Dissertationen zum Thema „Glycosidic bonds“

The problem of forming a glycosidic bond between ribose and the free nucleoside bases to produce beta-nucleosides under plausible prebiotic conditions is commonly referred to in origin of life research as The Nucleoside Problem. The lack of a general solution to this problem currently represents one of the largest stumbling blocks to the RNA world hypothesis and many other theories regarding the origin of life. Over thirty years ago the purine nucleosides were successfully synthesized by drying the fully-formed bases and ribose together in the presence of divalent metal ion salts. However, glycosidic bond formation by the pyrimidine bases has never been achieved under similar reaction conditions. This thesis describes the first plausible prebiotic synthesis of a pyrimidine nucleoside, demonstrated with the pyrimidine base analogue 2-pyrimidinone. Information provided by nucleoside-formation reaction involving 2-pyrimidinone and related pyrimidine bases should provide valuable insights into the possible mechanism by which glycosidic bond formation was accomplished on the prebiotic Earth.

En esta tesis se ha investigado el mecanismo catalítico de las glicosiltransferasas que retienen la configuración a través de simulaciones híbridas de mecánica cuántica/mecánica molecular (QM/MM) y dinámicas moleculares (MD). La investigación se centra en la evaluación de las propuestas mecanísticas, además de la identificación de los principales factores que contribuyen a la eficiencia catalítica. Para esto usamos dos enzimas, α1,4-N-Acetilhexosaminiltransferasa (EXTL2) y Glucosil-3-fosfoglicerato Sintasa (GpgS) de Mycobacterium Tuberculosis. Además, se llevaron a cabo simulaciones adicionales de modelos in silico construidos en base a la enzima nativa o mutante para desvelar los roles de los residuos que se encuentran en una de las caras del sustrato transferible (cara β del azúcar). Los resultados de GpgS se exponen en el Capítulo 4 y los resultados de EXTL2 se presentan a lo largo de los Capítulos 5 y 6. En el Capítulo 7, otras dos glicosiltransferasas retenedoras investigadas en una tesis previa del grupo (LgtC y α3GalT), junto a EXTL2 y GpgS son estudiadas y comparadas para analizar con mayor profundidad las características estructurales de la cara β del azúcar y sus implicaciones en el mecanismo catalítico. En este capítulo también se incluye una discusión general sobre los principales factores que modulan la eficiencia catalítica en cada enzima, dando de esta manera, una visión más completa y general acerca de las estrategias catalíticas realizadas por las glicosiltransferasas retenedoras. Finalmente, las conclusiones generales de este trabajo se resumen en el Capítulo 8. Parte de los resultados presentados en esta tesis pueden ser encontrados en las siguientes publicaciones: • Albesa-Jove, D.; Mendoza, F.; Rodrigo-Unzueta, A.; Gomollon-Bel, F.; Cifuente, J. O.; Urresti, S.; Comino, N.; Gómez, H.; Romero-Garcia, J.; Lluch, J. M.; Sancho-Vaello, E.; Biarnes, X.; Planas, A.; Merino, P.; Masgrau, L.; Guerin, M. E. Angew. Chem. Int. Ed. 2015, 54, 9898-9902. • Gomez, H.; Mendoza, F.; Lluch, J. M.; Masgrau, L. Advances in protein chemistry and structural biology 2015, 100, 225-254. • Mendoza, F.; Gomez, H.; Lluch, J. M.; Masgrau, L. Acs Catalysis 2016, 6, 2577-2589.
In the present thesis the catalytic mechanism of retaining glycosyltransferases has been investigated by means of hybrid quantum mechanics/molecular mechanics (QM/MM) and molecular dynamics (MD) simulations. The research is focused on the evaluation of the mechanistic proposals, as well as the identification of the main factors that contribute to the catalytic efficiency. For that we use two enzymes, α1,4-N-Acetylhexosaminyltransferase (EXTL2) and Mycobacterium Tuberculosis Glucosyl-3-phosphoglycerate Synthase (GpgS). Moreover, further simulations using in silico models built based on the native or mutant enzymes were carried out to unveil the roles of the residues located in one of the faces of the transferable substrate (β-face of the sugar). The results for GpgS are exposed in Chapter 4 and the results for EXTL2 are presented through Chapters 5 and 6. In Chapter 7, two other retaining glycosyltransferases investigated in a previous thesis of the group (LgtC and α3GalT), along with EXTL2 and GpgS are studied and compared to further analyse the structural features in the β-face of the sugar and its implications on the catalytic mechanism. A general discussion around the main factors modulating the catalytic efficiency in each enzyme is also included in this Chapter, providing in this way a more complete and general picture about the catalytic strategies performed by retaining glycosyltransferases. Finally, the general conclusions of this work are outlined in Chapter 8. Part of the results presented in this thesis is already published and can be found in the following papers: • Albesa-Jove, D.; Mendoza, F.; Rodrigo-Unzueta, A.; Gomollon-Bel, F.; Cifuente, J. O.; Urresti, S.; Comino, N.; Gómez, H.; Romero-Garcia, J.; Lluch, J. M.; Sancho-Vaello, E.; Biarnes, X.; Planas, A.; Merino, P.; Masgrau, L.; Guerin, M. E. Angew. Chem. Int. Ed. 2015, 54, 9898-9902. • Gómez, H.; Mendoza, F.; Lluch, J. M.; Masgrau, L. Advances in protein chemistry and structural biology 2015, 100, 225-254. • Mendoza, F.; Gómez, H.; Lluch, J. M.; Masgrau, L. Acs Catalysis 2016, 6, 2577-2589.

Les travaux rapportés dans ce mémoire concernent le développement de nouvelles réactions métallo-catalysées pour la création de liaison carbone-hétéroatome et carbone-carbone ainsi que leurs applications à la synthèse de produits biologiquement actifs. La première partie de ce manuscrit est consacrée à l'étude de la réactivité des sucres dans les couplages organométalliques. Des conditions ont été développées pour la création de la liaison C-S entre glycosyl thiols et partenaires arylés. De plus, la création de la liaison carbone azote de glycosyl amines avec des acides boroniques a été étudiée. Les produits synthétisés dans cette première partie ont été évalués pour leur potentiel d'inhibition de la Lectine A chez Pseudomonas aeruginosa, impliquée dans de sévères infections pulmonaires.La seconde partie de ce travail est dédiée à la création d'une série inédite d'analogues du 6BrCaQ, inhibiteurs de la Hsp90 ainsi que leur évaluation biologique. Cette nouvelle série est obtenue grâce à une nouvelle méthodologie de synthèse basée sur l'activation C-H entre un hétérocycle halogéné et son partenaire C-H activable. L'activité antiproliférative et l'inhibition de la Hsp90 ont été évaluées et seront présentées dans ce manuscrit
The work reported in this dissertation concerns the development of new metal-catalyzed reactions for the creation of carbon-heteroatom and carbon-carbon bonds as well as their applications to the synthesis of biologically active products.The first part of this manuscript is devoted to the study of the reactivity of sugars as nucleophiles in organometallic couplings. Conditions were developed for the creation of the C-S bond between glycosyl thiols and aryl partners. Moreover, the creation of the nitrogen carbon bond of glycosyl amine with boronic acids was studied. The products synthesized in this first part have been evaluated for their potential to inhibit the lectin A, in Pseudomonas aeruginosa related lung infections.The second part of this work is dedicated to the creation of a new series of 6BrCaQ analogues as Hsp90 inhibitors and their biological evaluation. This new series was synthetized through a new CH activation methodology. The antitumoral potential was evaluated and will be presented in this manuscript

Thesis (M. S.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2008.
Committee Chair: Hud, Nicholas V.; Committee Member: Fox, Ronald F.; Committee Member: Lynn, David G.; Committee Member: Powers, James C.; Committee Member: Wartell, Roger M.; Committee Member: Williams, Loren D.

L'etude des complexes du titre a ete envisagee selon deux approches: a) la premiere correspond a la synthese de composes mixtes modeles, avec differentes aglycones mono ou dimeriques, correspondant a des liaisons glycosidiques et a des liaisons ether benzylique. La sensibilite de ces liaisons vis-a-vis de l'oxydation a ete testee. B) la seconde est une etude biochimique. On prepare des polymeres a base d'alcool coniferylique a l'aide d'un extrait brut enzymatique en presence d'hydrates de carbone (polysaccharides ou nucleotides-oses) pour obtenir in vitro les complexes recherches

La première partie de ce manuscrit est consacrée à l'inhibition des glycosidases. Ces enzymes sont impliquées dans de très nombreux processus biologiques et, par voie de conséquence, dans de nombreuses maladies (diabète, cancer, maldadies lysosomales etc...). Outre l'intérêt de développer de nouveaux médicaments, les glycosides hydrolases font aussi figure de cibles de choix pour étudier les itinéraires conformationnels au cours de l'hydrolyse de la liaison glycosidique et en particulier au niveau de l'état de transition. La mise en parallèle de l'importance de l'orientation des liaisons C2-O2 et C3-O3 dans la stéréosélectivité des réactions de b-mannopyranosylation dirigées par les groupements 4,6-O-benzylidène, avec le rôle des interactions avec la liaison C3-O3 dans la catalyse enzymatique pour les b-mannosidases, nous a conduits à nous intéresser au développement de nouveaux glycoimidazoles, potentiellement inhibiteurs de ces enzymes. Nous nous sommes attachés à développer ce type de molécules car ils sont considérés, à l'heure actuelle, comme les meilleurs mimes de l'état de transition. La catalyse par les b-mannosidases passerait par un état de transition de type B2,5. Ce chemin conformationnel semble inhabituel mais serait le plus approprié au développement du caractère de double liaison entre le carbone anomérique et l'oxygène endocyclique ainsi qu'à la charge positive lorsque le système est proche de l'état de transition. De plus, il semblerait que la conformation B2,5 soit aussi privilégiée pour les a-mannosidases, ce qui renforce l'intérêt thérapeutique de synthétiser de nouveaux inhibiteurs de glycosidases. La stratégie de synthèse s'appuie sur des travaux de Vasella. Elle repose sur la construction du squelette bicyclique des tétrahydroimidazopyridines, via une réaction de cyclisation intramoléculaire d'oxoéthylamidines intermédiaires, qui permet d'obtenir de nouveaux glycoimidazoles en une quinzaine d'étapes. Dans la seconde partie, sont exposés les travaux concernant la préparation et l'utilisation en synthèse de glutaconaldéhydes oxydés en position 2 et de N-acylaminopentadiènals. Après avoir rappelé l'importance des glutaconaldéhydes et des aminopentadiènals dans les hypothèses de biogenèse des alcaloïdes marins de la famille des manzamines, nous avons vu que ces espèces pouvaient être considérés comme des intermédiaires possibles pour la synthèse d'alcaloïdes marins de la famille des pyrrole-2-aminoimidazoles (P-2-AI). La nouvelle méthode de préparation des sels de glutaconaldéhydes nous a permis de synthétiser un glutaconaldéhyde oxydé en position 2 et de commencer à étudier la réactivité des 2-alkoxy-N-acylaminopentadiènals. Le dernier chapitre de cette partie traite d'une méthode de préparation de N-acyl-5-aminopenta-2,4-diènals via l'ouverture de furfurylamines N-acylées développée en parallèle avec les travaux précédemment cités.

Glycoside hydrolase. (GH) family 31 contains enzymes that catalyze several different reactions on glycosides. These include a nucleophilic substitution reactions with net retention of stereochemistry, common to retaining glycosidases, as well as an unusual β-elimination reaction. Since structures and mechanism are expected to be conserved in the same gene family, the two mechanisms were expected to feature common aspects. Three different GH family 31 enzymes were therefore studied. A double displacement mechanism for the retaining α-glucosidase from Aspergillus niger was shown via trapping of a covalent glycosyl-enzyme intermediate with the mechanism based inactivator 5-fluoro-α-D-glucopyranosyl fluoride. The amino acid residue involved, Asp 224, was identified by LC MS/MS analysis of proteolytic digests. This residue is fully conserved in GH family 31 and has been suggested to be the catalytic nucleophile. An unusual GH family 31 enzyme, the α-1,4-glucan lyase from Gracilariopsis sp. (GLase) that cleaves the glycosidic bond of α-1,4-glucans via a net β-elimination reaction was also studied. The trapping of a covalent glycosyl-enzyme intermediate using 5-fluoro-(β-L-idopyranosyl fluoride, another mechanism based inactivator of α-glucosidases, strongly suggests that the mechanism also involves the formation of a covalent intermediate like that of α-glucosidases. The labeled amino acid residue was confirmed to be the highly conserved Asp 553, equivalent to Asp 224, the catalytic nucleophile in α-glucosidase from A. niger. A detailed mechanistic evaluation was also carried out. A classical bell shaped pH dependence of k[sub cat]/K[sub m] indicates two ionizable groups (pK[sub α1] =3.1, pK[sub α2] = 6.7). Brønsted relationships of log k[sub cat] versus K[sub m] and log (k[sub cat]/K[sub m]) versus pK[sub a] for a series of aryl glucosides both show a linear monotonic dependence on leaving group pK[sub a] with low β[sub 1g] values of -0.32 and -0.33, respectively. The combination of these low β[sub 1g] values with large a-secondary deuterium kinetic isotope effects (k[sub H]/k[sub D] = 1.16 ~ 1.19) on the first step indicate a transition state for the glycosylation step with substantial glycosidic bond cleavage and proton donation to the leaving group oxygen. Substantial oxocarbenium ion character at the transition state is also suggested by the potent inhibition afforded by acarbose and 1-deoxynojirimycin and by the substantial rate reduction afforded by adjacent fluorine substitution. For only one substrate, 5-fluoro-α-D-glucopyranosyl fluoride, was the second, elimination, step shown to be rate-limiting. The large a-secondary deuterium kinetic isotope effect (k[sub H]/k[sub D] = 1.23) at C1 and the small primary deuterium kinetic isotope effect (k[sub H]/k[sub D] = 1.92) at C2 confirm an E2 mechanism with considerable E1 character for this second step. This considerable structural and mechanistic similarity with retaining a-glucosidases is a clear example of the mechanistic plasticity of glycosidic bond cleavage through evolution. Finally, an unknown protein (yic1) whose sequence has high similarity with GH family 31 was cloned from E. coli. and shown to be an α-xylosidase. Two new mechanism-based inactivators for α-xylosidases, (5S)- and (5R)-5-fluoro-α-Dxylopyranosyl fluorides were synthesized and shown to inactivate this enzyme. The amino acid residue labeled by these inactivators was identified as the invariant catalytic aspartate residue Asp 416, demonstrating the integrity of the mechanism within this gene family.
Science, Faculty of
Chemistry, Department of
Graduate

Seven-membered cyclic sugars, namely, septanoses and septanosides, are less commonly known sugar homologues. Synthesis of septanoses arise an interest due to their configurational and conformational features and the attendant possibilities to explore their chemical and biological properties. Septanosides derivatives, mostly, deoxy-septanosides were synthesized, by many synthetic methodologies, such as, Knoevengal condensation, ring-closing metathesis, Bayer-Villeger oxidation and ring-expansion of 1,2-cyclopropanted glycals as key steps. Apart from septanosyl monosaccharides, septanoside containing di- and tri-saccharides were also performed using glycosylation and ring expansions. Another area of sustained interest is the studies of the stabilities of glycosidic bonds. Acid- and enzyme-catalyzed hydrolysis of glycosidic bond were investigated intensely in the case of pyranosides and furanosides. The explanation of the hydrolysis of such stereomeric sugars were rationalized on the basis of stereoelectronic effects, such as, (i) antiperiplanarity; (ii) synperiplanarity of lone-pair of electrons involed in the hydrolysis process; (iii) steric effects; (iv) field and hyperconjugative effects; (v) conformational effects; (vi) disarming torsional effects and (vii) substituent effects. Chapter 1 of the thesis describes a survey of (i) synthesis of deoxy-septanosides and septanoside-containing di-and tri-saccharides and (ii) acid-catalyzed hydrolysis of glycopyranosides. In a programme, it was desired to identify a new methodology for the synthesis of 2-deoxy-2-C-septanosides. Synthesis of various septanosides from 2-hydroxy glycals, namely, oxyglycals, involves intermediates, such as, vinyl halide (III) and diketone (IV) (Scheme 1). These intermediates were identified as precursors for the synthesis of desired 2-deoxy-2-C-septanosides. Scheme 1 reactions, namely, Heck, Suzuki and Sonogashira reaction for the formation of hither-to unknown septanoside, branching out at C-2. Heck coupling and Suzuki coupling reaction of bromo-oxepine was performed using activated alkenes, acrylates and substituted boronic acid, respectively, in presence of Pd(OAc)2, to furnish 2-deoxy-2-C-alkyl/aryl septanoside derivatives (Scheme 2). Scheme 2 2-deoxy-2-C-alkynyl septanoside derivatives (Scheme 3). Scheme 3 BnO OOMe BnO OOMePd(PPh3)2Cl2,CuIBr BnO R BnO DMF:THF:Et3N(3:2:1)BnO OBn 98 oC, 72 h BnO OBn R=Ph,SiMe3,C6H13 One of the 2-deoxy-2-C-alkyl septanoside derivative was converted to the corresponding protecting-group free 2-deoxy-2-C-alkyl septanoside, using hydrogenolysis (Pd/C, H2) and NaBH4-mediated reduction. Chapter 2 presents details of the synthesis of 2-deoxy-2-C-alkyl/aryl/alkynyl septanoside derivatives from a bromo-oxepine. Continuing the efforts to extend the ring-opening of oxyglycal derived gem-dihalo-1,2¬cyclopropanted sugar, a Lewis acid-catalyzed ring-opening was considered important. The presence of an additional substituent in C-2 of oxyglycal switches reactivity as compared to glycals. For example, ring-opening of glycal derived gem-dihalo-1,2-cyclopropane generates 2-C-branched pyranoside, whereas corresponding oxyglycal generates oxepines even when both the reactions were performed under a mild basic condition, illustrating a sufficient reactivity difference between a glycal and an oxyglycal. Thus, ring-opening reaction of gem-dichloro-1,2-cyclopropanted oxyglycal in the presence of a Lewis acid, hither-to unknown, was examined. In this event, it was found that ring-opening reaction led to chloro-oxepine derivatives in the presence of AgOAc, using alcohol as nucleophiles. Primary, secondary, unsaturated and aromatic alcohols were used in the ring-opening reaction. The ring-opening reaction was stereoselective and only α-anomer was obtained in a good yield in each case (Scheme 5). The counter-anion also reacted in an instance, so as to furnish O-acetyl chloro-oxepine during the ring-opening reaction. Scheme 5 The course of the reaction in the absence of alcohol led to afford only the O-acetyl chloro-oxepine (Scheme 6). Scheme 6 It became pertinent to compare the result this work with that of AgOAc-catalyzed ring-opening of glycal derived gem-dihalo-1,2-cyclopropanated sugar, which led to C-furanoside derivative, as reported by Harvey and co-workers. The sequence of reactions involved were protonation of the endo-cyclic oxygen, followed by ring-opening to generate resonance stabilized allylic ion, which rearranged to C-furanoside. In contrast, oxyglycal derived gem-dihalo-1,2-cyclopropane studied herein led to chloro-oxepine exclusively, without subsequent rearrangement. Ring-opening of glucal derived gem-dihalo-1,2-cyclopropanated sugars, followed by cyclization to C-furanoside were likely to have occurred, due to isomerisation of less-substituted endo-cyclic double bond at C2-C3 of oxepine to C1-C2 unsaturated vinyl ether. Such a reaction was related closely to the acid-catalyzed rearrangement in less-substituted oxepine systems. On the other hand, gem-dichloro-1,2¬cyclopropanated oxyglycal derived chloro-oxepine did not undergo such an isomerisation, possibly due to unsaturation being present at highly substituted C2-C3 carbons (Scheme 7). Thus, the presence of an additional oxy-substituent at C-2 in oxyglycal derived cyclopropane derivative plays a major role to control the reactivity, as compared to glycal derived cyclopropane derivatives. Scheme 7 without undergoing further reactions, was confirmed further by the following reactions: (i) RuCl3¬NaIO4 mediated oxidation; (ii) NaBH4 reduction and (iii) Pd/C mediated hydrogenolysis (Scheme 8). Scheme 8 1,2-cyclopropane to exclusive formation of chloro-oxepine in the presence of AgOAc. It was planned further to synthesize a 1,7-linked-α-D-diseptanoside, through the oxyglycal route. Ring-opening of oxyglycal derived gem-dihalo-1,2-cyclopropanated derivative with 6¬hydroxy glycal led to 1,7-α-linked disaccharide unit. The following reactions were performed in order to synthesize 1,7-linked-α-diseptanoside 2: (i) cyclopropanation of the glycal double bond; (ii) ring opening of the gem-dihalo cyclopropane; (iii) RuO4 mediated oxidation; (iv) NaBH4 reduction and (v) hydrogenolysis using Pd/C, H2 (Scheme 9). Similar methodology was used for the synthesis of monoseptanoside, namely, n-pentyl-D-glycero-D-galacto-septanoside. Scheme 9 1 Oxyglycal route was also used for the synthesis of 2-chloro-2-deoxy septanoside 3, using hydrogenolysis (Pd/C, H2) and NaBH4 mediated reduction of chloro-oxepine (Scheme 10). Scheme 10 A kinetic study of the hydrolytic stabilities of mono-and diseptanoside was undertaken using an acid-catalysis, in a subsequent investigation. In the course of studies, it was observed that glycosidic bond in the reducing-end hydrolyzed twice faster than that at the non-reducing end, whereas glycosidic bond in monosaccharide 1 hydrolyzed 1.5 times faster than of reducing-end glycosidic bond in diseptanoside 2. Further, it was found that the replacement of the C-2 hydroxyl group by a chloride group reduced the rate of hydrolysis (Table 1). Table 1. First order rate constants and thermodynamic parameters for the acid-catalyzed hydrolysis of glycosidic bond in septanosides 1, 2 and 3. Compound Rate of hydrolysis ΔH# ΔS# ΔG# (kobs) (104 s-1) (kcal/mol) (cal/mol K) (kcal/mol) 35 oC 45 oC 85 oC 90 oC a non-reducing end of 2. A computational study was conducted, in order to gain further insight into the hydrolysis, using B3LYP/6-311G* level theory in the Gaussian 09 program packages. Calculations using the PCM solvent model with water as the solvent showed that the orientation of hydroxylmethyl group plays an important role. In the case of 1, the gg conformer was calculated stable by 2.12 kcal/mol, as compared, to tg-conformer. In the gg conformation, the optimal positioning of the dipole C7-O7 stabilized the oxo-carbonium ion in the transition state (Figure 1). Also, hydroxyl group at C4 stabilized the transition state, through non-covalent interaction (Figure 1). The transition state for the hydrolysis of 1 was found to present activation barrier (∆G#) of 19.9 kcal/mol, which was in good agreement with value for 1 (∆G# = 23.26 kcal/mol), as calculated from Erying plot (Table 1). On the other hand, inductive effect of the chloride group, as well as, the tg-orientation of the hydroxymethyl group appeared to contribute to the slower rate of the hydrolysis. Figure 1. gg- and tg-conformations in the ground state of 1. Chapter 4 describes synthesis of 1,7-linked-α-D-diseptanoside, 2-chloro-2-deoxy septanoside and their acid-catalyzed hydrolysis studies. Solid-state and solution phase conformation of septanosides are rare at present even when solid-state structures of pyranoside and furanosides are known commonly, that provide rich information of covalent and non-covalent interactions. In this context, single crystal X-ray structural analysis of septanosides, namely, n-pentyl-2-chloro-2-deoxy-α-D-manno-sept-3-uloside 4 and p-bromo phenyl 4,5,7-tri-O-benzyl-β-D-glycero-D-talo-septanoside 5 were analyzed. It was observed that the solid-state structure of 4 adopted twist-chair conformation, namely, 5,6TC3,4, whereas 5 adopted O,1TC2,3 conformation (Figure 2). An analysis of non-covalent interactions revealed that a dense network of O−H···O and C−H···O stabilized the crystal lattice of 4, whereas O−H···O and C−H···π stabilized the crystal lattice of 5. Chapter 5 describes the detailed analysis of X-ray crystal structure of two septanoside derivatives including non-covalent interactions responsible for the stabilization of crystal lattice. Figure 2. ORTEP of 4 and 5 with displacement ellipsoids, at a 10 % and 50 % probability level. In summary, the thesis established the following major results: (i) synthesis of 2-deoxy-2-C¬alkyl/aryl septanoside from a bromo-oxepine, using organometallic C-C bond forming reactions; (ii) the ring-opening reaction of oxyglycal derived gem-dihalo-1,2-cyclopropane in the presence of AgOAc and the effect of additional C-2 oxy-substituent in the reactivity, in comparison to glycal; (iii) an oxyglycal route for the synthesis of 1,7-linked-α-D-diseptanoside, 2-chloro-2-deoxy septanoside and their acid-catalyzed hydrolysis studies and (iv) solid-state X-ray crystal structural analysis and computational analysis of the conformation and non-covalent interactions associated with the stabilization of crystal lattice. Overall, the studies presented in the thesis provide a new insight into the synthesis, acid-catalyzed hydrolysis and solid-state structural analysis of septanoside derivatives.

碩士
國立臺灣大學
食品科技研究所
91
Four different disaccharides, maltose, trehalose, cellobiose, and chitobiose were used to study the effect of glycosidic bond, α-1, 4, α-1, 1, β-1, 4 on the ozonolysis of disaccharides. The yields of monosaccharides from maltose and cellobiose were similar. That indicated theαorβ form of glycosidic bond did not contribute the difference in ozonolysis. The yield of monosaccharides from trehalose was higher than three from both maltose and cellobiose. The data indicated thatα-1, 1 of glycosidic bond was more favorable during ozonolysis.Chitobiose yield more monosacchadises than cellobiose. The presence of amino group enhanced the production of glucosamine. The degradation rate was increased with the ozone dosade and the initial concentration of disaccharides. At 70℃℃, the yield of glucose from trehalose at initial concentration of 0.5 % was higher than that at the initial concentration of 0.06 %. Nevertheless, the variation in ozone dosage did not alter the reaction orders in the range from 0.7 to 1.1. The analysis from FTIR revealed the presence of carboxylic acid. The formation and inhibitor of carboxylic acid are worthy of further studies.

博士
國立清華大學
化學系
97
摘 要 自然界存在之聚唾液酸醣苷鍵形式主要有三種，α(2→8)與α(2→9)醣鍵結形式，以及α(2→8)/ α(2→9)醣鍵結交錯出現形式。最近在醣生物學研究上指出，出現在細胞表面的α(2→8)和α(2→9) 醣鍵形式的雙醣及寡醣在生物方面上扮演重要的角色。然而這些醣類在溫和的酸性或鹼性條件下是不穩定的，而且容易被醣水解酶影響而水解。因此，硫鍵結之醣苷分子已經被提出用來增強被化學或酵素水解之醣苷鍵結的穩定度。 本論文的目標是發展便利的策略來合成硫鍵結之唾液酸寡糖抗原。我們發展了一個不對稱的異丁基雙硫鍵來當作變旋異位性(anomeric)硫原子的保護基，並利用此新方法來合成硫鍵結α(2→9)唾液酸寡糖。比較一般傳統在唾液酸變旋異位性硫原子的保護基，我們所使用的不對稱異丁基雙硫鍵保護基能夠承受在官能基轉換過程中而不會產生不飽和鍵的脫去產物。此外，不對稱異丁基雙硫鍵保護基可以有效率的去保護而在變旋異位中心產生具有硫醇的親核試劑，並且不會造成變旋異位中心的變旋異構化。我們實驗室藉由這個方法已經成功合成了4-,6-,8-硫鍵結α(2→9)唾液酸寡糖。 除此之外，藉由硫親核反應來發展合成硫鍵結之α(2→8)以及α(2→8)/α(2→9)三醣體，這些方法包含利用C2-硫基化之唾液酸醣苷分子當做親核試劑以及C8-碘基化之唾液酸醣苷分子當做活性化之親電試劑，來進行化學及立體選擇性之烷基化反應。此外，我們也發展了一個有效的轉移方法，在溫和的鹼性條件下將唾液酸醣苷分子七號位置的乙醯基官能基轉換至九號位置上。接著利用二氯二甲基矽烷和碘化鈉的條件下將乙醯基轉移之唾液酸醣苷分子在八號位置上進行碘基化反應。藉由這些方法，我們已經合成了硫鍵結α(2→8)唾液酸三醣體以及α(2→8)/ α(2→9)唾液酸三醣體。 由於已經成功合成了硫鍵結之α(2→9)唾液酸寡醣體，將合成的硫代抗原和載體蛋白(KLH)進行結合也在疫苗發展上被研究。藉由MHSu(6-maleimidohexanoic acid active ester)裝配在硫鍵結之α(2→9)唾液酸寡醣體，接著與硫基化之載體蛋白(KLH)進行結合，我們利用此新穎與有效的方法來製備碳水化合物結合疫苗。另外，我們也使用Ellmans試劑去定量有多少硫鍵結之α(2→9)唾液酸醣苷分子在載體蛋白(KLH)上面。這些方法可以普遍適用在合成的寡糖上面。

Pathogenesis originating from mycobacterial invasion on host cells is prevalent and is a major challenge in efforts towards overcoming the burden of mycobacterial diseases. Complex architecture of mycobacterium cell wall includes an assortment of glycolipids, phospholipids, glycopeptidolipids (GPLs), peptidoglycans, arabinogalactans, lipoarabinomannans and mycolic acid. Aided by thick cell wall envelope, mycobacteria are known to survive in hostile environment. As most antibiotics target the log phase of the bacteria, bacterial survival is also largely dependent on its stationary phase. Mycobacteria have evolved colonization by means of biofilm formation in the stationary phase, so as to survive under stress and hostile conditions. Biofilms are the specialized form of phenotype which makes bacteria several fold resistant to antibiotics. Development of inhibitors against biofilms remains a challenge due to the poor permeability of molecules and coordination among cells. The first part of Chapter 1 of the thesis describes the details of formation of biofilm in the stationary phase of bacteria and understanding the molecular level details for making the strategies to overcome antidrug resistance of mycobacteria. Among the cyclic hosts, cyclodextrins are prominent. Due to their unique structural and physical properties, cyclodextrins can form inclusion complexes with a wide range of guest molecules. Although synthetic modifications of cyclodextrins through hydroxy groups are very common, modifications at backbone continue to be a challenge. Backbone modified cyclodextrins using different organic moieties were developed and their altered cavity properties were explored in many instances. Chemical synthesis of cyclic oligosaccharides is, in general, involved (i) a cyclo-oligomerization of linear oligosaccharide precursor and (ii) an one-pot polycondensation of appropriately designed monomer under suitable reaction conditions. The second part of Chapter 1 deals with a literature survey of skeletal modification of cyclodextrins, their synthesis and binding abilities with different guest molecules. In my research programme, synthesis and studies of oligosaccharide glycolipids relevant to mycobacterial cell wall were undertaken. Arabinofuranoside trisaccharide glycolipids, containing β-anomeric linkages at the non-reducing ends and double hexadecyloxy lipid moieties, interconnected to the sugar moiety through a glycerol core, were synthesized (Figure 1). Arabinan trisaccharides 1 with lipidic chain and 3 without lipidic chain comprise β-(1→2), β-(1→3) anomeric linkages at the non-reducing end, whereas in the case of arabinan trisaccharides 2 and 4, β-(1→2), β-(1→5) linkages are present between the furanoside units. In the scheme of synthesis of trisaccharide glycolipids, monosaccharide derivative and lipidic portions were individually prepared first and were assembled subsequently to secure the target glycolipids. Incorporation of β-arabinofuranoside linkages in trisaccharide arabinofuranosides 1-4 was achieved by low temperature activation of silyl group protected conformationally locked thioglycoside donor 5 (Figure 1), in the presence of N-iodosuccinimide (NIS) and silver trifluoromethanesulfonate (AgOTf). Figure 1. Molecular structures of trisaccharides 3, 4 and glycolipids 1, 2 with β-arabinofuranoside linkages at the non-reducing end and glycosyl donor 5. Following the synthesis, the efficacies of synthetic glycolipids to interact with surfactant protein A (SP-A) were assessed by using surface plasmon resonance (SPR) technique, from which association-dissociation rate constants and equilibrium binding constants were derived. SP-A, a lung innate immune system component, is known to bind with glycolipids present in the cell surface of a mycobacterial pathogen. From the analysis of SPR studies with glycolipids 1, 2 and SP-A, the association rate constants (ka) were found to be in the range of 0.3 to 0.85 M−1 s−1, whereas the dissociation rate constants (kd) were varied between 2.21 and 3.2×10−3 s−1. The equilibrium constants (Ka) values were in the range of 93 and 274 M−1. Trisaccharides 3 and 4, without lipidic chains, were also assessed for their efficacies to interact with SP-A. The association constants for 3 were found to be in the range of 2,470 to 9,430 M−1, whereas for the derivative 4, Ka values varied between 25,600 and 76,900 M−1. The association and equilibrium binding constants for 3 and 4 were found to be significantly higher when compared to glycolipids 1 and 2. In conjunction with our previous report, the present study shows that arabinofuranoside glycolipids, with β-anomeric linkages bind to SP-A with lesser extent as compared to α-anomers. Further, the studies of trisaccharides and glycolipids in mycobacterial growth and sliding motility assays were performed with model organism M. smegmatis and it was found that the synthetic compounds affected both growth and motility and the extent was lesser than that of α-anomeric glycosides and glycolipids. Chapter 2 of the thesis describes the details of synthesis, biophysical and biological studies of arabinan trisaccharide glycolipids, with β-anomeric linkages at the non-reducing end. Continuing the synthesis and studies of arabinan oligosaccharides, a linear arabinomannan pentasaccharide and heptasaccharide glycolipids 6 and 10, containing α-(1→2) and α-(1→3) linkages between core arabinofuranoside units, as well as, a branched arabinomannan pentasaccharide and heptasaccharide glycolipids 7 and 11, with α-(1→2) and α-(1→5) linkages between core arabinofuranoside units, were synthesized (Figures 2 and 3). Figure 2. Molecular structures of arabinomannan glycolipids 6 and 7 and the corresponding oligosaccharides 8 and 9. In addition to glycolipids, arabinomannan pentasaccharides without lipidic chain 8 and 9 and arabinomannan heptasaccharides without lipidic chain 12 and 13, were also synthesized. Synthesis was performed using trichloroacetimidate and thioglycosides as glycosyl donors. A block condensation methodology was adopted by which disaccharide donor and monosaccharide acceptor were chosen to assemble the pentasaccharide, by a two-fold glycosylation. Monosaccharide acceptors with and without lipidic chain were used in the glycosylations for the synthesis of glycolipids and pentasaccharides, respectively. Similarly, a trisaccharide thioglycoside donor and monosaccharide acceptors were chosen for the double glycosylation to synthesize heptasaccharides in the presence of NIS and AgOTf. Figure 3. Molecular structures of arabinomannan heptasaccharide glycolipids 10, 11 and corresponding heptasaccharides 12 and 13. Subsequent to synthesis, activities of pentasaccharide glycolipids were assayed on M. smegmatis bacterial growth, sliding motilities and also the effects on mycobacterial biofilms. Profound effects were observed with the synthetic compounds, to reduce the mycobacterial growth, sliding motilities and biofilm structures. Whereas reduction up to ~50% occurred on mycobacterial growth, as much as, 70% reduction in the motilities of the bacteria was observed in the presence of the synthetic glycolipids, at 100 µg mL-1 concentration. At the same concentration, 80–85% reduction in the biofilm was observed. These effects were more pronounced with branched glycolipids than linear analogues. Chapter 3 of the thesis presents the synthesis of linear and branched arabinomannan penta- and heptasaccharide glycolipids and biological studies of arabinomannan pentasaccharide glycolipids with M. smegmatis. Cyclodextrins, the most abundant naturally-occurring cyclic oligosaccharides, are valuable synthetic hosts, primarily as a result of their properties to form inclusion complexes with guest molecules. In spite of voluminous literature on the application of cyclodextrins, through modifications of hydroxy groups, modifications at the backbone continue to be a challenge. Skeletal modifications using aromatic, triazole, diyne, thioether and disulfide moieties were developed, that helped to alter the cavity properties of cyclodextrins. A programme was undertaken to synthesize backbone modified cyclic oligosaccharide, which was achieved using a monomer wherein a one carbon insertion is conducted at C4 of a pyranose, such that the hydroxy moiety at C4 is replaced with a hydroxymethyl moiety. In an approach, a linear trisaccharide monomer was anticipated to provide cyclic oligosaccharides in multiples of such a monomer. In the event, a trisaccharide linear monomer 14 was found to afford a cyclic trisaccharide macrocycle 15, as the major cyclo-oligomer (Scheme 1). Subsequent solid state structural studies show that the molecule confers a perfect trigonal symmetry in the P3 space group, in a narrow cone shape and a brick-wall type arrangement of molecules, such a geometry is hither-to unknown to a cyclic oligosaccharide (Figure 4). Furthermore, binding abilities of cyclic trisaccharide with few organic bases, such as 1-aminoadamantane and hexamethylenetetramine, was evaluated by the means of isothermal titration calorimetry and it was found that such a cyclic trisaccharide exhibits strong binding affinities towards 1-aminoadamantane in aqueous solutions, as compared to the same with naturally-occurring β-cyclodextrin. Scheme 1 Apart from cyclic trisaccharide, synthesis of cyclic tetrasaccharide 17, containing alternative anomeric α-(1→4) and β-(1→4) linkages was also undertaken by one-pot cyclo-oligomerization in the suitable reaction condition, from an activated disaccharide thioglycoside monomer 16, having β-(1→4) linkage at the non-reducing end (Scheme 2). Chapter 4 describes the synthesis of cyclic oligosaccharides 15 and 17, as well as, the details of solid state structure and binding studies of cyclic trisaccharide 15. Scheme 2 Figure 4. (a) Stick model of the crystal structure of 15, as viewed along the crystallographic c-axis; (b) trigonal view from crystal packing; (c) packing diagram crystal lattice, as viewed along the crystallographic b-axis, and without solvent inclusion and (d) packing diagram included with methanol (grey) and water (red) solvents, as viewed along the crystallographic c-axis. Hydrogen atoms are omitted for clarity in (c and d). In summary, the thesis presents (i) synthesis, biophysical and biological studies of synthetic arabinan and arabinomannan glycolipids, and (ii) synthesis, solid-state structural analysis and binding studies of glycosidic bond expanded cyclic oligosaccharides. Synthetic trisaccharide arabinofuranoside glycolipids containing β-anomeric linkages at the non-reducing end showed binding affinity towards pulmonary surfactant protein A, as assessed by surface plasmon resonance technique, with comparatively lower extent as compared to synthetic glycolipids having α-anomeric linkages. Linear and branched arabinomannan penta- and heptasaccharide glycolipids, having α-anomeric linkages were synthesized and biological studies with non-pathogenic strain M. smegmatis were conducted with pentasaccharide glycolipids. It was found that arabinomannan glycolipids inhibited the growth and sliding motility of mycobacteria. Importantly, disruption of biofilm and significant reduction in biofilm formation was observed in the presence of the synthetic glycolipids. Glycosidic bond expanded cyclic trisaccharide with anomeric α-(1→4) linkages and cyclic tetrasaccharide with alternative anomeric α-(1→4) and β-(1→4) linkages were prepared from suitably designed trisaccharide and disaccharide monomer, respectively, by cyclo-oligomerization. Solid-state structural analysis and binding studies of cyclic trisaccharide in solution by isothermal titration calorimetry were also conducted. Cyclic trisaccharide possessed a bowl shape and brick-wall type of arrangement in the solid-state structure, whereas it exhibited stronger binding affinity towards 1-aminoadamantane as compared to β-cyclodextrin in aqueous solution. Overall, the results presented in the thesis provide a possibility to develop new types of synthetic glycolipids that can act as inhibitors of biofilm formation of mycobacteria, as well as, to develop newer types of cyclic oligosaccharide synthetic hosts that can modify binding abilities towards various guest compounds.