Enzymatic Synthesis and Molecular Modelling Studies of Rhamnose Esters Using Lipase from Pseudomonas stutzeri.

Rhamnolipids are becoming an important class of glycolipid biosurfactants. Herein, we describe for the first time the enzymatic synthesis of rhamnose fatty acid esters by the transesterification of rhamnose with fatty acid vinyl esters, using lipase from Pseudomonas stutzeri as a biocatalyst. The use of this lipase allows excellent catalytic activity in the synthesis of 4-O-acylrhamnose (99% conversion and full regioselectivity) after 3 h of reaction using tetrahydrofuran (THF) as the reaction media and an excess of vinyl laurate as the acyl donor. The role of reaction conditions, such as temperature, the substrates molar ratio, organic reaction medium and acyl donor chain-length, was studied. Optimum conditions were found using 35 °C, a molar ratio of 1:3 (rhamnose:acyldonor), solvents with a low logP value, and fatty acids with chain lengths from C4 to C18 as acyl donors. In hydrophilic solvents such as THF and acetone, conversions of up to 99-92% were achieved after 3 h of reaction. In a more sustainable solvent such as 2-methyl-THF (2-MeTHF), high conversions were also obtained (86%). Short and medium chain acyl donors (C4-C10) allowed maximum conversions after 3 h, and long chain acyl donors (C12-C18) required longer reactions (5 h) to get 99% conversions. Furthermore, scaled up reactions are feasible without losing catalytic action and regioselectivity. In order to explain enzyme regioselectivity and its ability to accommodate ester chains of different lengths, homology modelling, docking studies and molecular dynamic simulations were performed to explain the behaviour observed.

Efficient Synthesis of Muramic and Glucuronic Acid Glycodendrimers as Dengue Virus Antagonists.

Carbohydrates are involved in many important pathological processes, such as bacterial and viral infections, by means of carbohydrate-protein interactions. Glycoconjugates with multiple carbohydrates are involved in multivalent interactions, thus increasing their binding strengths to proteins. In this work, we report the efficient synthesis of novel muramic and glucuronic acid glycodendrimers as potential Dengue virus antagonists. Aromatic scaffolds functionalized with a terminal ethynyl groups were coupled to muramic and glucuronic acid azides by click chemistry through optimized synthetic strategies to afford the desired glycodendrimers with high yields. Surface Plasmon Resonance studies have demonstrated that the compounds reported bind efficiently to the Dengue virus envelope protein. Molecular modelling studies were carried out to simulate and explain the binding observed. These studies confirm that efficient chemical synthesis of glycodendrimers can be brought about easily offering a versatile strategy to find new active compounds against Dengue virus.

Acceptor Specificity of ß-N-Acetylhexosaminidase from Talaromyces flavus: A Rational Explanation.

Fungal ß-N-acetylhexosaminidases, though hydrolytic enzymes in vivo, are useful tools in the preparation of oligosaccharides of biological interest. The ß-N-acetylhexosaminidase from Talaromyces flavus is remarkable in terms of its synthetic potential, broad substrate specificity, and tolerance to substrate modifications. It can be heterologously produced in Pichia pastoris in a high yield. The mutation of the Tyr470 residue to histidine greatly enhances its transglycosylation capability. The aim of this work was to identify the structural requirements of this model ß-N-acetylhexosaminidase for its transglycosylation acceptors and formulate a structure-activity relationship study. Enzymatic reactions were performed using an activated glycosyl donor, 4-nitrophenyl N-acetyl-ß-d-glucosaminide or 4-nitrophenyl N-acetyl-ß-d-galactosaminide, and a panel of glycosyl acceptors of varying structural features (N-acetylglucosamine, glucose, N-acetylgalactosamine, galactose, N-acetylmuramic acid, and glucuronic acid). The transglycosylation products were isolated and structurally characterized. The C-2 N-acetamido group in the acceptor molecule was found to be essential for recognition by the enzyme. The presence of the C-2 hydroxyl moiety strongly hindered the normal course of transglycosylation, yielding unique non-reducing disaccharides in a low yield. Moreover, whereas the gluco-configuration at C-4 steered the glycosylation into the ß(1-4) position, the galacto-acceptor afforded a ß(1-6) glycosidic linkage. The Y470H mutant enzyme was tested with acceptors based on ß-glycosides of uronic acid and N-acetylmuramic acid. With the latter acceptor, we were able to isolate and characterize one glycosylation product in a low yield. To our knowledge, this is the first example of enzymatic glycosylation of an N-acetylmuramic acid derivative. In order to explain these findings and predict enzyme behavior, a modeling study was accomplished that correlated with the acquired experimental data.

Novel DNA Aptamers Against CCL21 Protein: Characterization and Biomedical Applications for Targeted Drug Delivery to T Cell-Rich Zones.

The chemokine (C-C motif) ligand 21 (CCL21) is a cytokine that attracts CCR7-positive cells to the T cell (paracortical) zone of lymph nodes by directional migration of these cells along the CCL21 gradient. In this article, we sought to mimic this chemotactic mechanism, by identifying a novel aptamer that binds CCL21 with high affinity. In vitro selection of DNA aptamers was performed by the Systematic Evolution of Ligands by Exponential Enrichment. Quantitative polymerase chain reaction (qPCR) and enzyme-linked oligonucleotide assay were used to screen for high-affinity aptamers against human and mouse CCL21 protein, respectively. Three such aptamers were identified. Surface plasmon resonance showed equilibrium dissociation constant (Kd) for these three aptamers in the nano to picomolar range. Cytotoxicity assays showed <10% toxicity in HEK293 and HL-60 cells. Last, in vivo biodistribution was successfully performed and CCL21 chemokine-binding aptamers were quantified within the draining lymph nodes and spleen using qPCR. Fluorescence microscopy revealed that one of the aptamers showed significantly higher presence in the paracortex than the control aptamer. The use of anti-CCL21 aptamers to mimic the chemotaxis mechanism thus represents a promising approach to achieve targeted delivery of drugs to the T cell-rich zones of the lymph node. This may be important for the treatment of HIV infection and the eradication of HIV reservoirs.