Carbohydrate-protein interactions are responsible for several biological functions. While these interactions maintain high levels of specificities, the binding strength of individual carbohydrate-protein recognitions are weak, with dissociation constants (Kd) ~10-3-10-6 M. In order to increase the binding strengths meaningful to physiological functions, multivalent, clustered patches of carbohydrate ligands are required. Synthetic glycoclusters contribute in a significant manner to understand the fine details of the weak carbohydrate-protein interactions. The extent of clustering of the ligands, spatial, topological orientations and the nature of the scaffolds are prominent issues to address the carbohydrate-protein interactions in general. Chapter 1 of the Thesis presents a summary of the synthetic cluster glycosides, mechanisms and energetics of their interactions with lectins. The presence of several ligands within the molecular scaffold is not sufficient, rather there exists a critical demand on the spatial disposition of the individual ligands in the multivalent ligand system to achieve enhanced binding affinities. In order to assess the multivalent effects, influence of linkers and the spatial disposition of the ligands, a systematic study was undertaken, involving a series of the most minimal of the multivalent sugar ligand system, namely, the bivalent sugar ligands. In a programme, it was desired to study the bivalent and monovalent sugar ligand-lectin interactions in a two-dimensional membrane model system. An appropriate model system was the Langmuir monolayer formations of the sugar ligands and their recognitions of the lectins at the interface. A series of bivalent and monovalent glycolipids were thus designed and synthesized. Molecular structure of the ligands utilized to study the lectins binding behavior at the air-water interface are presented in Figure 1. The sugar density dependent lectin binding at the air-water interface caused by the glycolipids was studied in detail. Prior to lectin binding studies, the monolayer behavior of the glycolipids (GL), non-sugars (NS) and their mixtures were assessed. It was observed that the apparent molecular areas of the mixed monolayers increased with increasing percentage of the glycolipid in the mixed monolayer. Interactions of the glycolipid mixed monolayers with lectin were assessed at a constant surface pressure of 10 mN/m. The adsorption kinetics of the lectin concanavalin A (Con A) with the mixed monolayers was monitored by the surface area variation (ΔA) as a function of time. The detailed studies showed a maximum increase in ΔA of 10% of the bivalent glycolipids in the mixed monolayer and a ΔA of 20% of the monovalent glycolipids (Figure 2). With both bivalent and monovalent glycolipids, change in the area per molecule had decreased progressively with higher percentage of the glycolipids in the monolayers. On the other hand, with ethylene glycol spacers, the relative responses and the amount of bound lectin increased. Figure 2. Ligand-lectin interactions at the air-water interface as a function of the percentage of (a) bivalent glycolipids and (b) monovalent glycolipids in the mixed monolayers. To verify the specificity of these interactions, the mannopyranoside non-specific lectin, namely, wheat germ agglutinin (WGA) was tested and there were no deviations in the ΔA for various ratios of the sugar–non-sugar mixed monolayers. The study established that (i) maximal binding of the lectin to the bivalent glycolipids occurred at lower sugar densities at the interface than that for the monovalent glycolipids and (ii) the surface presenting sparsely populated sugar residues are efficient for a lectin binding. Chapter 2 presents the details of synthesis and ligand-lectin interactions at the air-water interface, relevant in the two-dimensional membrane model system. A study of the multivalent effects originating through glycolipid micelles and their lectin interactions was undertaken in another programme. The kinetic studies of the glycolipid micelles-lectin interactions were conducted with the aid of surface plasmon resonance (SPR) technique. Prior to the SPR studies, the critical micellar concentration (CMC), aggregation number and the hydrodynamic diameter of each glycolipid (GL-1 to GL-6, Figure 1) micelles were determined. The glycolipid micelles were used as the analytes on a Con A immobilized surface. The sensorgrams obtained for the interaction of the various glycolipid micelles with Con A are presented in Figure 3. Figure 3. Sensorgrams obtained for the binding of various glycolipids micelles to a Con A immobilized surface, at a constant glycolipid concentration of 250 µM. The kinetic studies of the interactions were performed and the analysis showed that the bivalent analyte model provided a better fitting for the interaction sensorgrams. The analysis revealed that the ka1/kd1 values remained largely uniform for all the glycolipids, whereas the ka2/kd2 values were about two orders of magnitude larger for the bivalent glycolipid (GL-4 to GL-6) micelle-lectin interactions than for the monovalent series (GL-1 to GL-3) (Table 1). From the SPR studies, it emerged that the additional sugar unit in the bivalent glycolipid micelles provided a favorable complexation between the sugar ligand and the lectin. Further, the glycolipid micelles mediated layer-by-layer Con A multilayer formation was also studied by SPR and atomic force microscopy (AFM) methods. Chapter 3 provides the SPR studies of glycolipid micelles-lectin interactions. A study of the monomolecular recognitions of the mono- and bivalent sugar ligands 1-8 (Figure 4) to a lectin was undertaken subsequently. The kinetic studies of the bivalent vs monovalent ligands during lectin binding were conducted by employing the SPR technique, for which the sugar ligands 1-6 were used as the analytes on a lectin coated sensor surface. Figure 4. Structures of the mono- and bivalent sugar ligands 1-8 and the NS derivative. The following observations were made from the SPR analysis. (i) Within the mono- and bivalent series, the response units increased in the series 1–3 and 4–6; (ii) the equilibrium responses were attained within 105 seconds in the monovalent ligands and (iii) the association response gradually increased for the bivalent ligands 5 and 6 and reached an equilibrium after ~3 min. An important outcome of the kinetic studies was the identification of ka and kd for the monomolecular interactions, that were distinctly different for the bivalent ligands. Specifically, the ka was significantly faster and kd was slower for bivalent sugar ligands, in comparison to the monovalent sugar ligands (Table 2). Table 2. SPR derived kinetic parameters for the interactions of sugar ligand to a Con A immobilized surface at 25 oC. Isothermal titration calorimetry (ITC) studies were also conducted, in order to correlate the functional valencies and the thermodynamic parameters. The studies were conducted at ligand concentrations much below their CMCs. The general observations from the ITC studies were that the binding site saturations were slower for the monovalent sugar ligands, in comparison to the bivalent sugar ligands. It was observed that the binding affinities of bivalent ligands 5 and 6 enhanced ~5 times higher than the monovalent ligands 2 and 3 (Table 3). The effective linker length, which allowed the sugar ligands to be functionally active, was determined to be ~15 Å and this separation was necessary for the intermolecular cross-linking formation. The dynamic light scattering (DLS) study of the bivalent ligands 5 or 6-lectin complexes showed the presence of intermolecular cross-linked complexes that existed in solution from the initial stages of the binding process. Upon realizing the nanometric diameters of the sugar ligand-lectin complex, an attempt was undertaken to visualize the complexes by transmission electron micoscopy (TEM). In TEM, 4-Con A complex exhibited particle sizes in the range of 5-10 nm, matching nearly the size of the lectin alone. On the other hand, 5–Con A and 6–Con A complexes provided sizes varying between 20¬150 nm. These particle sizes corresponded to similar aggregate sizes derived from the DLS studies. Chaper 4 describes the kinetic, thermodynamic, DLS and TEM studies of sugar ligand-lectin intearctions. Table 3. Binding stoichiometries and thermodynamic parameters of the sugar ligand-Con A interactions at 25 oC.a Ligand n Ka (x 10 -4) ΔG ΔH TΔS 1 0.91 9.14 ( ± 0.75) -6.76 -3.39 3.37 2 1.01 5.76 (± 0.80) -6.49 -3.98 2.51 3 1.09 7.06 (± 1.23) -6.61 - 3.01 3.60 4 1.10 5.75 (± 0.27) -6.49 - 6.39 0.10 5 0.50 20.6 (± 1.7) -7.59 - 12.80 -5.21 6 0.47 37. 4 (± 2. 4) -7.61 -11.54 -3.93 7 1.03 0.86 (± 0.06) -5.36 -7.9 -2.62 8 1.05 2.48 (± 0.12) -5.99 -6.3 -0.32 MeαMan 1.04 0.79 (± 0.04) -5.27 -7.83 -2.56 Ka is in the unit of M-1; ΔG, ΔH and TΔS are in the units of kcal mol-1. Errors in ΔG are ~1-4%. Errors in ΔH are in the range of 1-8%. Errors in TΔS are in the range of 1-6 %. A study was undertaken further to assess the kinetic interactions of the tumor-associated T-antigen with a lectin. Synthesis of amine-tethered T-antigen and lactose derivatives (Figure 5) were accomplished and an assessment of their kinetic interactions with lectin peanut agglutinin (PNA) was conducted. Figure 5. Structures of the amine-tethered T-antigen, lactose and mannose derivatives. The lectin PNA was used as the analyte onto the sugar ligand immobilized surfaces. It was found that the interaction with T-antigen showed higher response units than the lactose derivative (Figure 6). The kinetic studies of PNA with immobilized T-antigen and the lactose derivatives demonstrated that the binding followed a bivalent analyte model of the interaction. The T-antigen derivative interacted with the lectin and relatively faster association (ka) and a slower dissociation (kd) were observed, in comparison to the lactose derivative. The ratio of second binding kinetic constants (ka2/kd2) was observed higher than the first binding kinetic constants (ka1/kd1). Further, the ITC studies were conducted, in order to provide the thermodynamic parameters governing the lectin-T-antigen interactions. The combined approach of SPR and ITC studies showed that the T-antigen derivative exhibited a higher binding affinity to PNA than the lactose derivative. Chapter 5 presents synthesis of the T-antigen and lactose derivatives and studies of their lectin interactions. In summary, the thesis provides a detailed insight into the kinetic and thermodynamic parameters of the bivalent sugar ligand-lectin interactions, in comparison to the monovalent sugar ligands. Langmuir monolayer formation of the sugar ligands and the assessment of their lectin binding at the air-water interface demonstrated that the surface presenting sparsely populated sugar residues are efficient for a lectin binding. The kinetic studies of various glycolipid micelles-lectin interactions showed that the additional sugar unit in the bivalent glycolipid micelles provided a favorable complexation between the sugar ligand and the lectin. The detailed monomolecular kinetic studies showed that the bivalent sugar ligands underwent a faster association (kon) and a slower dissociation (koff) of the ligand-lectin complexes. The ITC studies on sugar ligand-lectin interactions led to identify not only the thermodynamic parameters, but also the influence of the hydrophobic alkyl units and the linker moieties. The DLS and TEM characterizations of sugar ligand-lectin complexes showed the status of the complexation, sizes and the morphologies. The studies were extended further to tumor associated T-antigen-lectin interactions. Overall, the Thesis establishes the most minimal multivalent sugar ligands, namely, the bivalent sugar ligand-letin interactions. The studies presented in the Thesis should be useful to design multivalent sugar ligands for highly avid lectin interactions and also to raise possibilities for the construction of defined lectin oligomers, facilitated through the multivalent sugar ligand-lectin cross-linking interactions.

Xyloglucans are polysaccharides found as storage polymers in seeds and tubers, and as cross-linking glycans in the cell wall of plants. Their structure is complex with intricate branching patterns, which contribute to the physical properties of the polysaccharide including its binding to and interaction with other glycans such as cellulose. Xyloglucan is widely used in bulk quantities in the food, textile and paper making industries. With an increasing interest in technically more advanced applications of xyloglucan, such as novel biocomposites, there is a need to understand and control the properties and interactions of xyloglucan with other compounds, to decipher the relationship between xyloglucan structure and function, and in particular the effect of different branching patterns. However, due to the structural heterogeneity of the polysaccharide as obtained from natural sources, relevant studies have not been possible to perform in practise. This fact has stimulated an interest in synthetic methods to obtain xyloglucan mimics and analogs with well-defined structure and decoration patterns. Glycosynthases are hydrolytically inactive mutant glycosidases that catalyse the formation of glycosidic linkages between glycosyl fluoride donors and glycoside acceptors. Since its first conception in 1998, the technology is emerging as a useful tool in the synthesis of large, complex polysaccharides. This thesis presents the generation and characterisation of glycosynthases based on xyloglucanase scaffolds for the synthesis of well-defined homogenous xyloglucan oligo- and polysaccharides with regular substitution patterns.

Tesis por compendio / [ES] Los oligosacáridos de la leche humana (OLHs) y la parte glicana de los glicoconjugados son hidrolizados por las glicosil hidrolasas (GHs), las cuales son expresadas por la microbiota intestinal de niños lactantes promoviendo el establecimiento de una microbiota intestinal con beneficios para su salud. El objetivo de esta Tesis Doctoral consistió en la caracterización funcional de GHs de la microbiota intestinal de niños lactantes capaces de metabolizar OLHs y glicoconjugados, y el estudio de su relevancia biológica y su potencial biotecnológico. Se aislaron cepas bacterianas a partir de heces de niños lactantes.Solo las cepas del género Bifidobacterium metabolizaron alguno de los OLHs testados. Bifidobacterium infantis Y538 consumió eficientemente todos los OLHs testados, mientras que las dos cepas de Bifidobacterium dentium Y510 y Y521 solo metabolizaron lacto-N-tetraosa (LNT) y lacto-N-neotetraosa (LNnT). Se caracterizaron dos ß-galactosidasas de B. dentium Y510; Bdg42A mostró la mayor actividad en LNT, hidrolizándolo en galactosa y lacto-N-triosa (LNTII), mientras que Bdg2A mostró actividad contra lactosa, 6'-galactopiranósil-N-acetilglucosamina, N-acetillactosamina y LNnT. También se aislaron cepas bacterianas con actividad glicosidasa extracelular de las heces de lactantes. B. infantis E17 y E18, y Enterococcus faecalis E8 y E41 exhibieron actividad de endo-ß-N-acetilglucosaminidasa, liberando N-glicanos de glicoproteínas. La endo-ß-N-acetilglucosaminidasa EndoE de E. faecalis E8 deglicosiló eficientemente la proteína S1 del coronavirus 2 del síndrome respiratorio agudo severo (SARS-CoV-2). Tanto la EndoE silvestre como un mutante catalíticamente inactive mostraron actividad lectina frente a la proteína S1 y actividad neutralizante frente a la infección de un virus pseudotipado que presenta la proteína S de SARS-CoV-2 expresada. También se identificaron GHs putativas a través del análisis metagenómico de las heces de niños lactantes, pertenecientes a los géneros Bifidobacterium, Bacteroides, Ruminococcus, Actinomyces, Klebsiella, Phocaeicola y Streptococcus. Se seleccionaron diez ¿-L-fucosidasas GH29 (Fuc18, Fuc19A, Fuc30, Fuc35A, Fuc35B, Fuc39, Fuc193, Fuc1584, Fuc2358 y Fuc5372). Las ¿-L-fucosidasas Fuc18, Fuc19A, Fuc35B, Fuc39 y Fuc1584 mostraron actividad hidrolítica frente a enlaces de fucosa ¿-1,3/4 y Fuc35A, Fuc193 y Fuc2358 mostraron actividad enlaces de fucosa ¿-1,2/3/4/6. Fuc30 mostró actividad sobre la enlaces de fucosa ¿-1,6 mientras que Fuc5372 mostró preferencia por los enlaces ¿-1,2. Fuc2358 mostró actividad frente a glicoconjugados con lacto-N-fucopentaosa II, lacto-N-fucopentaosa III y contra la mucina. Fuc18, Fuc19A y Fuc39 eliminaron fucosa de neoglicoproteínas y de la glicoproteína ¿-1 ácida. Las ¿-L-fucosidasas aisladas fueron evaluadas por su capacidad para sintetizar fucosil-oligosacáridos (FUS) a través de reacciones de transfucosilación. Fuc2358 produjo rendimientos del 35% de 2'-fucosillactosa (2'FL) y también 3'-fucosillactosa (3'FL) y 1-fucosillactosa (1FL). Fuc5372 sintetizó 2'FL, 3'FL y 1FL, con una proporción más alta de 3'FL. Se llevó a cabo mutagénesis dirigida para aumentar los rendimientos de transfucosilación. Los mutantes Fuc2358-H132F, Fuc2358-F184H, Fuc2358-R301Q, Fuc2358-K286R y Fuc5372-R230K mostraron una mayor relación entre la 2'FL producida y el pNP-Fuc hidrolizado que sus respectivas enzimas silvestres. Además, se observó que los residuos F184 de Fuc2358 y W151 de Fuc5378 afectan a la regioselectividad de la transfucosilación, la fenilalanina aumentando la selectividad por los enlaces ¿-1,2 y el triptófano aumentando la selectividad por los enlaces ¿-1,3. Los resultados presentados muestran la diversidad de GHs presentes en la microbiota intestinal de niños lactantes y expanden el conocimiento sobre su especificidad, contribuyendo al conocimiento del posible papel de las GHs en la colonización bacteriana del tracto gastrointestinal y, además, muestra su potencial biotecnológico / [CA] Els oligosacàrids de la llet humana (OLHs) i la part glicana de glicoconjugats són hidrolitzats per les glicosil hidrolases (GHs), les quals són expressades per la microbiota intestinal de xiquets lactants, promovent l'establiment d'una microbiota intestinal amb beneficis per a la seua salut. L'objectiu d'aquesta Tesi Doctoral va ser la caracterització funcional de GHs de la microbiota intestinal de xiquets lactants capaços de metabolitzar OLHs i glicoconjugats i l'estudi de la seua rellevància biològica i el seu potencial biotecnològic. Es van aïllar soques bacterianes a partir de les femtes de xiquets lactants. Només els soques del gènere Bifidobacterium van metabolitzar algun dels OLHs testats. Bifidobacterium infantis Y538 va consumir eficientment tots els OLHs testats. Les dos soques de Bifidobacterium dentium Y510 i Y521 sol van metabolitzar lacto-N-tetraosa (LNT) i lacto-N-neotetraosa (LNnT).Es van caracteritzar dos ß-galactosidasas de B. dentium Y510; Bdg42A va exhibir la major activitat enfront de LNT, hidrolitzant-la en galactosa i lacto-N-triosa (LNTII), mentre que Bdg2A va mostrar activitat enfront de lactosa, 6'-galactopiranósil-N-acetilglucosamina, N-acetillactosamina i LNnT. També es van aïllar soques bacterianes amb activitat glicosidasa extracelul·lar. B. infantis E17 i E18, i Enterococcus faecalis E8 i E41 van exhibir activitat endo-ß-N-acetilglucosaminidasa, alliberant N-glicans de glicoproteïnes. La endo-ß-N-acetilglucosaminidasa EndoE de E. faecalis E8 va deglicosilar eficientment la proteïna S1 del coronavirus 2 del síndrome respiratori agut greu (SARS-CoV-2).Tant la EndoE salvatge com un mutant catalíticament inactiu van mostrar activitat lectina enfront de la proteïna S1 i activitat neutralitzador enfront de la infecció d'un virus pseudotipat que presenta la proteïna S de SARS-CoV-2 expressada. També es van identificar GHs putatives a través de l'anàlisi metagenómic de la femta de xiquets lactants, pertanyents als gèneres Bifidobacterium, Bacteroides, Ruminococcus, Actinomyces, Klebsiella, Phocaeicola i Streptococcus. Es van seleccionar deu ¿-L-fucosidasas GH29 (Fuc18, Fuc19A, Fuc30, Fuc35A, Fuc35B, Fuc39, Fuc193, Fuc1584, Fuc2358 i Fuc5372). Les ¿-L-fucosidasas Fuc18, Fuc19A, Fuc35B, Fuc39 i Fuc1584 van mostrar activitat hidrolítica enfront d'enllaços de fucosa ¿-1,3/4 i Fuc35A, Fuc193 i Fuc2358 van mostrar activitat enllaços de fucosa ¿-1,2/3/4/6. Fuc30 va mostrar activitat enfront d'enllaços de fucosa ¿-1,6 mentre que Fuc5372 va mostrar preferència pels enllaços ¿-1,2. Fuc2358 va mostrar activitat enfront de glicoconjugats amb lacto-N-fucopentaosa II, lacto-N-fucopentaosa III i contra la glicoproteïna de la mucina. Fuc18, Fuc19A i Fuc39 van eliminar fucosa de neoglicoproteïnes i de la glicoproteïna ¿-1 àcida. Les ¿-L-fucosidasas aïllades van ser avaluades per la seua capacitat per a sintetitzar fucosil-oligosacàrids (FUS) mediant reaccions de transfucosilació. Fuc2358 va produir rendiments del 35% de 2'-fucosillactosa (2'FL) i també 3'-fucosillactosa (3'FL) i 1-fucosillactosa (1FL). Fuc5372 va sintetitzar 2'FL, 3'FL i 1FL, amb una proporció més alta de 3'FL. Es va dur a terme mutagénesis dirigida per a augmentar els rendiments de transfucosilación. Els mutants Fuc2358-H132F, Fuc2358-F184H, Fuc2358-R301Q, Fuc2358-K286R i Fuc5372-R230K van mostrar una major relació entre la 2'FL produïda i el pNP-Fuc hidrolitzat que els seus respectius enzims salvatges.A més, els residus F184 de Fuc2358 i W151 de Fuc5378 afecten la regioselectivitat de la transfucosilación; la fenilalanina augmenta la selectivitat pels enllaços ¿-1,2 i el triptòfan augmenta la selectivitat pels enllaços ¿-1,3. Els resultats presentats mostren la diversitat de GHs presents en la microbiota intestinal de xiquets lactants i expandixen el coneixement sobre la seua especificitat, contribuint al coneixement del possible paper de les GHs en la colonització bacteriana del tracte gastrointestinal i, a més, mostra el seu potencial biotecnològic. / [EN] Human milk oligosaccharides (HMOs) and the glycan portion of glycoconjugates are hydrolyzed by glycoside hydrolases (GHs) that are expressed by the neonatal intestinal microbiota, promoting the establishment of an intestinal microbiota with health benefits for infants. The objective of this Doctoral Thesis consisted of the functional characterization of GHs from the intestinal microbiota of breastfed infants capable of metabolizing HMOs and glycoconjugates and the study of their biological relevance and their biotechnological potential. Bacterial strains were isolated from breastfed infant faeces, showing that only Bifidobacterium genus strains metabolized any of the HMOs tested. Bifidobacterium infantis Y538 efficiently consumed all tested HMOs, while the two strains isolated from Bifidobacterium dentium Y510 and Y521 only metabolized lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT). Two ß-galactosidases from B. dentium Y510 were characterized; Bdg42A exhibited the highest activity on LNT, hydrolyzing it into galactose and lacto-N-triose (LNTII), while Bdg2A displayed activity against lactose, 6'-galactopyranosyl-N-acetylglucosamine, N-acetyllactosamine and LNnT. Bacterial strains with extracellular glycosidase activity were also isolated from breastfed infant faeces. B. infantis E17 and E18, and Enterococcus faecalis E8 and E41 exhibited endo-ß-N-acetylglucosaminidase activity, releasing N-glycans from glycoproteins. The endo-ß-N-acetylglucosaminidase EndoE from E. faecalis E8 efficiently deglycosylated the spike S1 protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Both the EndoE wild-type and a catalytically inactive mutant exhibited lectin activity towards the S1 protein and neutralizing activity against SARS-CoV-2 S pseudotyped virus infection. Putative GHs were also identified through metagenomic analysis of breastfed infant faeces, belonging to Bifidobacterium, Bacteroides, Ruminococcus, Actinomyces, Klebsiella, Phocaeicola, and Streptococcus genera. Ten ¿-L-fucosidases GH29 (Fuc18, Fuc19A, Fuc30, Fuc35A, Fuc35B, Fuc39, Fuc193, Fuc1584, Fuc2358, and Fuc5372) were selected. The ¿-L-fucosidases Fuc18, Fuc19A, Fuc35B, Fuc39, and Fuc1584 showed hydrolytic activity on ¿-1,3/4-linked fucose and Fuc35A, Fuc193 and Fuc2358 showed activity on ¿-1,2/3/4/6-linked fucose. Fuc30 displayed activity only on ¿-1,6-linked fucose, and Fuc5372 showed a preference for ¿-1,2-linked fucose. Fuc2358 displayed activity against glycoconjugates carrying lacto-N-fucopentaose II, lacto-N-fucopentaose III and against the mucin glycoprotein. Fuc18, Fuc19A, and Fuc39 removed fucose from neoglycoproteins and human ¿-1 acid glycoprotein. The isolated ¿-L-fucosidases were evaluated for their capacity to synthesize fucosyl-oligosaccharides (FUS) through transfucosylation reactions. Fuc2358 produced 35 % yields of 2'-fucosyllactose (2'FL) and also 3'-fucosyllactose (3'FL) and 1-fucosyllactose (1FL). Fuc5372 synthesized 2'FL, 3'FL and 1FL, with a higher proportion of 3'FL. Site-directed mutagenesis was conducted to increase the transglycosylation yields. Mutants Fuc2358-H132F, Fuc2358-F184H, Fuc2358-R301Q, Fuc2358-K286R and Fuc5372-R230K showed a higher ratio between 2'FL yields and hydrolyzed pNP-Fuc than their respective wild-type enzymes. The transfucosylation activity results also showed that the residues F184 of Fuc2358 and W151 of Fuc5378 affect transfucosylation regioselectivity, with phenylalanine increasing the selectivity for ¿-1,2 linkages and tryptophan for ¿-1,3 linkages. The results presented in this doctoral thesis illustrate the diversity of GHs in the intestinal microbiota of breastfed infants and have expanded our knowledge of their specificities, which could contribute to a better understanding of the possible role of GHs in the bacterial colonization of the infant gastrointestinal tract and presents significant biotechnological potential. / This work is part of the Grant PID2020-115403RB (C21 and C22) funded by the Spanish Ministry of Science and Innovation (MICIN)/Spanish State Research Agency (AEI)/10.13039/501100011033. The study was also supported by Valencian Government grant AICO/2021/033. EMM-G was supported by the Grant PRE2018-085768 funded by MICIN/AEI/10.13039/501100011033 and by “ESF Investing in your future”. / Moya Gonzálvez, EM. (2024). Isolation, Functional Characterization and Biotechnological Applications of Glycoside Hydrolases from the Intestinal Microbiota of Breastfed Infants [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/203171 / Compendio

Nous avons démontré l’utilité du groupement protecteur tert-butylsulfonyle (N-Bus) pour la chimie des acides aminés et des peptides. Celui-ci est préparé en deux étapes, impliquant la réaction d’une amine avec le chlorure de tert-butylsulfinyle, suivie par l’oxydation par du m-CPBA, pour obtenir les tert-butylsulfonamides correspondants avec d’excellents rendements. Le groupement N-Bus peut être clivé par traitement avec 0.1 N TfOH/DCM/anisole à 0oC en 10h pour régénérer le sel d’ammonium. Une variété d’acides aminés N-Bus protégés ainsi que d’autres aminoacides peuvent alors être utilisés pour préparer divers dipeptides et tripeptides. A l’exception du groupe N-Fmoc, les conditions de déprotection du groupe N-Bus clivent également les groupements N-Boc, N-Cbz et O-Bn. Une déprotection sélective et orthogonale des groupes N-Boc, N-Cbz, N-Fmoc et O-Bn est également possible en présence du groupe protecteur N-Bus. Le nouvel acide aminé non-naturel (3R, 2R) 3–méthyl-D-leucine (β-Me-Leu) et son régioisomère 2-méthyle ont été synthétisés par ouverture d’une N-Ts aziridine en présence d’un excès de LiMe2Cu. Chacun des régioisomères du mélange (1:1,2) a été converti en la méthylleucine correspondante, puis couplé à l’acide D-phényllactique puis au motif 2-carboxyperhydroindole 4-amidinobenzamide en présence de DEPBT. Des élaborations ultérieures ont conduit à des analogues peptidiques non-naturels d’aeruginosines telles que la chlorodysinosine A. Les deux analogues ont ensuite été évalués pour leur activité inhibitrice de la thrombine et la trypsine. La présumée aeruginosine 3-sulfate 205B et son anomère β ont été synthétisés avec succès à partir de 5 sous-unités : la 3-chloroleucine, l’acide D-phényllactique, le D-xylose, le 2-carboxy-6-hydroxyoctahydroindole et l’agmatine. La comparaison des données RMN 1H et 13C reportées avec celles obtenues avec l’aeruginosine synthétique 205B révèle une différence majeure pour la position du groupe présumé 3'-sulfate sur l’unité D-xylopyranosyle. Nous avons alors synthétisés les dérivés méthyl-α-D-xylopyranosides avec un groupement sulfate à chacune des positions hydroxyles, afin de démontrer sans ambiguïté la présence du sulfate en position C-4' par comparaison des données spectroscopiques RMN 1H et 13C. La structure de l’aeruginosine 205B a alors été révisée. Une des étapes-clés de cette synthèse consiste en la formation du glycoside avec le groupe hydroxyle en C-6 orienté en axial sur la sous-unité Choi. Le 2-thiopyridylcarbonate s’est avéré une méthode efficace pour l’activation anomérique. Le traitement par AgOTf et la tétraméthylurée en solution dans un mélange éther-DCM permet d’obtenir l’anomère α désiré, qui peut alors être aisément séparé de l’anomère β par chromatographie / We have demonstrated the usefulness of tert-butylsulfonyl (N-Bus) protecting group in amino acid and peptide chemistry. It is formed in a 2-step procedure involving reaction of an amine with tert-butylsulfinyl chloride, followed by oxidation with m-CPBA to obtain the corresponding tert-butyl- sulfonamides in excellent yields. The N-Bus group can be cleaved to regenerate the corresponding amino salt in 0.1 N TfOH/DCM/anisole at 0 oC for 10 h. A variety of N-Bus protected amino acids and other common amino acids can be used to form dipeptides and tripeptides. With the exception of the N-Fmoc group, the conditions required for the N-Bus group cleavage also cleaved the N-Boc, N-Cbz and O-Bn groups. Selective and orthogonal deprotection of N-Boc, N-Cbz, N-Fmoc and O-Bn groups could be achieved in the presence of the N-Bus protecting group. The new unnatural amino acids (3R, 2R) 3–methyl-D-leucine (β-Me-Leu) and its 2-methyl regioisomer were synthesized by ring opening of an N-Ts aziridine intermediate with excess LiMe2Cu. The 1:1.2 mixture of regioisomers were each converted to the corresponding methyl leucines, then coupled to D-phenyllactic acid, followed by coupling with 2-carboxyperhydroindole 4-amidino-benzamide core in the presence of DEPBT. Further elaboration led to linear peptidic unnatural analogues of known aeruginosins such as chlorodysinosin A. The two analogues were also evaluated in enzymatic assays for their inhibitory activity against thrombin and trypsin. The presumed 3-sulfated aeruginosin 205B and its β–anomer were successfully synthesized from 5 subunits: 3-chloroleucine, D-phenyllactic acid, D-xylose, 2-carboxy-6-hydroxyoctahydroindole, and agmatine. Comparison of 1H and 13C NMR reported data with that of synthetic aeruginosin 205B revealed a disturbing discrepancy with regard to the position of the presumed 3'-sulfate on the D-xylopyranosyl unit. We synthesized methyl α-D-xylopyranosides with sulfates at each of the hydroxyl groups and conclusively demonstrated the the presence of a C-4'-sulfate by comparison of the 1H and 13C NMR spectroscopic data. Thus, the structure of aeruginosin 205B should be revised. One of the key steps in the synthesis is glycoside formation of the axially oriented C-6 hydroxyl group in the Choi subunit. The 2-thiopyridyl carbonate was a suitable method for anomeric activation, followed by treatment with AgOTf and tetramethylurea in ether-DCM solution to give the desired α-anomer, which was easily separable from the β-anomer by column chromatography.

Les glucides constituent la classe de molécules organiques la plus abondante et ceux-ci jouent des rôles cruciaux dans divers processus biologiques. De part leur importance médicinale, la préparation des désoxy-sucres, des C-glycosides et des C-disaccharides est devenue un sujet de pointe en synthèse organique. De façon générale, cette thèse décrit une nouvelle synthèse de novo des 4-désoxy hexopyrannoses en plus de la préparation de C-glycosides biologiquement actifs. De plus, une attention particulière a été portée à la préparation de novo de 4-désoxy-C-disaccharides. Dans un premier temps, le catalyseur de Cr(III) de Jacobsen et un complexe binaphtol/titane ont été utilisés pour réaliser des hétéro-Diels-Alder énantiosélectives. Les dihydropyrannes ainsi générés ont été transformés en 4-désoxy hexopyrannoses présents dans la nature. De cette façon, un dérivé de l’acide ézoaminuroïque, un précurseur de la désosamine et de la néosidomycine, a été préparé suivant cette approche de novo. De plus, à titre comparatif, la néosidomycine a également été fabriquée selon une approche chiron, à partir du méthyl alpha-D-mannopyrannoside. Finalement, une évaluation biologique préliminaire de la néosidomycine a été effectuée sur une la concanavaline-A (Chapitre 2). Dans un deuxième temps, une allylation stéréosélective sur un aldéhyde lié via des liens C-C à une unité mannoside a permis de générer un alcool homoallylique. Cette dernière fonctionnalité a été transformée en 4-désoxy hexopyrannose de configuration D ou L. De cette façon, la préparation de pseudo 4-désoxy-C-disaccharides, de 4-désoxy-C-disaccharides et de pseudo 4-désoxy aza-C-disaccharides a facilement été réalisée. Les rapports diastéréoisomériques de la réaction d’allylation ont été déterminés en plus de la configuration absolue des nouveaux centres stéréogéniques formés. La transformation des alcools homoallyliques en pyrannes poly hydroxylés ou en lactames poly hydroxylés a été réalisée, en plus de la déprotection de certains membres de cette famille pour une évaluation biologique préliminaire sur la concanavaline-A (Chapitre 3). Finalement, la synthèse de C-glycosides biologiquement actifs a été réalisée selon deux volets: i) préparation de 3-C-mannopyrannosyl coumarines et ii) synthèse de C-galactosides, inhibiteurs de la lectine PA-IL. Pour ce faire, le couplage de Heck a été utilisé à partir d’un ester alpha,bêta-insaturé, attaché à une unité glycosidique via des liens C-C, pour générer un dérivé glycosyl cinnamate de méthyle. Cependant, lorsque le 2-iodophénol est utilisé comme partenaire de Heck, la coumarine correspondante a été isolée. Les dérivés C-galactopyrannosyl cinnamates de méthyle représentent de bons inhibiteurs monovalents de la PA-IL avec un Kd aussi bas que 37 micro M (Chapitre 4). / Carbohydrates represent a large family of organic molecules that play key roles in various biological processes. Due to their medicinal importance, preparation of deoxy-sugars, C-glycosides and C-disaccharides have become an important topic in organic synthesis. Mostly, this thesis presents a new de novo synthesis of 4-deoxy hexopyranoses, along with the preparation of biologically relevant C-glycosides. Moreover, a special attention has been focussed on the de novo synthesis of 4-deoxy-C-disaccharides. Firstly, Jacobsen Cr(III) catalyst and a binaphthol/titanium complex have been used to catalyze enantioselective hetero-Diels-Alder reactions. The dihydropyran thus formed has been transformed into naturally occurring 4-deoxy hexopyranoses. Therefore, the ezoaminuroic acid core, a desosamine precursor and neosidomycin have been prepared following a de novo approach. Moreover, as a comparative study, neosidomycin has also been synthesized using a chiron approach from methyl alpha-D-mannopyranoside. Finally, a preliminary biological evaluation of neosidomycin has been applied on concanavalin-A (Chapter 2). Secondly, homoallylic alcohols have been generated from a stereoselective allylation on aldehyde linked via C-C bonds to a mannoside residue. Then, the homoallylic alcohols have been transformed into 4-deoxy hexopyranoses in various configurations (D or L). Thereby, the syntheses of pseudo 4-deoxy-C-disaccharides, 4-deoxy-C-disaccharides and pseudo 4-deoxy aza-C-disaccharides have been easily performed. Determinations of the diastereoisomeric ratio of the allylation reactions along with the absolute configuration of the newly formed chiral center have been easily achieved. Various members of this new family have been deprotected for a preliminary biological evaluation on concanavalin-A (Chapter 3). Finally, the syntheses of relevant C-glycosides have been realized regarding two aspects: i) 3-C-mannopyranosyl coumarin synthesis and ii) synthesis of C-galactosides as PA-IL inhibitors. Methyl glycosyl cinnamates have been isolated using a Heck coupling on alpha,beta-insaturated ester linked to glycosidic moieties via C-C bonds. However, when 2-iodophenol is used as a Heck partner, the corresponding coumarins have been isolated. C-Galactosyl methyl cinnamate derivatives represent good monovalent inhibitors with Kd as low as 37 micro M against PA-IL (Chapter 4).

Le développement d'une médecine personnalisée nécessite la mise au point de sondes permettant la détection des marqueurs d'une pathologie au stade le plus précoce possible. Ce mémoire décrit la synthèse et l'évaluation de sondes ciblées permettant de réaliser des images en IRM ou en scintigraphie. Ces sondes sont construites par fonctionnalisation d'un châssis moléculaire préparé par une réaction à trois composants. La propargylamine résultante possède plusieurs points d'ancrage pour introduire les ligands destinés au ciblage et deux groupes chélatants pour l'imagerie. Ce châssis possède aussi en position terminale un groupement réactif pour le fixer sur des petites molécules polyfonctionnelles, des dendrimères ou des protéines. Une technique de criblage à flux rapide, basée sur l'utilisation des puces à petites molécules, a été utilisée pour la détection des ligands. Les molécules déposées sur la puce proviennent soit des C-glycosides complexes préparés selon le concept de la synthèse orientée vers la diversité, soit de la chimiothèque du laboratoire. Ces puces ont permis l'identification de ligands de plusieurs protéines comme la protéine VPR de la capside du virus du sida. Un criblage secondaire par RMN a confirmé l'interaction protéine-petite molécule. Les sondes portant un flavonoïde pour cibler les cellules tumorales de l'endothélium ont été traitées par une solution de sel de gadolinium puis leurs propriétés ont été évaluées par IRM. Les premiers résultats indiquent que ces composés offrent un contraste de 2 à 4 fois supérieurs aux agents de contraste usuellement utilisés. Leur évaluation biologique sera réalisée prochainement.

ABSTRACT The xyloglucan endo-transglycosylase/hydrolase (XTH) genes are found in all vascular and some nonvascular plants. The XTH genes encode proteins which comprise a subfamily of glycoside hydrolase (GH) family 16 in the Carbohydrate-Active enZYmes (CAZY) classification. The XTH gene products are believed to play intrinsic role in cell wall modification during growth and development throughout the lifetime of the plant. In the present investigation, biochemical and reverse genetic approaches were used to better understand the functions of individual members of the XTH gene family of two important plants: the model organism Arabidopsis thaliana and the grain crop barley (Hordeum vulgare). A phylogenetic tree of the xyloglucan-active enzymes of GH16 has previously been constructed, where enzymes with similar activities have been shown to cluster together. Several members of phylogenetic Group I/II and III-B, predicted to exhibit xyloglucan endo-transglycosylase activity (EC 2.4.1.207) and members of Group III-A, predicted to exhibit xyloglucan endo-hydrolase activity (EC 3.2.1.151), were included to analyze the functional diversity of XTH gene products. A heterologous expression system using the yeast Pichia pastoris was found to be effective for recombinant protein production with a success rate of ca. 50%. XTH gene products were obtained in soluble and active forms for subsequent biochemical characterization. In order to be able to screen larger numbers of protein producing clones, a fast and easy method is required to identify clones expressing active protein in high enough amounts. Thus, a miniaturized XET/XEH assay for high-throughput analysis was developed, which was able to identify activities with good precision and with a reduced time and materials consumption and a reduced work load. Enzyme kinetic analysis indicated that the XET or XEH activity of all XTH gene products characterized in the present study corresponded to predictions based on the previously revised phylogenetic clustering. To gain insight into the biological function of the predominant XEHs AtXTH31 and AtXTH32, which are highly expressed in rapidly developing tissues, a reverse genetic approach was employed using T-DNA insertion lines of the A. thaliana Columbia ecotype. Genotypic and phenotypic characterization, together with in situ assays of XET and XEH activities, in single- and double-knock-out mutants indicated that these Group III-A enzymes are active in expanding tissues of the A. thaliana roots and hypocotyl.  Although suppression of in muro XEH activity was clearly observed in the double-knock-out, no significant growth phenotype was observed, with the exception that radicle emergence appeared to be faster than in the wild type plants. Keywords: Arabidopis thaliana, Hordeum vulgare, plant cell wall, xyloglucan, glycoside hydrolase family 16, xyloglucan endo-transglycosylase/hydrolase gene family, xyloglucan endo-transglycosylase, xyloglucan endo-hydrolase, heterologous protein expression, Pichia pastoris, T-DNA insertion, in situ XET/XEH assay, high-throughput screening / QC 20110114

This thesis examines the cytochrom P450 (CYP) drug metabolizing enzyme inhibition and antimicrobial properties of 46 common food plants available in the Canadian Market and the inhibitory properties of 17 traditional Cree antidiabetic medicines on aldose reductase. Inhibitory activity profiles of CYP 3A4, 3A5, 3A7 and 2D6 were created for the 46 samples. The most active plants in the CYP inhibition assay were the spices, belonging to the Apiaceae and Lamiaceae. Similarly, the most active plants in the antimicrobial assay were also the Apiaceae and Lamiaceae. Swine lens homogenate was tested as a novel model for the aldose reductase inhibition assay. Several Cree plants selected for the aldose reductase study showed a high activity, primarily in samples which also contained high levels of phenolics. A positive correlation was observed between total phenolics content and aldose reductase inhibition r2=0.44, p=0.05. Crude extracts of Rhododendron groenlandicum exhibited inhibitory activities of 35.11 ± 0.16 %. The subfractionation and HPLC analysis of R. groenlandicum revealed high levels of phenolics compounds including, catechin, epicatechin, quercetin and quercetin glycosides. This study found that medicinal and food plants contain phytochemicals that may have both beneficial and detrimental biological effects. / Nous avons étudié dans cette thèse les capacités de 46 plantes comestibles, disponibles sur le marché canadien, à inhiber le cytochrome P450 (CYP), enzyme responsable du métabolisme des médicaments, les propriétés antimicrobiennes, et les propriétés inhibitrices de l'aldose réductase à partir de 17 médicaments antidiabétiques traditionnellement utilisés par les Cris. Les profils de l'activité inhibitrice du CYP 3A4, 3A5, 3A7 et 2D6 ont été réalisés pour les 46 plantes à l'étude. Les plantes les plus actives dans le test d'inhibition du CYP furent les épices, plantes appartenant aux familles des Apiaceae et Lamiaceae. De même, les plantes les plus actives dans le bioessai antimicrobien furent aussi les plantes de ces deux mêmes familles. Un homogénat de cristallin de porc a été utilisé comme modèle nouveau pour le test d'inhibition de l'aldose réductase. Plusieurs plantes, utilisées par la nation Cri, qui ont été sélectionnées pour l'étude ont montré une forte activité inhibitrice de l’aldose réductase, principalement dans les échantillons qui contenaient des teneurs élevées en composés phénoliques. Une corrélation positive a été observée entre la teneur totale en composés phénoliques et l'inhibition de l'aldose réductase (r2 = 0.44, p = 0.05). Des extraits bruts de Rhododendron groenlandicum ont montré des activités inhibitrices de 35.11 ± 0.16%. Le sous-fractionnement et l'analyse HPLC de R. groenlandicum ont aussi révélé des teneurs élevées des composés phénoliques, incluant la catéchine, l'épicatéchine, la quercétine et les glycosides de quercétine. Cette étude a montré que les plantes médicinales et alimentaires contiennent des composés phytochimiques qui peuvent avoir à la fois des effets biologiques bénéfique et préjudiciable.

This thesis examines the cytochrom P450 (CYP) drug metabolizing enzyme inhibition and antimicrobial properties of 46 common food plants available in the Canadian Market and the inhibitory properties of 17 traditional Cree antidiabetic medicines on aldose reductase. Inhibitory activity profiles of CYP 3A4, 3A5, 3A7 and 2D6 were created for the 46 samples. The most active plants in the CYP inhibition assay were the spices, belonging to the Apiaceae and Lamiaceae. Similarly, the most active plants in the antimicrobial assay were also the Apiaceae and Lamiaceae. Swine lens homogenate was tested as a novel model for the aldose reductase inhibition assay. Several Cree plants selected for the aldose reductase study showed a high activity, primarily in samples which also contained high levels of phenolics. A positive correlation was observed between total phenolics content and aldose reductase inhibition r2=0.44, p=0.05. Crude extracts of Rhododendron groenlandicum exhibited inhibitory activities of 35.11 ± 0.16 %. The subfractionation and HPLC analysis of R. groenlandicum revealed high levels of phenolics compounds including, catechin, epicatechin, quercetin and quercetin glycosides. This study found that medicinal and food plants contain phytochemicals that may have both beneficial and detrimental biological effects. / Nous avons étudié dans cette thèse les capacités de 46 plantes comestibles, disponibles sur le marché canadien, à inhiber le cytochrome P450 (CYP), enzyme responsable du métabolisme des médicaments, les propriétés antimicrobiennes, et les propriétés inhibitrices de l'aldose réductase à partir de 17 médicaments antidiabétiques traditionnellement utilisés par les Cris. Les profils de l'activité inhibitrice du CYP 3A4, 3A5, 3A7 et 2D6 ont été réalisés pour les 46 plantes à l'étude. Les plantes les plus actives dans le test d'inhibition du CYP furent les épices, plantes appartenant aux familles des Apiaceae et Lamiaceae. De même, les plantes les plus actives dans le bioessai antimicrobien furent aussi les plantes de ces deux mêmes familles. Un homogénat de cristallin de porc a été utilisé comme modèle nouveau pour le test d'inhibition de l'aldose réductase. Plusieurs plantes, utilisées par la nation Cri, qui ont été sélectionnées pour l'étude ont montré une forte activité inhibitrice de l’aldose réductase, principalement dans les échantillons qui contenaient des teneurs élevées en composés phénoliques. Une corrélation positive a été observée entre la teneur totale en composés phénoliques et l'inhibition de l'aldose réductase (r2 = 0.44, p = 0.05). Des extraits bruts de Rhododendron groenlandicum ont montré des activités inhibitrices de 35.11 ± 0.16%. Le sous-fractionnement et l'analyse HPLC de R. groenlandicum ont aussi révélé des teneurs élevées des composés phénoliques, incluant la catéchine, l'épicatéchine, la quercétine et les glycosides de quercétine. Cette étude a montré que les plantes médicinales et alimentaires contiennent des composés phytochimiques qui peuvent avoir à la fois des effets biologiques bénéfique et préjudiciable.

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