4 / Chapter II.3 --- Mechanism of Enzyme Catalysed Reaction --- p.8 / Chapter II.4 --- Types of Glycosidase Inhibitors --- p.12 / Chapter II.5 --- Cyclophellitol and Aminocyclitols / Chapter II.5.1 --- General background on cyclophellitol --- p.17 / Chapter II.5.2 --- Mode of inhibition of cyclophellitol --- p.20 / Chapter II.5.3 --- General background on aminocyclitols --- p.24 / Chapter Chapter III --- Characterization of Synthetic Glycosidase Inhibitors / Chapter III.1 --- Covalent-based Inactivator (Cyclophellitol and its Analogues) / Chapter III.1.1 --- Introduction --- p.28 / Chapter III.1.2 --- Materials --- p.32 / Chapter III.1.3 --- Methods / Chapter III.1.3.1 --- Inhibitory assay of commercially available glycosidases --- p.33 / Chapter III.1.3.2 --- Partial purification of β-D-mannosidase from A. oryzae --- p.34 / Chapter III.1.3.3 --- Protein assay in purification of β-D-mannosidase --- p.38 / Chapter III.1.3.4 --- Inhibitory assay for partially purified β-D- mannosidase (A . oryzae) --- p.38 / Chapter III.1.3.5 --- Influence of dialysis on glycosidase inhibition --- p.39 / Chapter III.1.3.6 --- Inactivation experiment on glycosidases --- p.39 / Chapter III.1.4 --- Results / Chapter III.1.4.1 --- Inhibitory activities of cyclophellitol and its analogues against glycosidases --- p.41 / Chapter III.1.4.2 --- Effect of dialysis on glycosidase inhibition --- p.44 / Chapter III.1.4.3 --- The kinetic studies of glycosidase inactivation --- p.47 / Chapter III. 1.5 --- Discussion --- p.50 / Chapter III.1.6 --- Further studies --- p.55 / Chapter III.2 --- Reversible Competitive Inhibitors (Aminocyclitols) / Chapter III.2.1 --- Introduction --- p.56 / Chapter III.2.2 --- Materials --- p.58 / Chapter III.2.3 --- Methods / Chapter III.2.3.1 --- Assay of glucoside hydrolase inhibition activity --- p.60 / Chapter III.2.3.2 --- Glucose oxidase method for determination of released D-glucose --- p.60 / Chapter III.2.3.3 --- Inhibitory assay of aminocyclitols on other glycosidases --- p.61 / Chapter III.2.3.4 --- Influence of dialysis on the glycosidase inhibition --- p.62 / Chapter III.2.3.5 --- Lineweaver-Burk plot --- p.63 / Chapter III.2.4 --- Results / Chapter III.2.4.1 --- Inhibitory activities of valiolamine and related aminocyclitols against six glycosidases --- p.64 / Chapter III.2.4.2 --- Characterization the aminocyclitols as reversible competitive inhibitors --- p.69 / Chapter III.2.5 --- Discussion --- p.80 / Chapter Chapter IV --- Isolation of the Naturally Occurring Glycosidase Inhibitor from Mushrooms / Chapter IV.1 --- Introduction --- p.83 / Chapter IV.2 --- Materials --- p.84 / Chapter IV.3 --- Methods / Chapter IV.3.1 --- Preparation of Ganoderma lucidum --- p.86 / Chapter IV.3.2 --- Preparation of V. volvacea --- p.86 / Chapter IV.3.3 --- Inhibitory assay of aqueous extract of mushrooms on glycosidases --- p.87 / Chapter IV.3.4 --- Anthrone method for determination of reducing sugars --- p.87 / Chapter IV.3.5 --- Flash liquid chromatography for purification of putative inhibitors in G. lucidum --- p.88 / Chapter IV.4 --- Results / Chapter IV.4.1 --- Prescreening of Inhibitory effects of Various Fungal Extracts --- p.90 / Chapter IV.4.2 --- Inhibitory Effects of Partially Purified G. lucidum Extract on Glycosidase --- p.92 / Chapter IV.4.3 --- Effect of Endogenous Substrates on Glycosidase Activities --- p.93 / Chapter IV.4.4 --- Results of Liquid Column Chromatography --- p.93 / Chapter IV.4.5 --- Structure Determination and Characterization of purified compounds --- p.95 / Chapter IV.4.6 --- Inhibitory Activities of Compounds A and B against Brewers yeast a- glucosidase --- p.96 / Chapter IV.5 --- Discussion --- p.98 / Chapter Chapter V --- Conclusions --- p.113 / References --- p.116

Os carboidratos são moléculas diversas e complexas que são empregadas por organismos vivos em funções biológicas, como eventos energéticos, estruturais e de sinalização. L-Fucose é um monossacarídeo presente em diversos grupos biológicos, como mamíferos, insetos e plantas. Umas das modificações póstraducionais mais conhecidas contendo L-Fucose são os antígenos do sistema sanguíneo ABO e o leite humano. Alterações no padrão de fucosilação estão relacionadas a patologias como câncer e fucosidose. Essas alterações estão relacionadas ao balanço entre as atividades de α-L-fucosidases e fucosiltransferases. As -L-fucosidases são glicosídeo hidrolases que catalisam a hidrólise de ligações entre resíduos de L-Fucose ligados a outras moléculas. Fucosiltransferases são glicosiltransferases que transferem L-Fucose de GDPfucose para um substrato receptor específico. Embora a importância do metabolismo da fucose, existem poucas informações sobre este assunto em Arthropoda. A literatura indica que fucose, α-L-fucosidases e fucosiltransferases estão envolvidas na interação parasita-hospedeiro em carrapatos, sugerindo que o metabolismo da fucose é essencial para Arachnida. Nosso objetivo foi estudar o metabolismo de fucose em duas espécies de Arachnida: a aranha Nephilingis cruentata e o carrapato Amblyomma sculptum. Ele foi realizado através da caracterização das fucosidases nativas e recombinantes, estrutura e análise in silico, mutagênese sitio-dirigida, padrão de expressão, especificidade e efeito nas células tumorais. Além disso, analisamos as sequências de fucosiltransferases e a expressão por qPCR. As enzimas envolvidas em caminhos metabólicos de fucose também foram investigadas. NcFuc e AsFuc têm um pH ótimo de 5, são inibidas por fucose e fuconojirimicina e apresentam processo de oligomerização dependente de pH. Nós produzimos com sucesso as formas recombinantes dessas enzimas, e elas apresentam as mesmas propriedades cinéticas das formas nativas. A hidrólise de substratos naturais por NcFucr e AsFucr sugerem especificidades diferentes, e ambas conseguiram remover resíduos de fucose de celulares tumorais, reduzindo a invasão celular. Além disso, elas catalisaram reações de transfucosilação. A produção recombinante permitiu a identificação dos resíduos D214 e E59 como díade catalítica em NcFuc. As análises filogenéticas, os dados cinéticos, a modelagem molecular e a especificidade sugerem que os sítios ativos das α-Lfucosidases são diferentes em cada espécie de Arachnida e indicaram que o significado fisiológico da remoção de fucose é diferente entre os organismos. Os ensaios de qPCR evidenciaram que, embora as α-L-fucosidases possam ser enzimas lisossômicas, elas são principalmente expressas no sistema digestório em Arachnida e estão envolvidas na digestão. Representantes de todas as famílias conhecidas de fucosiltransferases foram identificados nos dados do transcriptoma de aranha. No entanto, POFUT1 também foi identificado no nível proteômico e sua análise de expressão indicou uma maior expressão no MG. Isso pode estar relacionado à regeneração de células após a secreção de enzimas digestivas. A análise transcriptômica e proteômica indica que o Arachnida usa vias salvage e de novo para a síntese de fucose. Considerando todos os dados obtidos, concluímos 11 que o metabolismo da fucose está relacionado à digestão em Arachnida, uma vez que eles podem obter a fucose da dieta devido à presença de α-L-fucosidases muito ativas. / Carbohydrates are diverse and complex molecules being employed by living organisms in biological functions such as energetic, structural and signalling events. L-Fucose is a monosaccharide component of many glycans present in a variety of biological groups, such as mammals, insects, and plants. Some of the best-known examples of post-translational modified molecules containing L-Fucose are the ABO blood antigen system and human milk. Changes in the fucosylation pattern are related to pathologies like cancer and fucosidosis. These changes are related to the balance between the activities of α-L-fucosidases and fucosyltransferases. α-Lfucosidases are glycoside hydrolases that catalyse the hydrolysis of glycosidic bonds between residues of L-Fucose to other molecules. Fucosyltransferases are glycosyltransferases which transfer L-Fucose from a GDP-fucose to a specific acceptor. Although the importance of fucose metabolism, there is few information about this subject in Arthropoda. Literature indicates that fucose, α-L-fucosidases and fucosyltransferases are involved in host-pathogen interaction in ticks, suggesting that fucose metabolism is essential to Arachnida. Our aim was to study the metabolism of fucose in two Arachnida species: the spider Nephilingis cruentata and the tick Amblyomma sculptum. This was accomplished through the characterization of native and recombinant fucosidases, structure and in silico analyses, site-directed mutagenesis, expression pattern, specificity and, effect on tumour cells. Besides that, we analysed fucosyltransferases sequences and expression by qPCR. The enzymes involved in fucose metabolic pathways were also investigated. NcFuc and AsFuc have a pH optimum of 5.0, are competitively inhibited by fucose and fuconojirimycin and present an oligomerisation process pH dependent. We successfully produced the recombinant form of these enzymes, and they have the same kinetic properties of native forms. Natural substrate hydrolysis by NcFucr and AsFucr suggest different specificities and they were able to remove fucose residues from tumour cell lines, reducing cell invasion. Moreover, they catalysed transfucosylation reactions. The recombinant production allowed the identification of the residues D214 and E59 as the catalytic dyad in NcFuc. Phylogenetic analysis, kinetic data, molecular modelling, and specificity assays suggest that α-L-fucosidase active sites are different to each Arachnida species and indicated that the physiological significance of fucose removal is different among organisms qPCR assays evidenced that although fucosidases might be lysosomal enzymes, they are mainly expressed at the digestive system in Arachnida and are involved in digestion. Representatives of all known families of fucosyltransferases were identified in the spider MG transcriptome data. However, POFUT1 was also identified at the proteomic level and its expression analysis indicated a higher expression at MG. This might be related to the regeneration of cells after secretion of digestive enzymes. Transcriptomic and proteomic analysis indicate that Arachnida uses both salvage and de novo pathways to fucose synthesis. Considering all the obtained data we concluded that fucose metabolism is related to digestion in Arachnida since they are able to salvage fucose from diet due to the presence of very active α-L-fucosidases.

The major beta-hexosaminidase isozymes in humans are Hex A (deficient in Tay-Sachs disease, TSD), an alphabeta heterodimer and Hex B (deficient in Sandhoff disease) a betabeta homodimer. Hex S, the alphaalpha homodimer is physiologically unstable. Mature alpha and beta subunits share 60% sequence identity. The beta subunit active site hydrolyzes neutral substrates. The alpha subunit active site hydrolyzes neutral (4MUG) and charged substrates (4MUGS, GM2 ganglioside). Only Hex A hydrolyzes the natural substrate, GM2 ganglioside, in the presence of the GM2 activator protein (AP). / We investigated regions of the alpha and beta subunits involved in AP binding, subunit dimerization, and substrate specificity. We constructed four chimeric cDNAs: alpha1--259beta292--544 , alpha1--118beta152--544, beta 1--418alpha387--529, and beta1--151 alpha119--259beta292--544 (subscripts refer to amino acid residues). Chimeric cDNAs were expressed in a TSD neuroglial cell line, which produces no endogenous alpha subunits. The chimeric isozymes were chromatofocused and assayed for hydrolysis of (a) 4MUG, (b) 4MUGS and (c) GM2 ganglioside. / Transfection of the cDNA constructs lead to expression of homodimeric and heterodimeric chimeric proteins, albeit at lower yields than transfection of wild alpha-cDNA. All of the chimeric proteins hydrolyzed 4MUG but none were active towards 4MUGS or GM2 ganglioside. These results suggest that (a) all constructs contained sufficient information to form both heterodimeric and homodimeric chimeric proteins, (b) the chimeras lacked the alpha-subunit sequence necessary for the hydrolysis of charged substrates.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 5 uppsatser.

The exoglucanase from Cellulomonas fimi catalyses the hydrolysis of cellobiose units from the non-reducing terminus of cello-oligosaccharides with overall retention of anomeric configuration. Its mechanism of action is therefore thought to involve a double displacement reaction, involving as the first step, formation of a glycosyl-enzyme intermediate (glycosylation) and as a second step, the hydrolysis of this intermediate (deglycosylation). This mechanism is investigated here through the study of the kinetics of hydrolysis of aryl β-glucosides and aryl β-cellobiosides and by employing the mechanism-based irreversible inactivators, 2', 4'-dinitrophenyl 2-deoxy-2-fluoro-β-D-glucoside (2F-DNPG) and 2", 4"-dinitrophenyl 2-deoxy-2-fluoro-β-D-cellobioside (2F-DNPC). The study with the aryl β-glucosides revealed that this enzyme is indeed active on glucosides, a feature that had previously been undetected. A linear relationship was found to exist between the logarithm of Vmax for hydrolysis and the phenol pKa as well as between the logarithm of Vmax/Krn and me phenol pKa, showing that glycosylation is both the rate determining step and the first irreversible step for all substrates. The reaction constant calculated, ρ = 2.21, indicates a considerable amount of charge build up at the transition state of glycosylation. The linear free energy relationship study of the aryl β-cellobiosides revealed no significant dependence of the logarithm of Vmax on the pKa of the phenol, indicating that deglycosylation is rate determining. However, the slight downward trend in this Hammett plot at higher pKa values may suggest that the rate determining step is changing from deglycosylation to glycosylation. However, the logarithm of Vmax/Km does correlate with the pKa of the phenol, thus showing that the first irreversible step is glycosylation. The reaction constant (ρ = 0.60) which reflects the development of charge at the glycosylation transition state for the cellobiosides is less than that calculated for the glucosides, thus suggesting a glycosylation transition state with either a greater degree of acid catalysis or less C-O bond cleavage than that for the glucosides. The inactivators, 2F-DNPC and 2F-DNPG, are believed to inactivate the exoglucanase by binding to the enzyme and forming covalent glycosyl-enzyme intermediates. The inactivated-enzyme was stable in buffer but reactivated in the presence of a suitable glycosyl-acceptor such as cellobiose, presumably via a transglycosylation reaction. These results indicate that covalent 2F-glycosyl-exoglucanase intermediates are stable and are catalytically competent to turn over to product, thus supplying further evidence for the Koshland mechanism. The exoglucanase is inactivated more rapidly by 2F-DNPC than by 2F-DNPG. However, both inactivated forms of the enzyme reactivated at comparable rates in the presence of cellobiose, showing that the second glucosyl unit present on the cellobiosides increases the rate of glycosylation relative to that found for the glucosides but not the rate of deglycosylation. The stable covalent nature of the 2F-glycosyl-enzyme intermediates provided an excellent opportunity to identify the enzymic nucleophile. This was accomplished by radiolabelling the exoglucanase with a tritiated analogue of 2F-DNPG cleaving the protein into peptides and purifying the radiolabelled peptides. Sequencing of this peptide resulted in the identification of the active site nucleophile as glutamic acid residue 274. This residue was found to be highly conserved in this family of β-glycanases, further indicating its importance in catalysis. / Science, Faculty of / Chemistry, Department of / Graduate

No description available.

Cette thèse traite de la conception et mise en œuvre d’espaceurs auto-effondrables novateurs pour une utilisation dans des sondes d’activité enzymatique in vivo.La première partie détaille la synthèse et la validation in vitro d’espaceurs cyclisant, couplant l’activité d’un aminopeptidase à la libération d’un phénol. Les sondes fluorogènes modulaires basées sur ces espaceurs 1,2-diamine sont très robustes (demi-vie > 560 h), mais sont rapidement enzymatiquement hydrolysées, et puis relâchent rapidement (demi-temps ~ 3 min) un fluorophore ESIPT insoluble et exceptionnellement photostable.Ces sondes ont une excellente sensibilité (rapport signal:contrôle > 3000:1), et fournissent la première démonstration d’un système macroscopiquement binaire éteint–ALLUMÉ, pour la libération de phénols sous activité d’aminopeptidases. Ce système pourrait permettre de faire de l’imagerie moléculaire ultra-sensible d’une gamme d’exopeptidases. Ces espaceurs pourraient également servir dans des sondes comportant d’autres fluorophores phénoliques, dans des promédicaments de phénols/alcools activés par des peptidases spécifiques (thérapies ciblées), ou comme adaptateurs chimiques en générale.La deuxième partie détaille les synthèses de deux familles d’espaceurs tautomérisant/éliminant pour utilisation dans des sondes magnétogènes d’activité de glycosidases. Les premières architectures substrat-espaceur basées sur des 2-furanols et des carbimidates cycliques ont été explorées. Notamment, des glycosides 2-furanoliques ont été abordés comme espaceurs énergétiques alternatifs aux quinone méthydes, et des carbimidates ont été explorés comme espaceurs pour ligands modèles des sondes promagnétiques. / This thesis concerns the design and implementation of novel auto-immolative spacers for use in probes for enzymatic activity in vivo.The first part relates the development and in vitro validation of cyclisation spacers which couple the action of an aminopeptidase to the release of a phenol. The modular three-component fluorogenic probes based on these 1,2-diamine spacers are very robust (halflife > 560 h), but are also rapidly enzymatically processed, and quickly (halftime ~3 min) release an exceptionally photostable, insoluble ESIPT fluorophore. The probes have excellent detection sensitivity relative to current methods (signal to control ratio > 3000:1), and provide the first demonstration of a macroscopically binary off–ON system for phenol-releasing probes of aminopeptidase activity. The probe system may allow the exceptionally sensitive, ESIPT-based molecular imaging of a range of exopeptidases. The spacers may also be applied in off ON peptidase probes of other phenolic fluorophores, to peptidase-specific phenol/alcohol prodrugs for targeted therapy, or more generally in chemical adapter technologies.In the second part, two novel families of auto-immolative elimination/tautomerisation spacers were designed for use in three-component off ON magnetogenic probes sensing glycosidase activity. The first known substrate-spacer designs based on 2-hydroxyfurans and on carbimidates were explored. Notably, 2 furanol glycosides were synthesised in pursuit of high-energy alternatives to quinone methides, and a general method for preparing model carbimidate-bearing ligands for pro-magnetic probes was elaborated.

Malgré les efforts extrêmement importants réalisés pour la recherche de nouveaux agents anticancéreux, les traitements par chimiothérapie ne permettent toujours pas de traiter efficacement un grand nombre de tumeurs. En effet, les molécules utilisées cliniquement ne sont généralement pas sélectives des tissus cancéreux et la destruction des tissus sains qu'elles engendrent provoque de lourds effets secondaires. Le développement de nouvelles stratégies thérapeutiques plus sélectives représente donc un intérêt majeur en chimie médicinale. Dans ce cadre, nous avons développé plusieurs vecteurs non toxiques conçus pour reconnaître des spécificités liées à la malignité puis libérer de manière contrôlée des agents cytotoxiques exclusivement au niveau de la tumeur. Dans le cadre de cette thèse, le concept de vecteur glucuronylé a été adapté à la MMAE et à la cyclopamine. Ces vecteurs pourront être sélectivement activé par la B-glucuronidase présente en concentration importante dans les zones nécrotiques de nombreuses tumeurs solides et ainsi libérer ces agents anticancéreux au niveau des tissus cancéreux. Des études préliminaires visant à évaluer l'efficacité de ces composés in-vitro et in-vivo ont montré la validité de cette approche. Dans un deuxième temps, un vecteur glucuronylé de la doxorubicine pouvant se lier à l'albumine plasmatique a été conçu. Une étude réalisée chez la souris a montré que ce composé possède une efficacité thérapeutique similaire à celle de la doxorubicine et ne provoque pas les effets secondaires constatés lors du traitement avec la drogue seule. La troisième partie est consacrée au développement du premier vecteur activable par la B-galactosidase ut / Despite several years of intensive research devoted to the discovery of novel anticancer agents, chemotherapy is still not entirely effective for the treatment of many solid tumors. Most of the drugs used clinically act by anti-proliferative mechanisms and lack any intrinsic selectivity, leading to severe adverse effects due to the destruction of normal tissues. Therefore, the development of more selective therapeutic approaches has become a major goal in medicinal chemistry. Within this framework, we have developed several nontoxic drug carriers designed for both the efficient recognition of malignant specificities and the controlled release of anti-neoplastic agents exclusively at the tumour site. First, we applied the concept of glucuronide prodrugs to MMAE and Cyclopamine in order to deliver these anticancer drugs at the tumor site. Indeed, glucuronide prodrugs can be selectively activated by B-glucuronidase present in high concentration in necrotic area of numerous solid tumors. Preliminary in vitro and in vivo evaluations of these prodrugs demonstrated the potential of this approach. Second, we studied the first B-glucuronidase-responsive albumin-binding prodrug designed for the selective delivery of doxorubicin at the tumor site. In vivo experiment conducted in mice show that this compound inhibits tumor growth in a similar manner to doxorubicin whilst avoiding side effects induced by the free drug. We also developed the first B-galactosidase-responsive drug delivery system suitable for the treatment of solid tumors in PMT. Such a targeting system can be selectively activated by lysosomal B-galactosidase located inside malignant cells expressing a specific tumor-a