Characterization of carboxylated cellulose nanofibrils and oligosaccharides from Kraft pulp fibers and their potential elicitor effect on the gene expression of Capsicum annuum.

Biomass-derived oligo- and polysaccharides may act as elicitors, i.e., bioactive molecules that trigger plant immune responses. This is particularly important to increase the resistance of plants to abiotic and biotic stresses. In this study, cellulose nanofibrils (CNF) gels were obtained by TEMPO-mediated oxidation of unbleached and bleached kraft pulps. The molecular structures were characterized with ESI and MALDI MS. Analysis of the fine sequences was achieved by MS and MS/MS of the water-soluble oligosaccharides obtained by acid hydrolysis of the CNF gels. The analysis revealed the presence of two families: one corresponding to homoglucuronic acid sequences and the other composed by alternating glucose and glucuronic acid units. The CNF gels, alone or with the addition of the water-soluble oligosaccharides, were tested on Chili pepper (Capsicum annuum). Based on the characterization of the gene expression with Next Generation Sequencing (NGS) of the C. annuum's total messenger RNA, the differences in growth of the C. annuum seeds correlated well with the downregulation of the pathways regulating photosynthesis. A downregulation of the response to abiotic factors was detected, suggesting that these gels would improve the resistance of the C. annuum plants to abiotic stress due to, e.g., water deprivation and cold temperatures.

DP2, a Carbohydrate Derivative, Enhances In Vitro Osteoblast Mineralisation.

Bone fracture healing is a complex biological process involving four phases coordinated over time: hematoma formation, granulation tissue formation, bony callus formation, and bone remodelling. Bone fractures represent a significant health problem, particularly among the elderly population and patients with comorbidities. Therapeutic strategies proposed to treat such fractures include the use of autografts, allografts, and tissue engineering strategies. It has been shown that bone morphogenetic protein 2 (BMP-2) has a therapeutic potential to enhance fracture healing. Despite the clinical efficacy of BMP-2 in osteoinduction and bone repair, adverse side effects and complications have been reported. Therefore, in this in vitro study, we propose the use of a disaccharide compound (DP2) to improve the mineralisation process. We first evaluated the effect of DP2 on primary human osteoblasts (HOb), and then investigated the mechanisms involved. Our findings showed that (i) DP2 improved osteoblast differentiation by inducing alkaline phosphatase activity, osteopontin, and osteocalcin expression; (ii) DP2 induced earlier in vitro mineralisation in HOb cells compared to BMP-2 mainly by earlier activation of Runx2; and (iii) DP2 is internalized in HOb cells and activates the protein kinase C signalling pathway. Consequently, DP2 is a potential therapeutical candidate molecule for bone fracture repair.

Octanoyl esterification of low molecular weight sulfated galactan enhances the cellular uptake and collagen expression in fibroblast cells.

Low molecular weight sulfated galactan (LMSG) supplemented with octanoyl ester (Oct-LMSG) demonstrated superior wound healing activity compared to the unsupplemented LMSG in a fibroblast wound model. To test the hypothesis that the increased bioactivity of Oct-LMSG may depend on its penetration into the plasma membrane, its cellular uptake was investigated and collagen production in fibroblast cells was assessed for the first time. The cellular uptake of Oct-LMSG was examined using indirect immunofluorescence and a confocal laser scanning microscope. In addition, the degree of fibroblast activation associated with this uptake was evaluated. The results indicated increased LMSG internalization in fibroblasts treated with Oct-LMSG. Transmission electron micrographs revealed the ultrastructure of active protein production in fibroblasts upon treatment with Oct-LMSG. In addition, Oct-LMSG upregulated the expression of type I collagen mRNA and proteins, as well as related signaling molecules involved in collagen synthesis, including collagen type I α1 chain (Col1A1), Col1A2, phosphorylated (p)-Smad2/3 and p-Smad4. The current findings support the notion that the supplementation of LMSG with octanoyl enhanced its cellular uptake into fibroblasts and, as a result, regulated the expression of type I collagen in fibroblasts via the activation of the Smad signaling pathway. This study demonstrates the therapeutic potential of Oct-LMSG in promoting tissue regeneration.

Sulfated Galactans from Gracilaria fisheri with Supplementation of Octanoyl Promote Wound Healing Activity In Vitro and In Vivo.

Sulfated galactans (SG) isolated from Gracilaria fisheri is partially degraded (DSG), and subsequentially supplemented with octanoyl (DSGO) and sulfate (DSGS) groups. The molecular weights of DSG, DSGO, and DSGS are 7.87, 152.79, and 97.07 kDa, respectively. The modification is confirmed using FTIR and NMR, while in vitro wound healing activity is assessed using scratched wound fibroblasts. The results reveal that DSGO exhibits highest percentage of wound closure in scratched fibroblast L929 cells. Furthermore, DSGO is able to promote proliferation and accelerate migration of scratched fibroblasts, which correspond to the regulation of proteins and mRNA (Ki67, p-FAK, vimentin, and E-cadherin) determined by Western blotting and qPCR analysis. The superior wound healing activity of DSGO is also confirmed in excision wound of rats. The results demonstrate that DSGO significantly enhances the percentage of wound closure, re-epithelialization, and collagen arrangement, increases α-smoth muscle actin (α-SMA) and vimentin expression, and decreases that of tumor necrosis factor-α (TNF-α) at the wound site. The results suggest that degraded SG supplemented with medium-chain fatty acids of octanoyl group may pass through the membrane, subsequently activating the mediators associated with proliferation and migration of fibroblasts, which can potentially lead to the promotion of wound healing activity.

Using NMR to Dissect the Chemical Space and O-Sulfation Effects within the O- and S-Glycoside Analogues of Heparan Sulfate.

Heparan sulfate (HS), a sulfated linear carbohydrate that decorates the cell surface and extracellular matrix, is ubiquitously distributed throughout the animal kingdom and represents a key regulator of biological processes and a largely untapped reservoir of potential therapeutic targets. The temporal and spatial variations in the HS structure underpin the concept of "heparanome" and a complex network of HS binding proteins. However, despite its widespread biological roles, the determination of direct structure-to-function correlations is impaired by HS chemical heterogeneity. Attempts to correlate substitution patterns (mostly at the level of sulfation) with a given biological activity have been made. Nonetheless, these do not generally consider higher-level conformational effects at the carbohydrate level. Here, the use of NMR chemical shift analysis, NOEs, and spin-spin coupling constants sheds new light on how different sulfation patterns affect the polysaccharide backbone geometry. Furthermore, the substitution of native O-glycosidic linkages to hydrolytically more stable S-glycosidic forms leads to observable conformational changes in model saccharides, suggesting that alternative chemical spaces can be accessed and explored using such mimetics. Employing a series of systematically modified heparin oligosaccharides (as a proxy for HS) and chemically synthesized O- and S-glycoside analogues, the chemical space occupied by such compounds is explored and described.

Increased Sulfation in Gracilaria fisheri Sulfated Galactans Enhances Antioxidant and Antiurolithiatic Activities and Protects HK-2 Cell Death Induced by Sodium Oxalate.

Urolithiasis is a common urological disease characterized by the presence of a stone anywhere along the urinary tract. The major component of such stones is calcium oxalate, and reactive oxygen species act as an essential mediator of calcium oxalate crystallization. Previous studies have demonstrated the antioxidant and antiurolithiatic activities of sulfated polysaccharides. In this study, native sulfated galactans (N-SGs) with a molecular weight of 217.4 kDa from Gracilaria fisheri were modified to obtain lower molecular weight SG (L-SG) and also subjected to sulfation SG (S-SG). The in vitro antioxidant and antiurolithiatic activities of the modified substances and their ability to protect against sodium oxalate-induced renal tubular (HK-2) cell death were investigated. The results revealed that S-SG showed more pronounced antioxidant activities (DPPH and O2- scavenging activities) than those of other compounds. S-SG exhibited the highest antiurolithiatic activity in terms of nucleation and aggregation, as well as crystal morphology and size. Moreover, S-SG showed improved cell survival and increased anti-apoptotic BCL-2 protein in HK-2 cells treated with sodium oxalate. Our findings highlight the potential application of S-SG in the functional food and pharmaceutical industries.

Synthesis of new sulfated disaccharides for the modulation of TLR4-dependent inflammation.

Natural sulfated glycans are key players in inflammation through TLR4 activation; therefore synthetic exogenous sulfated saccharides can be used to downregulate inflammation processes. We have designed and synthesized new sulfated compounds based on small and biocompatible carbohydrates that are able to cross the BBB. A suitable protected donor and acceptor, obtained from a unique precursor, have been stereoselectively glycosylated to give an orthogonally protected cellobiose disaccharide. Selective deprotection and sulfation allowed the syntheses of four differentially sulfated disaccharides, which have been characterized by NMR, HRMS and MS/MS. Together with their partially protected precursors, the new compounds were tested on HEK-TLR4 cells. Our results show the potential of small oligosaccharides to modulate TLR4 activity, confirming the need for sulfation and the key role of the 6-sulfate groups to trigger TLR4 signalization.

One-Pot Synthesis of Asymmetrically Difunctionalized Oligomaltosides by Cyclodextrin Ring Opening.

The synthesis of pure difunctionalized hexa-, hepta- and octamaltosides was performed by one-pot chemical reaction from perbenzoylated cyclodextrin. Oligomaltosides with azide, propargyl or allyl on reducing end and an unprotected hydroxyl group on non-reducing end were obtained from perbenzoylated α-, ß- and γ-cyclodextrin with 12 to 48 % yields.

A simple procedure to obtain a medium-size oligogalacturonic acids fraction from orange peel and apple pomace wastes.

Pectin oligosaccharides, which can be obtained from fruit wastes, have proven their potential as plant immune-system elicitors. Although the precise size of active species is still under investigation, medium size oligosaccharides have been reported as the most active. Three defined oligogalacturonic acid (OGAs) mixtures were produced from commercial pectin, orange peel and apple pomace residues. The methodology developed involves two sequential acid treatments followed by stepwise ethanol precipitation. Without the need of chromatographic separations, three different fractions were obtained. The fractions were analyzed by high performance anion exchange chromatography (HPAEC) and were completely characterized by mass spectrometry, showing that the small size, medium size and large size fractions contained OGAs of degree of polymerization 3 to 9, 6 to 18, and 16 to 55, respectively.

Crystal structures of peanut lectin in the presence of synthetic ß-N- and ß-S-galactosides disclose evidence for the recognition of different glycomimetic ligands.

Carbohydrate-lectin interactions are involved in important cellular recognition processes, including viral and bacterial infections, inflammation and tumor metastasis. Hence, structural studies of lectin-synthetic glycan complexes are essential for understanding lectin-recognition processes and for the further design of promising chemotherapeutics that interfere with sugar-lectin interactions. Plant lectins are excellent models for the study of the molecular-recognition process. Among them, peanut lectin (PNA) is highly relevant in the field of glycobiology because of its specificity for ß-galactosides, showing high affinity towards the Thomsen-Friedenreich antigen, a well known tumor-associated carbohydrate antigen. Given this specificity, PNA is one of the most frequently used molecular probes for the recognition of tumor cell-surface O-glycans. Thus, it has been extensively used in glycobiology for inhibition studies with a variety of ß-galactoside and ß-lactoside ligands. Here, crystal structures of PNA are reported in complex with six novel synthetic hydrolytically stable ß-N- and ß-S-galactosides. These complexes disclosed key molecular-binding interactions of the different sugars with PNA at the atomic level, revealing the roles of specific water molecules in protein-ligand recognition. Furthermore, binding-affinity studies by isothermal titration calorimetry showed dissociation-constant values in the micromolar range, as well as a positive multivalency effect in terms of affinity in the case of the divalent compounds. Taken together, this work provides a qualitative structural rationale for the upcoming synthesis of optimized glycoclusters designed for the study of lectin-mediated biological processes. The understanding of the recognition of ß-N- and ß-S-galactosides by PNA represents a benchmark in protein-carbohydrate interactions since they are novel synthetic ligands that do not belong to the family of O-linked glycosides.

Synthesis of defined oligohyaluronates-decorated liposomes and interaction with lung cancer cells.

In this work hyaluronic acid (HA) oligosaccharides with degree of polymerization (DP) 4, 6 and 8, obtained by enzymatic depolymerization of HA, were conjugated to a PEG-phospholipid moiety. The products (HA-DP4, HA-DP6 and HA-DP8) were used to prepare decorated liposomes. The cellular uptake of HA-DP4, HA-DP6 and HA-DP8-decorated fluorescently labelled liposomes was significantly higher (12 to 14-fold) in lung cancer cell lines with high CD44 expression than in those with low CD44 expression, suggesting a receptor-mediated entry of HA-conjugated formulations. Competition assays showed that the uptake followed this rank order: HA-DP8>HA-DP6>HA-DP4 liposomes. Moreover, they are capable of a faster interaction with CD44, followed by phagocytosis, than HA liposomes obtained from HA of higher molecular weight (4800 and 14800 Da). HA-DP4, HA-DP6 and HA-DP8-liposomes did not show cytotoxicity or inflammatory effects. Overall, we propose our new HA-DP oligosaccharides as biocompatible and effective tools for a potential drug delivery to CD44-positive cells.

The uncommon strong inhibition of α-glucosidase by multivalent glycoclusters based on cyclodextrin scaffolds.

The homeostasis disruption of d-glucose causes diabetes, a dramatic chronic disease worldwide. Type 1 diabetes is a successfully treatable form, where blood d-glucose is regulated by insulin treatment. In contrast type 2 diabetes, the non-insulin dependent kind, is problematic. The control of the d-glucose blood level via intestinal α-d-glucosidase inactivation can be achieved by using competitive inhibitors, such as iminosugars (e.g. acarbose) or sulfonium sugar derivatives (e.g. salacinol). Recently, an unprecedented result showed that multivalent diamond nanoparticles grafted with unmodified sugars displayed α-glucosidase inhibition at low micromolar concentrations. Herein we describe the synthesis of multivalent glycoclusters using cyclodextrins (CDs) as scaffolds and an assessment of their role as inhibitors of α-d-glucosidase. The glycoclusters were efficiently obtained from per-azido α, ß and γ-CD derivatives and propargyl glycosides using click-chemistry under microwave irradiation. The methodology was successfully applied to various protected and non-protected propargylated monosaccharides, including both O- and S-glycosides, giving clear evidence of its versatility. The targeted 6-per-glycosylated CDs were isolated in moderate to excellent yields (30-90%) by silica gel chromatography. The results showed inhibition of α-glucosidase from Saccharomyces cerevisiae with IC50 values in the 32-132 µM range, lower than that of acarbose (IC50 = ∼250 µM), a well-known competitive inhibitor used in the clinical treatment of type 2 diabetes. Preliminary experiments suggest a mixed-type non-competitive inhibition mode for these new glycoclusters.

High resolution MALDI-TOF-MS and MS/MS: Application for the structural characterization of sulfated oligosaccharides.

Sulfated oligosaccharides are involved in important biological events that are often modulated by specific sequences and sulfation patterns, but their structural analysis remains challenging. Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) analysis of three different sulfated oligosaccharides (Fondaparinux, the octasulfated pentasaccharide, a disulfated heparin-derived tetrasaccharide 1, and an octasulfated maltoheptaose) 2 was performed using the 2-(4-hydroxyphenylazo)benzoic acid-tetramethylguanidinium (HABA-TMG2) matrix. High resolution mass spectrometry of the main ions observed was successful, and this was complemented by tandem mass spectrometry (MS/MS) analysis for structural assessment. Despite sulfate losses, fully sulfated molecular ions were observed and these allowed the determination of oligosaccharide structures: UA-GlcNAc-UA(2S)-AnhMan(6S) for compound 1 and (Glc6S)6-Glc (1S,6S) for compound 2.

Solid-phase synthesis of molecularly imprinted polymer nanolabels: Affinity tools for cellular bioimaging of glycans.

Hyaluronic acid (HA) is a glycosaminoglycan that plays many roles in health and disease and is a key biomarker of certain cancers. Therefore, its detection at an early stage, by histochemical methods, is of importance. However, intracellular HA can be masked by other HA-binding macromolecules, rendering its visualization somehow problematic. We show that fluorescent molecularly imprinted polymer nanogels (MIP-NPs), can localize and detect intracellular HA. MIP-NPs were synthesized by solid-phase synthesis on glass beads (GBs). GBs were functionalized with terminal alkyne groups on which an azide derivative of the template molecule glucuronic acid was immobilized via click chemistry. Immobilization via the anomeric carbon left the template's carboxyl moiety free to enable strong stoichiometric electrostatic interactions with a benzamidine-based functional monomer, to confer selective recognition to the MIP-NPs. Due to the two-point orientation of the template, the resulting MIP-NPs were endowed with improved binding site homogeneity and specificity, reminiscent of monoclonal antibodies. These synthetic antibodies were then applied for probing and staining HA, of which glucuronic acid is a substructure (epitope), on human epidermal cells. Their excellent sensitivity, small size and water compatibility, enabled the MIP-NPs to visualize HA, as evidenced by confocal fluorescence micrographs.

Biological impact of octyl d-glucopyranoside based surfactants.

Development of many branches of industry has stimulated the search for new, effective surfactants with interesting properties. Potential use of alkyl glucose derivatives on a large scale, raises questions about the possible risks associated with their entry into the natural environment. To be able to evaluate this risk, the aim of the study was to determine the physicochemical properties of octyl d-glucopyranoside and its three derivatives: N-(octyl d-glucopyranosiduronyl)aspartic acid, N-(octyl d-glucopyranosiduronyl)glicyne and octyl d-glucopyranosiduronic acid. Moreover, their biodegradability by pure bacterial strains and biocenosis present in river water was examined. While descriptions of sugar-based surfactants on microbial cells are limited, the essential element of the study was to determine the effect of surfactants on cell surface properties of microorganisms isolated from activated sludge and compare it to the effects of the petroleum based surfactants and the surfactants produced from renewable materials. The results obtained indicate that physicochemical properties of surface active agents differ depending on the presence of functional groups in the surfactants molecules. What is more, the presence of amino acid substituent in the derivatives of octyl d-glucopyranoside resulted in a slight decrease in the surfactants biodegradation efficiency, in comparison to the compounds that did not contain such a substituent, prolonging this process from 5 to 10 days. Interestingly, even relatively slightly different derivatives modified the cell surface properties in a different way. Importantly, the surfactants based on octyl d-glucopyranoside have less negative impact on environmental microorganism and better biodegradability than the surfactant synthesized from petroleum products.

Synthesis and insecticidal activities of novel solanidine derivatives.

BACKGROUND: Potato (Solanum tuberosum) is the fourth culture in the world and is widely used in the agri-food industries. They generate by-products in which α-chaconine and α-solanine, the two major solanidine-based glycoalkaloids of potato, are present. As secondary metabolites, they play an important role in the protection system of potato and are involved in plant protection against insects. To add value to these by-products, we described here new glycoalkaloids that could have phytosanitary properties. RESULTS: Solanidine, as a renewable source, was modified with an azido linker and coupled by copper-catalyzed alkyne azide cycloaddition to alkynyl derivatives of the monosaccharides found in the natural potato glycoalkakoids: D-glucose, D-galactose and L-rhamnose. The efficacy of our compounds was evaluated on the potato aphid Macrosiphum euphorbiae. The synthetic compounds have stronger aphicidal properties against nymphs than unmodified solanidine. They also showed strong aphicidal activities on adults and a negative impact on fecundity. CONCLUSION: Our synthetic neoglycoalkaloids affected Macrosiphum euphorbiae survival at the nymphal stage as well as at the adult stage. Furthermore, they induced a decrease in fecundity. Our results show that chemical modifications of by-products may afford new sustainable compounds for crop and plant protection. © 2018 Society of Chemical Industry.

Oligogalacturonic Acid Inhibits Vascular Calcification by Two Mechanisms: Inhibition of Vascular Smooth Muscle Cell Osteogenic Conversion and Interaction With Collagen.

OBJECTIVE: Cardiovascular diseases constitute the leading cause of mortality worldwide. Calcification of the vessel wall is associated with cardiovascular morbidity and mortality in patients having many diseases, including diabetes mellitus, atherosclerosis, and chronic kidney disease. Vascular calcification is actively regulated by inductive and inhibitory mechanisms (including vascular smooth muscle cell adaptation) and results from an active osteogenic process. During the calcification process, extracellular vesicles (also known as matrix vesicles) released by vascular smooth muscle cells interact with type I collagen and then act as nucleating foci for calcium crystallization. Our primary objective was to identify new, natural molecules that inhibit the vascular calcification process. APPROACH AND RESULTS: We have found that oligogalacturonic acids (obtained by the acid hydrolysis of polygalacturonic acid) reduce in vitro inorganic phosphate-induced calcification of vascular smooth muscle cells by 80% and inorganic phosphate-induced calcification of isolated rat aortic rings by 50%. A specific oligogalacturonic acid with a degree of polymerization of 8 (DP8) was found to inhibit the expression of osteogenic markers and, thus, prevent the conversion of vascular smooth muscle cells into osteoblast-like cells. We also evidenced in biochemical and immunofluorescence assays a direct interaction between matrix vesicles and type I collagen via the GFOGER sequence (where single letter amino acid nomenclature is used, O=hydroxyproline) thought to be involved in interactions with several pairs of integrins. CONCLUSIONS: DP8 inhibits vascular calcification development mainly by inhibition of osteogenic marker expression but also partly by masking the GFOGER sequence-thereby, preventing matrix vesicles from binding to type I collagen.

Synthesis of ß-galactosylamides as ligands of the peanut lectin. Insights into the recognition process.

The synthesis of mono and divalent ß-galactosylamides linked to a hydroxylated chain having a C2 symmetry axis derived from l-tartaric anhydride is reported. Reference compounds devoid of hydroxyl groups in the linker were also prepared from ß-galactosylamine and succinic anhydride. After functionalization with an alkynyl residue, the resulting building blocks were grafted onto different azide-equipped scaffolds through the copper catalyzed azide-alkyne cycloaddition. Thus, a family of structurally related mono and divalent ß-N-galactopyranosylamides was obtained and fully characterized. The binding affinities of the ligands towards the model lectin PNA were measured by the enzyme-linked lectin assay (ELLA). The IC50 values were significantly higher than that of galactose but the presence of hydroxyl groups in the aglycone chain improved lectin recognition. Docking and molecular dynamics experiments were in accordance with the hypothesis that a hydroxyl group properly disposed in the linker could mimic the Glc O3 in the recognition process. On the other hand, divalent presentation of the ligands led to lectin affinity enhancements.

Kinetics of the incorporation of the main phenolic compounds into the lignan macromolecule during flaxseed development.

The main flax lignan, secoisolariciresinol diglucoside, is stored in a macromolecule containing other ester-bound phenolic compounds. In this study, NMR and HPLC-UV analyses were performed on flaxseeds harvested at different developmental stages to identify and quantify the main phenolic compounds produced during seed development. Extraction was carried out with or without alkaline hydrolysis to determine if these molecules accumulate in the lignan macromolecule and/or in a free form. Monolignol glucosides accumulate in a free form up to 9.85mg/g dry matter at the early developmental stages. Hydroxycinnamic acid glucosides and flavonoid accumulate (up to 3.18 and 4.07mg/g dry matter, respectively) in the later developmental stages and are ester-bound in the lignan macromolecule. Secosiolariciresinol diglucoside accumulates (up to 28.65mg/g dry matter) in the later developmental stages in both forms, mainly ester-bound in the lignan macromolecule and slightly in a free form.

Multivalent sialylation of ß-thio-glycoclusters by Trypanosoma cruzi trans sialidase and analysis by high performance anion exchange chromatography.

The synthesis of multivalent sialylated glycoclusters is herein addressed by a chemoenzymatic approach using the trans-sialidase of Trypanosoma cruzi (TcTS). Multivalent ß-thio-galactopyranosides and ß-thio-lactosides were used as acceptor substrates and 3'-sialyllactose as the sialic acid donor. High performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was shown to be an excellent technique for the analysis of the reaction products. Different eluting conditions were optimized to allow the simultaneous resolution of the sialylated species, as well as their neutral precursors. The TcTS efficiently transferred sialyl residues to di, tri, tetra and octa ß-thiogalactosides. In the case of an octavalent thiolactoside, up to six polysialylated compounds could be resolved. Preparative sialylation reactions were performed using the tetravalent and octavalent acceptor substrates. The main sialylated derivatives could be unequivocally assigned by MALDI mass spectrometry. Inhibition of the transfer to the natural substrate, N-acetyllactosamine, was also studied. The octalactoside caused 82 % inhibition of sialic acid transfer when we used equimolar concentrations of donor, acceptor and inhibitor.