Carbohydrate Residues Research Articles

A two-step approach to the hydrothermal gasification of carbohydrate-rich wastes: Process design and economic evaluation

A two-step approach to hydrothermal gasification of carbohydrate-rich wastes and wastewaters is a promising route for H2 production and simultaneously as a water clean-up technology. Experimental data and kinetic models are used to further develop the process for industrial scale. In this work, a preliminary process design is conducted in order to assess the market potential of the two-step process. For stabilisation, two cases are considered: the use of excess vs. stoichiometric H2, and for gasification, the utilisation of sequential reactors for gasification housing Pt and Ru catalysts is compared to a single reactor with Pt alone. A total of four options are conceptually designed and economically evaluated. Using state-of-the-art insights and process techniques and the current market scenario, a minimum H2 selling price of 3.4 $ kg−1 was obtained.A sensitivity study showed that the feedstock price, concentration and quantity, played a crucial role in the selling price of H2. These variables are all correlated and are dependent on the industry from where the feedstock is obtained. Industrial wastewater streams rich in carbohydrate residues and associated with gate fees were found to be promising feedstock for the process.Further advancement in the areas of catalyst development (hydrothermal stability, affordability) as well as increased H2 yields are necessary in order to improve the economics of this process on industrial scale.

Colorimetric Sensor Array Based on Wulff-Type Boronate Functionalized AgNPs at Various pH for Bacteria Identification

The efficient identification of bacteria is of considerable significance in clinical diagnosis. Herein, a novel colorimetric sensor array was developed for the detection and identification of bacteria based on the specific affinity and electrostatic interaction between Wulff-type 4-mercaptophenylboronic acid (MPBA)-mercaptoethylamine (MA) cofunctionalized AgNPs (MPBA-MA@AgNPs) and bacteria at various pH. In the neutral and alkaline conditions, AgNPs tended to be dispersed due to the specific affinity between cis-diol residues contained in carbohydrate-rich compositions on the bacterial cell surface and MPBA. Bacterial cells have different carbohydrate compositions on their surface. The differential binding affinity of MPBA on the surface of AgNPs to cis-diol residues of various carbohydrates resulted in a different color change of AgNPs, which could be tuned by pH. On the contrary, AgNPs tended to be aggregated due to the electrostatic interaction between positively charged MA and negatively charged bacteria under acidic conditions. Therefore, using various pH buffer solutions as the sensing elements and MPBA-MA@AgNPs as the indicator, bacteria could be differentiated from each other by their own color response patterns. Moreover, the complex bacteria mixtures could be well discriminated. The method is simple, efficient, and visual and has a potential application in pathogen diagnosis.

Lectin histochemical study of the quill sebaceous gland in the dorsal skin of the Sunda porcupine (Hystrix javanica)

Abstract. Prawira AY, Novelina S, Farida WR, Darusman HS, Agungpriyono S. 2019. Lectin histochemical study of the quill sebaceous gland in the dorsal skin of the Sunda porcupine (Hystrix javanica). Biodiversitas 20: 2677-2684. In the Sunda Porcupine skin, the sebaceous glands of the quill follicles are multi-lobed alveolar and better developed than those of hair follicles. Using lectin histochemistry, we have studied the distribution of sugar-binding in the sebaceous glands of quills in comparison with those of hairs in six adult Sunda Porcupines. The skin samples from the thoracodorsal and lumbosacral regions were collected by biopsy procedure and processed for histology, histochemistry, and lectin histochemistry. The results showed that the lectin binding patterns are similar in secretory acinar parts of both types of sebaceous glands. The acini and excretory duct contained neutral carbohydrate and sugar residues. The acini also contained alpha D-mannose sugar residue, while the non-secretory excretory duct and debris in the lumen contained alpha-D-mannose, alpha-L-fucose, and α>β-N-acetylgalactosamine, and complex type N-glycan (oligosaccharide) sugar residues, as well. The present findings allowed us to suggest, that in Sunda Porcupine functions of the sebaceous glands of quills are more complex and active compared to those of the hairs.

Microanatomy of the American Malaria Vector Anopheles aquasalis (Diptera: Culicidae: Anophelinae) Midgut: Ultrastructural and Histochemical Observations.

The mosquito gut is divided into foregut, midgut, and hindgut. The midgut functions in storage and digestion of the bloodmeal. This study used light, scanning (SEM), and transmission (TEM) electron microscopy to analyze in detail the microanatomy and morphology of the midgut of nonblood-fed Anopheles aquasalis females. The midgut epithelium is a monolayer of columnar epithelial cells that is composed of two populations: microvillar epithelial cells and basal cells. The microvillar epithelial cells can be further subdivided into light and dark cells, based on their affinities to toluidine blue and their electron density. FITC-labeling of the anterior midgut and posterior midgut with lectins resulted in different fluorescence intensities, indicating differences in carbohydrate residues. SEM revealed a complex muscle network composed of circular and longitudinal fibers that surround the entire midgut. In summary, the use of a diverse set of morphological methods revealed the general microanatomy of the midgut and associated tissues of An. aquasalis, which is a major vector of Plasmodium spp. (Haemosporida: Plasmodiidae) in America.

Intrinsic dynamic behavior of enzyme:substrate complexes govern the catalytic action of \u03b2-galactosidases across clan GH-A

The conformational itineraries taken by carbohydrate residues in the catalytic subsite of retaining glycoside hydrolases (GHs), harness the link between substrate conformation and reactivity. GHs’ active sites may be described as a combination of subsites dedicated to the binding of individual sugar residues and to catalysis. The three-dimensional structure of GH:carbohydrate complexes has demonstrated that carbohydrate ring conformation changes in an ordered manner during catalysis. Here we demonstrate in silico that a link exists between subsite binding dynamics and substrate specificity for β-galactosidases from clan GH-A families GH1, GH2, GH35, GH42 and GH59. Different oligosaccharides were docked in the active site of reference β-galactosidase structures using Vina-Carb. Subsequent molecular dynamics (MD) simulations revealed that these enzymes favor a high degree of flexibility and ring distortion of the substrate the lytic subsite −1. Although the β-galactosidase families examined are structurally and mechanistically related, distinct patterns of ring distortion were unveiled for the different families. For β-galactosidases, three different family-dependent reaction itineraries (1S3 → 4H3‡ → 4C1, 1,4B → 4H3/ 4E‡ → 4C1, and 1S5 → 4E/ 4H5‡ → 4C1) were identified, all compatible with the antiperiplanar lone pair hypothesis (ALPH) for the hydrolysis of β-glycosides. This comparative study reveals the fuzzy character of the changes in carbohydrate ring geometry prior to carbohydrate hydrolysis.

Abstract 581: Determining the influence of galectin-3 binding properties on its suppression of tumor antigen-specific CD8+ T cells

Abstract Improving the efficacy of immunotherapeutic cancer treatments requires increased understanding of the factors that contribute to a suppressive tumor microenvironment (TME). Recently, galectin-3 has emerged as an important player in the development and progression of cancer, favoring immune suppression and dysfunction at disease sites. While this protein is consistently identified as a tumor-promoting factor, its unique properties make the mechanisms of its functions difficult to study, and impede the development of therapies against it. Galectin-3 binds β-galactoside carbohydrate residues through a highly-conserved C-terminal domain (CRD) characteristic of mammalian galectins. It is made unique by its ability to self-associate into homo-oligomers through a disordered N-terminal domain (NTD). We have previously shown that galectin-3 knockout in a mouse model of tumor tolerance improves effector function of antitumor CD8+ T cells and increases duration of tumor-free survival, suggesting a suppressive role for galectin-3 in antitumor immunity. Our current work aims to elucidate how the distinct biochemical properties of galectin-3 contribute to this suppression. To this end, we are developing a new assay to model the suppression of CD8+ T cells by galectin-3 in vitro. Our assay utilizes TCR transgenic CD8+ T cells specific for rodent Erbb2, the Her-2/neu (Neu) oncogene. These cells are isolated from either normal transgenic mice or those deficient in galectin-3. The T cells are stimulated in culture, then co-cultured with NT2.5 Neu-expressing tumor cells. We use flow cytometry to analyze the levels of CD8+ effector molecules interferon gamma (IFNγ) and Granzyme B, as well as cell surface binding of galectin-3 after co-culture. To further elucidate the roles of carbohydrate binding and self-association in this process, we have developed recombinant mutant galectin-3 proteins deficient in carbohydrate binding through CRD point mutation, lacking the ability to self-associate through NTD truncation, or both. By introducing these recombinant proteins into the co-culture with normal or galectin-3 deficient transgenic T cells, we expect to be able to learn whether galectin-3-mediated suppression is more dependent on carbohydrate binding, self-association, or both properties in synergy. Current therapies targeting galectin-3 focus only on its carbohydrate binding properties through use of small molecules and glycomimetics, but these have been of limited efficacy. We hypothesize that self-association of galectin-3 through the NTD is an equally important factor in its unique ability among galectins to contribute to an immunosuppressive TME. Learning how these properties each contribute to the mechanism of galectin-3-mediated immunosuppression will allow us to refine strategies for breaking tumor tolerance, contributing to improved outcomes for cancer immunotherapies. Citation Format: Alexandra B. Pucsek, Todd D. Armstrong, Elizabeth M. Jaffee. Determining the influence of galectin-3 binding properties on its suppression of tumor antigen-specific CD8+ T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 581.

Почему бензилиденовая защитная группа непредпочтительна для гликозидов формилфенолов?

Benzylidene protecting groups are widely used in carbohydrate chemistry, as a rule, to protect the hydroxyl groups at O-4 and O-6 positions of the carbohydrate residue. With varying degrees of selectivity, 4,6-benzylidene protection allows introduction of other functional groups at O-2 and O-3 positions of sugars and is easily removed from the molecule under acidic conditions. We have previously proposed a scheme for obtaining acyl derivatives of natural glycoside salicin (glycoside of salicylic alcohol) from the intermediate glycoside helicin (glycoside of salicylic aldehyde). The aldehyde group can be reduced to alcohol during the final synthesis stages. Moreover, this group does not require additional protection during the modification of the carbohydrate residue, since it does not participate in acylation reactions, unlike the hydroxymethylene group of salicin. For the temporary protection of O-4 and O-6 hydroxyls of helicin, it was proposed to use a benzylidene protecting group. In this paper, we set out to study the reactivity of helicin in the reaction of 4,6-benzylidene formation, as well as to establish factors affecting such processes. Attempts were undertaken to introduce a benzylidene protecting group into a helicin molecule in two ways: using benzaldehyde dimethyl acetal, as well as using benzaldehyde and zinc chloride. In the former case, the conversion of the original glycoside was not observed, while the second reaction resulted in the product of an unidentified structure. Possible reactions occurring with the reagents applied were modelled using quantum-chemical calculations. It is found that the inability of benzaldehyde dimethyl acetal and benzaldehyde to react on 4,6 hydroxyls of glucose is explained by the competing reaction of the aldehyde group in the structure of helicin. Moreover, the catalysis with zinc chloride most likely produces a helicin dimer. Thus, it is found that benzylidene protection has certain limitations and cannot be applied in the case of glycosides containing aromatic aldehydes as the aglycone part.

Galactosyl carbohydrate residues on hematopoietic stem/progenitor cells are essential for homing and engraftment to the bone marrow

The role of carbohydrate chains in leukocyte migration to inflamed sites during inflammation and trafficking to the lymph nodes under physiological conditions has been extensively characterized. Here, we report that carbohydrate chains also mediate the homing and engraftment of hematopoietic stem/progenitor cells (HSPCs) to the bone marrow (BM). In particular, we found that transplanted BM cells deficient in β-1,4-galactosyltransferase-1 (β4GalT-1) could not support survival in mice exposed to a lethal dose of irradiation. BM cells obtained from mice deficient in β4GalT-1 showed normal colony-forming activity and hematopoietic stem cell numbers. However, colony-forming cells were markedly rare in the BM of recipient mice 24 h after transplantation of β4GalT-1-deficient BM cells, suggesting that β4GalT-1 deficiency severely impairs homing. Similarly, BM cells with a point mutation in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase gene, encoding a key enzyme in sialic acid biosynthesis, showed mildly impaired homing and engraftment abilities. These results imply that the galactosyl, but not sialyl residues in glycoproteins, are essential for the homing and engraftment of HSPCs to the BM. These findings suggest the possibility of modifying carbohydrate structures on the surface of HSPCs to improve their homing and engraftment to the BM in clinical application.

Programmable Synthesis of 2-Deoxyglycosides.

Control of glycoside bond stereochemistry is the central challenge in the synthesis of oligosaccharides. 2-Deoxyglycosides, which lack a C2 substituent to guide stereoselectivity, are among the most difficult classes of glycoside bond constructions. Here we present a method to synthesize 2-deoxysaccharides with specified glycoside bond stereochemistry using a nucleophilic carbohydrate residue and the synthetic equivalent of an alcohol electrophile. Because the configuration of the nucleophile can be precisely controlled, both α- and β-glycosides can be synthesized from the same starting material in nearly all cases examined. Stereoselectivities in these reactions are often greater than 50:1 and yields typically exceed 70%. This strategy is amenable to the stereocontrolled syntheses of trisaccharide diastereomers, and a tetrasaccharide. This method may be extensible to other classes of carbohydrates.

Increased Clinical Sensitivity and Specificity of Plasma Protein N-Glycan Profiling for Diagnosing Congenital Disorders of Glycosylation by Use of Flow Injection-Electrospray Ionization-Quadrupole Time-of-Flight Mass Spectrometry.

Congenital disorders of glycosylation (CDG) represent 1 of the largest groups of metabolic disorders with >130 subtypes identified to date. The majority of CDG subtypes are disorders of N-linked glycosylation, in which carbohydrate residues, namely, N-glycans, are posttranslationally linked to asparagine molecules in peptides. To improve the diagnostic capability for CDG, we developed and validated a plasma N-glycan assay using flow injection-electrospray ionization-quadrupole time-of-flight mass spectrometry. After PNGase F digestion of plasma glycoproteins, N-glycans were linked to a quinolone using a transient amine group at the reducing end, isolated by a hydrophilic interaction chromatography column, and then identified by accurate mass and quantified using a stable isotope-labeled glycopeptide as the internal standard. This assay differed from other N-glycan profiling methods because it was free of any contamination from circulating free glycans and was semiquantitative. The low end of the detection range tested was at 63 nmol/L for disialo-biantennary N-glycan. The majority of N-glycans in normal plasma had <1% abundance. Abnormal N-glycan profiles from 19 patients with known diagnoses of 11 different CDG subtypes were generated, some of which had previously been reported to have normal N-linked protein glycosylation by carbohydrate-deficient transferrin analysis. The clinical specificity and sensitivity of N-glycan analysis was much improved with this method. Additional CDGs can be diagnosed that would be missed by carbohydrate-deficient transferrin analysis. The assay provides novel biomarkers with diagnostic and potentially therapeutic significance.

Building and rebuilding N-glycans in protein structure models.

N-Glycosylation is one of the most common post-translational modifications and is implicated in, for example, protein folding and interaction with ligands and receptors. N-Glycosylation trees are complex structures of linked carbohydrate residues attached to asparagine residues. While carbohydrates are typically modeled in protein structures, they are often incomplete or have the wrong chemistry. Here, new tools are presented to automatically rebuild existing glycosylation trees, to extend them where possible, and to add new glycosylation trees if they are missing from the model. The method has been incorporated in the PDB-REDO pipeline and has been applied to build or rebuild 16 452 carbohydrate residues in 11 651 glycosylation trees in 4498 structure models, and is also available from the PDB-REDO web server. With better modeling of N-glycosylation, the biological function of this important modification can be better and more easily understood.

Abrogation of Endogenous Glycolipid Antigen Presentation on Myelin-Laden Macrophages by D-Sphingosine Ameliorates the Pathogenesis of Experimental Autoimmune Encephalomyelitis.

Background: Although myelin is composed of mostly lipids, the pathological role of myelin lipids in demyelinating diseases remains elusive. The principal lipid of the myelin sheath is β-galactosylceramide (β-Galcer). Its α-anomer (α-Galcer) has been demonstrated to be antigenically presented by macrophages via CD1d, a MHC class I-like molecule. Myelin, which is mostly composed of β-Galcer, has been long considered as an immunologically-inert neuron insulator, because the antigen-binding cleft of CD1d is highly α-form-restricted.Results: Here, we report that CD1d-mediated antigenic presentation of myelin-derived galactosylceramide (Mye-GalCer) by macrophages contributed significantly to the progression of experimental autoimmune encephalomyelitis (EAE). Surprisingly, this presentation was recognizable by α-Galcer:CD1d-specific antibody (clone L363), but incapable of triggering expansion of iNKT cells and production of iNKT signature cytokines (IFNγ and IL-4). Likewise, a synthesized analog of Mye-Galcer, fluorinated α-C-GalCer (AA2), while being efficiently presented via CD1d on macrophages, failed to stimulate production of IFNγ and IL-4. However, AA2 significantly exacerbated EAE progression. Further analyses revealed that the antigenic presentations of both Mye-GalCer and its analog (AA2) in α-form via CD1d promoted IL-17 production from T cells, leading to elevated levels of IL-17 in EAE spinal cords and sera. The IL-17 neutralizing antibody significantly reduced the severity of EAE symptoms in AA2-treated mice. Furthermore, D-sphingosine, a lipid possessing the same hydrophobic base as ceramide but without a carbohydrate residue, efficiently blocked this glycolipid antigen presentation both in vitro and in spinal cords of EAE mice, and significantly decreased IL-17 and ameliorated the pathological symptoms.Conclusion: Our findings reveal a novel pathway from the presentation of Mye-GalCer to IL-17 production, and highlight the promising therapeutic potential of D-sphingosine for the human disorder of multiple sclerosis.

Site-specific roles of N-linked oligosaccharides in recombinant eel follicle-stimulating hormone for secretion and signal transduction

Eel follicle-stimulating hormone (eelFSH) is composed of a common α-subunit and a hormone specific β-subunit, both of which contain two N-linked carbohydrate residues. We characterized the biologically active single chains by fusing the α-subunit to the carboxyl terminal region of the eelFSH β-subunit. Expression vectors were constructed and the biological activity of the recombinant hormones (rec-hormones) was characterized using Chinese hamster ovary (CHO) K1 cells expressing the eelFSH receptor gene. Mutagenesis of the individual and double glycosylated sites was performed to determine the functions of the oligosaccharide chains on signal transduction. The absence of the Asn22 (eelFSHβΔ22/α) and Asn5.22 (eelFSHβΔ5.22/α) N-linked oligosaccharide chain in the eelFSH β-subunit completely reduced the secretion level in the medium and cell lysate of CHO-K1 cells. The expression levels of eelFSHβ/α wild-type in CHO suspension (CHO-S) cells was approximately 4-fold higher in CHO-k1 cells. The molecular weight of rec-eelFSHβ/α wild-type by western blotting analysis was found to be 34 kDa. Mutants (β/αΔ56, β/αΔ79, and βΔ5/α) lacking single oligosaccharide sites showed molecular weights that were reduced by approximately 10%. The digestion of N-linked oligosaccharides using PNGaseF treatment showed that the molecular weights of all mutants were reduced to 27-kDa. The oligosaccharide chains in rec-eelFSHβ/α wild-type were modified to a molecular weight of approximately 7–10 kDa in CHO-K1 and CHO-S cells. Oligosaccharide site deletions at positions Asn56 and Asn79 on the α-subunit and Asn5 on the β-subunit were found to play an essential role in cAMP signal transduction through the eelFSH receptor. The EC50 values of Asn56 and Asn5 resulted in a significant decrease in potency to 64% and 53% of the wild type, respectively. Specifically, the removal of the carbohydrates at Asn79 of the α-subunit (β/αΔ79) was drastically reduced to 53.8% of the wild-type levels in maximum response. These results have allowed for the identification of the site-specific roles of carbohydrate residues in eel FSH. Our data suggest that N-linked oligosaccharide chains play a pivotal role in biological activity through the eelFSH receptor as suggested in similar studies of other mammalian FSH hormones.

Chemical composition analysis and structural features of banana rachis lignin extracted by two organosolv methods

Two organosolv methods involving Formic acid/Acetic (FA) acid and Sulphuric acid/Ethanol (SE) solvent mixtures were investigated for lignin extraction from banana rachis biomass residues. Different heating methods were also applied during each extraction process, respectively direct conduction heating and microwave heating. The chemical composition and structural features of the extracted lignin fractions were further analyzed by ATR-FTIR, TGA, elemental analysis and 13C NMR methods. SE extraction method showed a higher extraction yield (58.7%) and allowed also to obtain a lignin fraction with higher purity (76.5% Vs 71.0% for FA lignin). In addition, SE extraction method allowed a higher pulp yield which meant a better selectivity for lignin extraction thanks to the microwave heating method. SE lignin also showed a higher thermal stability due to its higher purity and higher density. The higher molecular weight found for FA lignin residues (7622.7g/mol Vs 5957.7g/mol for SEL) was suspected to be due to co-extracted carbohydrate residues bounded to extracted lignin macromolecules. These results allowed us to establish the SE extraction method (Sulphuric acid/Ethanol/water solvent with microwave heating) as effective for lignin extraction from banana rachis straw.

Differential Glycosylation Expression in Injured Rat Spinal Cord Treated with Immunosuppressive Drug Cyclosporin-A.

Glycosylation is ubiquitous throughout the central nervous system and altered following spinal cord injury (SCI). The glial scar that forms following SCI is composed of several chondroitin sulfate proteoglycans, which inhibit axonal regrowth. Cyclosporin-A (CsA), an immunosuppressive therapeutic, has been proposed as a potential treatment after SCI. We investigated CsA treatment in the spinal cord of healthy, contusion injured, and injured CsA-treated rats. Lectin histochemistry using fluorescently labeled lectins, SBA, MAA, SNA-I, and WFA, was performed to identify the terminal carbohydrate residues of glycoconjugates within the spinal cord. SBA staining decreased in gray and white matter following spinal cord injury, whereas staining was increased at the lesion site in CsA-treated animals, indicating an increase in galactose and N-acetylgalactosamine terminal structures. No significant changes in MAA were observed. WFA staining was abundant in gray matter and observed to increase at the lesion site, in agreement with increased expression of chondroitin sulfate proteoglycans. SNA-I-stained blood vessels in all spinal cord regions and dual staining identified a subpopulation of astrocytes in the lesion site, which expressed α-(2,6)-sialic acid. Glycosylation were altered in injured spinal cord treated with CsA, indicating that glycosylation and alteration of particular carbohydrate structures are important factors to consider in the examination of the environment of the spinal cord after injury.

Post-Glycosylation Diversification (PGD): An Approach for Assembling Collections of Glycosylated Small Molecules.

Many small molecule natural products with antibiotic and antiproliferative activity are adorned with a carbohydrate residue as part of their molecular structure. The carbohydrate moiety can act to mediate key interactions with the target, attenuate physicochemical properties, or both. Facile incorporation of a carbohydrate group on de novo small molecules would enable these valuable properties to be leveraged in the evaluation of focused compound libraries. While there is no universal way to incorporate a sugar on small molecule libraries, techniques such as glycorandomization and neoglycorandomization have made signification headway toward this goal. Here, we report a new approach for the synthesis of glycosylated small molecule libraries. It puts the glycosylation early in the synthesis of library compounds. Functionalized aglycones subsequently participate in chemoselective diversification reactions distal to the carbohydrate. As a proof-of-concept, we prepared several desosaminyl glycosides from only a few starting glycosides, using click cycloadditions, acylations, and Suzuki couplings as diversification reactions. New compounds were then characterized for their inhibition of bacterial protein translation, bacterial growth, and in a T-cell activation assay.

Expeditious Synthesis of C-Glycosyl Barbiturate Ligands of Bacterial Lectins: From Monomer Design to Glycoclusters and Glycopolymers.

The approach developed here offers a straightforward and efficient access to β- C-glycosyl barbiturate ligands, spanning from glycomimetics to multivalent C-neoglycoconjugates, with the aim of deciphering structural parameters impacting the binding to pathogenic lectins. We reinvestigated the Knoevenagel condensation of barbituratic acid on protecting-group free carbohydrates and successfully designed sodium and 5,5-disubstituted N, N-dimethyl barbiturate forms of D-galactose, L-fucose, melibiose, 2'-fucosyllactose, and maltose and evaluated their binding affinity by isothermal titration calorimetry with LecA (galactose-binding lectin) and LecB (fucose-binding lectin) from Pseudomonas aeruginosa and RSL (fucose-binding lectin) from Ralstonia solanacearum. The barbiturate ring was shown to be detrimental for binding to LecA ( KD in mM range) and even more to LecB (noninteraction) while RSL is much more tolerant especially in the presence of an aromatic group ( KD in μM range). However, distancing the barbiturate ring from the recognition carbohydrate residue by using oligosaccharides increased affinity up to low micromolar range. Extension of our convenient synthetic approach led in two steps to melibiose-based C-glycosyl barbiturate cluster and C-glycosyl barbiturate glycopolymers exhibiting a dramatic enhancement of binding avidity for LecA.

Protein Glycosylation through Sulfur Fluoride Exchange (SuFEx) Chemistry: The Key Role of a Fluorosulfate Thiolactoside.

Protein glycosylation is the most complex post-translational modification process. More than 50 % of human cells proteins are glycosylated, whereas bacteria such as E. coli do not have this modification machinery. Indeed, the carbohydrate residues in natural proteins affect their folding, immunogenicity, and stability toward proteases, besides controlling biological properties and activities. It is therefore important to introduce such structural modification in bioengineered proteins lacking the presence of carbohydrate residues. This is not trivial as it requires reagents and conditions compatible with the protein's stability and reactivity. This work reports on the introduction of lactose moieties in two natural proteins, namely ubiquitin (Ub) and l-asparaginase II (ANSII). The synthetic route employed is based on the sulfur(VI) fluoride exchange (SuFEx) coupling of a lactose tethered arylfluorosulfate (Lact-Ar-OSO2 F) with the ϵ-NH2 group of lysine residues of the proteins. This metal-free click SuFEx reaction relies on the properties of the fluorosulfate employed, which is easily prepared in multigram scale from available precursors and reacts chemoselectively with the ϵ-NH2 group of lysine residues under mild conditions. Thus, iterative couplings of Lact-Ar-OSO2 F to Ub and ANSII, afforded multiple glycosylations of these proteins so that up to three and four Lact-Ar-OSO2 groups were introduced in Ub and ANSII, respectively, via the formation of a sulfamoyl (OSO2 -NH) linkage.

Screening of a Library of Oligosaccharides Targeting Lectin LecB of Pseudomonas Aeruginosa and Synthesis of High Affinity Oligoglycoclusters.

The Gram negative bacterium Pseudomonas aeruginosa (PA) is an opportunistic bacterium that causes severe and chronic infection of immune-depressed patients. It has the ability to form a biofilm that gives a selective advantage to the bacteria with respect to antibiotherapy and host defenses. Herein, we have focused on the tetrameric soluble lectin which is involved in bacterium adherence to host cells, biofilm formation, and cytotoxicity. It binds to l-fucose, d-mannose and glycan exposing terminal fucose or mannose. Using a competitive assay on microarray, 156 oligosaccharides and polysaccharides issued from fermentation or from the biomass were screened toward their affinity to LecB. Next, the five best ligands (Lewisa, Lewisb, Lewisx, siayl-Lewisx and 3-fucosyllactose) were derivatized with a propargyl aglycon allowing the synthesis of 25 trivalent, 25 tetravalent and 5 monovalent constructions thanks to copper catalyzed azide alkyne cycloaddition. The 55 clusters were immobilized by DNA Directed immobilization leading to the fabrication of a glycocluster microarray. Their binding to LecB was studied. Multivalency improved the binding to LecB. The binding structure relationship of the clusters is mainly influenced by the carbohydrate residues. Molecular simulations indicated that the simultaneous contact of both binding sites of monomer A and D seems to be energetically possible.

Diagnostics and Therapy of Human Diseases - Focus on Sialidases.

Sialic acid residues that make part of the cell surface repertoire of carbohydrate residues are implicated in various physiological processes and human pathologies. Sialidases, or neuraminidases, are the enzymes that are able to cleave and release the sialic acid residues, while trans-sialidases can transfer the residues from donor to acceptor molecules. They are important for processing the surface glycolipids and glycoproteins. Therapeutic potential of pharmacological sialidases inhibition is currently actively studied. Knowledge and expertise gained from genetic defects leading to human sialidase deficiency can be used for designing such drugs. In this review, we discuss the current progress in studying sialidases and their inhibitors and the relevance of these studies to developing novel therapeutic approaches. In vitro studies suggest that some sialidase inhibitors might be useful therapeutics for treating sialidosis, cancer, infections, immune diseases, atherosclerosis and other pathologies. Consequently, there is a field for further research and development. A thorough investigation of human sialidases is therefore crucial to human health.