Phosphorylation of polysaccharides: A review on the synthesis and bioactivities.

Polysaccharides are macromolecules obtained from a wide range of sources and are known to have diverse biological activities. The biological activities of polysaccharides depend on their structure and physicochemical properties, including water solubility, monosaccharide composition, degree of branching, molecular structure, and molecular weight. Phosphorylation is a commonly used chemical modification method that improves the physicochemical properties of native polysaccharides, thus enhancing their biological activity, or even imparting novel biological activity. Therefore, phosphorylated polysaccharides have attracted increasing attention owing to their antioxidant, antitumor, antiviral, immunomodulatory, and hepatoprotective effects. In this review, we have discussed recent advances in the phosphorylation of polysaccharides, and the methods used for phosphorylation, structural characterization, and determination of biological activities, to provide a theoretical basis for the use of polysaccharides. The structure-activity relationship of phosphorylated polysaccharides and their use in the food and pharmaceutical industries needs to be further studied.

Advances in the preparation and assessment of the biological activities of chitosan oligosaccharides with different structural characteristics.

Chitosan oligosaccharides (COSs) are widely used biopolymers that have been studied in relation to a variety of abnormal biological activities in the food and biomedical fields. Since different COS preparation technologies produce COS compounds with different structural characteristics, it has not yet been possible to determine whether one or more chito-oligomers are primarily responsible for the bioactivity of COSs. The inherent biocompatibility, mucosal adhesion and nontoxic nature of COSs are well documented, as is the fact that they are readily absorbed from the intestinal tract, but their structure-activity relationship requires further investigation. This review summarizes the methods used for COS preparation, and the research findings with regard to the antioxidant, anti-inflammatory, anti-obesity, bacteriostatic and antitumour activity of COSs with different structural characteristics. The correlation between the molecular structure and bioactivities of COSs is described, and new insights into their structure-activity relationship are provided.

Recent developments in chitosan encapsulation of various active ingredients for multifunctional applications.

Microencapsulation being an emerging technique has provided effective solution to the challenges faced by pharmaceutical, cosmetic, food agriculture and textile industries to deliver ingredients in their active forms to the target sites. Chitosan is a non-toxic, biodegradable and biocompatible amino polysaccharide which makes it useful for the encapsulation of various active ingredients with potential applications. Chitosan coating on food products, for example, gives them protection from possible antimicrobial attacks, antioxidants and extended shelf life. Likewise, its coating on pharmaceutics has valuable applications in preserving drug and their targeted delivery. In this review, we discuss the formation of chitosan, its properties, microencapsulation process, micro-capsular morphologies, selection of core and shell materials in addition to the process of chitosan encapsulation of various active ingredients and their applications in various fields of science and technology.

Fast and Low-Cost Purification Strategy for Oligosaccharide Synthesis Based on a Hop-On/Off Carrier.

A feasible and convenient strategy for oligosaccharide synthesis, which realizes reaction in solution while product purification occurs only by solid-liquid filtration, has been developed. By using a hop-on/off carrier (polytetrafluoroethylene particle), rapid synthesis of tumor-associated antigen Globo-H hexasaccharide has been successfully achieved within 5 steps in 48% overall yield without any intermediate purification by column chromatography. Also, global deprotection, including the cleavage of the tag, proceeded simultaneously only by one-step hydrogenolysis.

Combined Experimental and Molecular Simulation Study of Insulin-Chitosan Complexation Driven by Electrostatic Interactions.

Protein-polysaccharide complexes constructed via self-assembly methods are often used to develop novel biomaterials for a wide range of applications in biomedicine, food, and biotechnology. The objective of this work was to investigate theoretically and to demonstrate via constant-pH Monte Carlo simulations that the complexation phenomenon between insulin (INS) and the cationic polyelectrolyte chitosan (CS) is mainly driven by an electrostatic mechanism. Experimental results obtained from FTIR spectra and ζ-potential determinations allowed us to complement the conclusions. The characteristic absorption bands for the complexes could be assigned to a combination of signals from CS amide I and INS amide II. The second peak corresponds to the interaction between the polymer and the protein at the level of amide II. INS-CS complexation processes not expected when INS is in its monomeric form, but for both tetrameric and hexameric forms, incipient complexation due to charge regulation mechanism took place at pH 5. The complexation range was observed to be 5.5 < pH < 6.5. In general, when the number of INS units increases in the simulation process, the solution pH at which the complexation can occur shifts toward acidic conditions. CS's chain interacts more efficiently, i.e. in a wider pH range, with INS aggregates formed by the highest monomer number. The charge regulation mechanism can be considered as a previous phase toward complexation (incipient complexation) caused by weak interactions of a Coulombic nature.

De novo structural determination of mannose oligosaccharides by using a logically derived sequence for tandem mass spectrometry.

Carbohydrates play important roles in biological recognition processes. However, determining the structures of carbohydrates remains challenging because of their complexity. A simple tandem mass spectrometry-based method for determining the structure of underivatized mannose tetrasaccharides was demonstrated. This method employed the multistage low-energy collision-induced dissociation (CID) of sodium adducts in an ion trap, a logically derived sequence (LODES) from the dissociation mechanism for deciding the sequence of CID, and a specially prepared disaccharide spectrum database. Through this method, the linkages, anomeric configurations, and branch locations of carbohydrates could be determined without the prior assumption of possible structures. We validated this method by blind test of all the commercial available mannose tetrasaccharides. We showed that the structure of a given tetrasaccharide can be determined from 928 isomers by using only three to six appropriately selected CID mass spectra according to the proposed procedure. This method is simple and rapid and has the potential to be applied to other hexoses and oligosaccharides larger than tetrasaccharides. The CID procedures can be built in a computer-controlled mass spectrometer for automatic structural determination of underivatized oligosaccharides. Graphical abstract.

On the effect of intramolecular H-bonding in the configurational assessment of polyhydroxylated compounds with computational methods. The hyacinthacines case.

Hyacinthacines are important members of the pyrrolizidine family, with several compounds having ambiguous, revised or unverified structures. Herein we thoroughly explored the performance DP4 and DP4+ for the in silico stereoassignment of hyacinthacines A1, A2 and five synthetic isomers. The results suggested that the quality of the predictions strongly depended on the conformational landscape provided by DFT energies, with five compounds correctly assigned. In the two cases incorrectly classified we found that the source of the problem was conformational in nature, with spurious conformations being considerably over-stabilized by intramolecular H-bondings. We showed that neglecting such shapes resulted in a noteworthy improvement, with all compounds correctly assigned in high confidence (>99.9%).

Affinity Calculations of Cyclodextrin Host-Guest Complexes: Assessment of Strengths and Weaknesses of End-Point Free Energy Methods.

The end-point methods like MM/PBSA or MM/GBSA estimate the free energy of a biomolecule by combining its molecular mechanics energy with solvation free energy and entropy terms. On the one hand, their performance largely depends on the particular system of interest, and despite numerous attempts to improve their reliability that have resulted in many variants, there is still no clear alternative to improve their accuracy. On the other hand, the relatively small cyclodextrin host-guest complexes, for which high-quality binding calorimetric data are usually available, are becoming reference models for testing the accuracy of free energy methods. In this work, we further assess the performance of various MM/PBSA-like approaches as applied to cyclodextrin complexes. To this end, we select a set of complexes between ß-cyclodextrin and 57 small organic molecules that has been previously studied with the binding energy distribution analysis method in combination with an implicit solvent model ( Wickstrom, L.; He, P.; Gallicchio, E.; Levy, R. M. J. Chem. Theory Comput. 2013 , 9 , 3136 - 3150 ). For each complex, a conventional 1.0 µs molecular dynamics simulation in explicit solvent is performed. Then we employ semiempirical quantum chemical calculations, several variants of the MM-PB(GB)SA methods, entropy estimations, etc., to assess the reliability of the end-point affinity calculations. The best end-point protocol in this study, which combines DFTB3 energies with entropy corrections, yields estimations of the binding free energies that still have substantial errors (RMSE = 2.2 kcal/mol), but it exhibits a good prediction capacity in terms of ligand ranking ( R2 = 0.66) that is close to or even better than that of rigorous free energy methodologies. Our results can be helpful to discriminate between the intrinsic limitations of the end-point methods and other sources of error, such as the underlying energy and continuum solvation methods.

Robust Assessment of Molecular Size Distribution of Meningococcal AC Vaccine.

The molecular size of meningococcal polysaccharides is an important physicochemical parameter that is related to immunogenicity and efficacy. A simple method for size-exclusion chromatography was developed, optimized, and applied for safe and rapid fractionation of meningococcal polysaccharide AC vaccine. Pooling of the fractions collected from size-exclusion chromatography was investigated and evaluated, rather than analyzing each fraction separately, for determining the percentages of meningococcal polysaccharide A and C that were eluted before the distribution coefficient of 0.5. Pooling is preferred rather than analyzing each fraction individually, as it is easily handled, faster, simpler, less expensive, more accurate, safe, and applicable. The developed method was validated and successfully applied for the determination of meningococcal polysaccharide vaccine serotype A and C in quality-control and commercial samples.

Azahar: a PyMOL plugin for construction, visualization and analysis of glycan molecules.

Glycans are key molecules in many physiological and pathological processes. As with other molecules, like proteins, visualization of the 3D structures of glycans adds valuable information for understanding their biological function. Hence, here we introduce Azahar, a computing environment for the creation, visualization and analysis of glycan molecules. Azahar is implemented in Python and works as a plugin for the well known PyMOL package (Schrodinger in The PyMOL molecular graphics system, version 1.3r1, 2010). Besides the already available visualization and analysis options provided by PyMOL, Azahar includes 3 cartoon-like representations and tools for 3D structure caracterization such as a comformational search using a Monte Carlo with minimization routine and also tools to analyse single glycans or trajectories/ensembles including the calculation of radius of gyration, Ramachandran plots and hydrogen bonds. Azahar is freely available to download from http://www.pymolwiki.org/index.php/Azahar and the source code is available at https://github.com/agustinaarroyuelo/Azahar .

Glucosinolate composition of Colombian accessions of mashua (Tropaeolum tuberosum Ruíz & Pavón), structural elucidation of the predominant glucosinolate and assessment of its antifungal activity.

BACKGROUND: The content of individual and total glucosinolates in 65 mashua tuber accessions (Tropaeolum tuberosum) from the germplasm bank at Universidad Nacional de Colombia was determined by reverse phase high-performance liquid chromatography on enzymatically desulfated extracts. The predominant glucosinolate was identified and the possible structure of the glucosinolate present in lower proportion was postulated from evidence obtained by high-performance liquid chromatography/mass spectrometry, 1 H and 13 C nuclear magnetic resonance and bi-dimensional experiments. The biological action of the hydrolysis products generated from the glucosinolates in the accessions that showed a higher content of these compounds was assessed in the presence of Fusarium oxysporum f. sp. dianthi, Rhizoctonia solani and Phytophthora infestans. RESULTS: The total content of glucosinolates ranged between >3.00 × 10-1 and 25.8 µmol g-1 dry matter. p-Methoxybenzyl glucosinolate was identified as the predominant glucosinolate in Colombian mashua accessions; besides, the possible presence of p-hydroxybenzyl glucosinolate was postulated. In vitro assays established an important fungal growth inhibition of the potato pathogen P. infestans. CONCLUSION: The biological action from p-methoxybenzyl glucosinolate and p-hydroxybenzyl glucosinolate found in Colombian mashua accessions depends on their concentration, with the Tt30 accession, characterized for showing the highest content of glucosinolates, being the most promising to control the assessed pathogens. © 2016 Society of Chemical Industry.

Comparative Assessment of Glycosylation of a Recombinant Human FSH and a Highly Purified FSH Extracted from Human Urine.

Glycosylation is an important PTM and is critical for the manufacture and efficacy of therapeutic glycoproteins. Glycan significantly influences the biological properties of human follicle-stimulating hormone (hFSH). Using a glycoproteomic strategy, this study compared the glycosylation of a putative highly purified FSH (uhFSH) obtained from human urine with that of a recombinant human FSH (rhFSH) obtained from Chinese hamster ovary (CHO) cells. Intact and subunit masses, N-glycans, N-glycosylation sites, and intact N- and O-glycopeptides were analyzed and compared by mass spectrometry. Classic and complementary analytical methods, including SDS-PAGE, isoelectric focusing, and the Steelman-Pohley bioassay were also employed to compare their intact molecular weights, charge variants, and specific activities. Results showed that highly sialylated, branched, and macro-heterogeneity glycans are predominant in the uhFSH compared with those in rhFSH. The O-glycopeptides of both hFSHs, which have not been described previously, were characterized herein. A high degree of heterogeneity was observed in the N-glycopeptides of both hFSHs. The differences in glycosylation provide useful information in elucidating and in further investigation the critical glycan structures of hFSH.

A molecular modeling approach to understand the structure and conformation relationship of (GlcpA)Xylan.

The structure and conformation relationships of a heteropolysaccharide (GlcpA)Xylan in terms of various molecular weights, Xylp/GlcpA ratio and the distribution of GlcpA along xylan chain were investigated using computer modeling. The adiabatic contour maps of xylobiose, XylpXylp(GlcpA) and (GlcpA)XylpXylp(GlcpA) indicated that the insertion of the side group (GlcpA) influenced the accessible conformational space of xylobiose molecule. RIS-Metropolis Monte Carlo method indicated that insertion of GlcpA side chain induced a lowering effect of the calculated chain extension at low GlcpA:Xylp ratio (GlcpA:Xylp = 1:3). The chain, however, became extended when the ratio of GlcpA:Xylp above 2/3. It was also shown that the spatial extension of the polymer chains was dependent on the distribution of side chain: the random distribution demonstrated the most flexible structure compared to block and alternative distribution. The present studies provide a unique insight into the dependence of both side chain ratio and distribution on the stiffness and flexibility of various (GlcpA)Xylan molecules.

Docking polysaccharide to proteins that have a Tryptophan box in the binding pocket.

Protein-carbohydrate interactions (PCIs) involve a variety of essential biological processes such as cell recognition and migration, metabolism processes and immunological reactions, which are important for securing functions of living organisms. Due to the polysaccharide structural diversity and dynamics flexibility, PCIs can be very difficult for experimental measurement and computer prediction. Here we report a simple method for docking polysaccharide to proteins whose binding pockets have a Tryptophan box. The method samples polysaccharide conformations using constraint conditions imposed by the box, evaluate the conformation energies based on a knowledge-based potential function, and finds the best docking structures using the conventional Monte Carlo simulated annealing technique. We applied the method to dock polysaccharides with 2 to 4 monomers to three carbohydrate-binding proteins, whose pockets have clear aromatic residue-defined binding channels. The predictions found correct carbohydrate binding conformations with atomic RMSD of 1.1-1.6 Å from X-ray crystal structures. The calculation can be performed in ordinary PC and only cost a couple of minutes for a single docking. Our method, when combined with other docking programs, provides a reliable start conformation for further accurate simulation of PCIs.

Xylooligosaccharides: an economical prebiotic from agroresidues and their health benefits.

Oligosaccharides and dietary fibres are non-digestible food ingredients that preferentially stimulate the growth of prebiotic Bifidobacterium and other lactic acid bacteria in the gastro-intestinal tract. Xylooligosaccharides (XOS) provide a plethora of health benefits and can be incorporated into several functional foods. In the recent times, there has been an over emphasis on the microbial conversion of agroresidues into various value added products. Xylan, the major hemicellulosic component of lignocellulosic materials (LCMs), represents an important structural component of plant biomass in agricultural residues and could be a potent bioresource for XOS. On an industrial scale, XOS can be produced by chemical, enzymatic or chemo-enzymatic hydrolysis of LCMs. Chemical methods generate XOS with a broad degree of polymerization (DP), while enzymatic processes will be beneficial for the manufacture of food grade and pharmaceutically important XOS. Xylooligomers exert several health benefits, and therefore, have been considered to provide relief from several ailments. This review provides a brief on production, purification and structural characterization of XOS and their health benefits.

Conformational analysis of oligosaccharides and polysaccharides using molecular dynamics simulations.

Complex carbohydrates usually have a large number of rotatable bonds and consequently a large number of theoretically possible conformations can be generated (combinatorial explosion). The application of systematic search methods for conformational analysis of carbohydrates is therefore limited to disaccharides and trisaccharides in a routine analysis. An alternative approach is to use Monte-Carlo methods or (high-temperature) molecular dynamics (MD) simulations to explore the conformational space of complex carbohydrates. This chapter describes how to use MD simulation data to perform a conformational analysis (conformational maps, hydrogen bonds) of oligosaccharides and how to build realistic 3D structures of large polysaccharides using Conformational Analysis Tools (CAT).

The carbohydrate-binding promiscuity of Euonymus europaeus lectin is predicted to involve a single binding site.

Euonymus europaeus lectin (EEL) is a carbohydrate-binding protein derived from the fruit of the European spindle tree. EEL was first identified for its erythrocyte agglutinating properties and specificity for B and H blood groups. However, a detailed molecular picture of the structural basis of carbohydrate recognition by EEL remains to be developed. In this study, we performed fluorescence titrations of a range of carbohydrates against EEL. Binding of EEL to a wide range of carbohydrates was observed, including a series of blood group-related carbohydrates, mannosides, chitotriose and sialic acid. Affinity was strongest for carbohydrates with H-related structures and the B trisaccharide. A homology model of EEL was produced from templates identified using the HHPred server, which employs hidden Markov models (HMMs) to identify templates. The HMM approach identified that the best templates for EEL were proteins featuring a ricin B-like (R-type) fold. Separate templates were used to model the core and binding site regions of the lectin. Through the use of constrained docking and spatial comparison with a template ligand, binding modes for the carbohydrate ligands were predicted. A relationship between the experimental binding energies and the computed binding energies of the selected docked poses was determined and optimized. Collectively, our results suggest that EEL utilizes a single site for recognition of carbohydrates terminating in a variety of monosaccharides.

Synthesis of N-glycan units for assessment of substrate structural requirements of N-acetylglucosaminyltransferase III.

N-Acetylglucosaminyltransferase (GnT) III is a glycosyltransferase which produces bisected N-glycans by transferring GlcNAc to the 4-position of core mannose. Bisected N-glycans are involved in physiological and pathological processes through the functional regulation of their carrier proteins. An understanding of the biological functions of bisected glycans will be greatly accelerated by use of specific inhibitors of GnT-III. Thus far, however, such inhibitors have not been developed and even the substrate-binding mode of GnT-III is not fully understood. To gain insight into structural features required of the substrate, we systematically synthesized four N-glycan units, the branching parts of the bisected and non-bisected N-glycans. The series of syntheses were achieved from a common core trimannose, giving bisected tetra- and hexasaccharides as well as non-bisected tri- and pentasaccharides. A competitive GnT-III inhibition assay using the synthetic substrates revealed a vital role for the Manß(1-4)GlcNAc moiety. In keeping with previous reports, GlcNAc at the α1,3-branch is also involved in the interaction. The structural requirements of GnT-III elucidated in this study will provide a basis for rational inhibitor design.

Molecular signature of kappa-carrageenan mimics chondroitin-4-sulfate and dermatan sulfate and enables interaction with arylsulfatase B.

The common food additive kappa-carrageenan (κ-CGN) is a sulfated polysaccharide that resembles chondroitin-4-sulfate (C4S) and dermatan sulfate (DS). All have a sulfate group on C4 of a glycoside (galactose for CGN and N-acetylgalactosamine for C4S), and the sulfate-bearing glycoside is linked in a ß-1,4-configuration to an unsulfated, six-carbon sugar (galactose for CGN, glucuronate for C4S and iduronate for DS). The enzyme arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfate) is the highly selective enzyme that removes the four-sulfate group from the nonreducing terminus of C4S and DS, thereby regulating subsequent degradation. In this report, κ-CGN is shown to be a substrate for recombinant human ARSB (rhARSB). Sulfate was generated from both C4S and κ-CGN following incubation with rhARSB. Exposure of human colonic epithelial cells to κ-CGN, but not to C4S, produced reactive oxygen species (ROS) and increased interleukin (IL)-8 secretion. The ROS production from κ-CGN was reduced by exposure to rhARSB, but increased by competition from C4S or DS, but not from chondroitin-6-sulfate. Prior treatment of either lambda- or iota-CGN with rhARSB had no impact on ROS, IL-8 or inorganic sulfate production, demonstrating a specific effect of the molecular configuration of κ-CGN. By mimicry of C4S and DS and by interaction with ARSB, κ-CGN can directly interfere with the normal cellular functions of C4S, DS and ARSB. Since C4S and DS are present in high concentration in tissues, the impact of κ-CGN exposure may be due to some extent to interference with the normal biological functions of ARSB, C4S and DS.