Enzyme-catalyzed synthesis of natural and unnatural polysaccharides

Synthesis of natural and unnatural polysaccharide was achieved via “enzymatic polymerization” by utilizing a glycoside hydrolase as catalyst. Particularly, hyaluronan, chondroitin, and their derivatives belonging to glycosaminoglycans have been prepared using sugar oxazoline monomers designed on the basis of the concept “transition-state analogue substrate”. The oxazoline derivatives of N-acetylhyalobiuronate [GlcAβ(1→3)GlcNAc] and N-acetylchondrosine [GlcAβ(1→3)GalNAc], which have the repeating disaccharide structures of hyaluronan and chondroitin, respectively, were successfully polymerized by the catalysis of hyaluronidase, giving rise to synthetic hyaluronan and chondroitin. Their 2-substituted oxazoline derivatives were also polymerized to the corresponding N-acylated hyaluronan and chondroitin derivatives. Furthermore, N-acetylchondrosine oxazoline derivatives sulfated at the C4, the C6, and both the C4 and C6 of the GalNAc unit were catalyzed by hyaluronidase; the monomer sulfated at the C4 was polymerized to chondroitin 4-sulfate with well-defined structure, whereas the other two monomers were exclusively hydrolyzed to the corresponding disaccharides. These different kinds of natural and unnatural polysaccharides having relatively high molecular weights were produced in all cases by the catalysis of hyaluronidase. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 5014–5027, 2006     

Direct quantification of deoxynivalenol glucuronide in human urine as biomarker of exposure to the <Emphasis Type="Italic">Fusarium</Emphasis> mycotoxin deoxynivalenol

The direct quantification of deoxynivalenol glucuronide (DON-GlcA) by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and its application as a biomarker of exposure to the Fusarium mycotoxin deoxynivalenol (DON) is reported. Usually, DON exposure is estimated from dietary average intakes or by measurement of the native toxin in urine after enzymatic hydrolysis with β-glucuronidase. These methods are time-consuming, expensive, and fail to determine the ratio of DON to DON-GlcA in a simple one-step procedure. One of the main reasons for the use of indirect methods is the unavailability of DON-GlcA standards. Consequently, DON-3-O-glucuronide (D3GlcA) was synthesized and used to develop a method allowing quantification of both DON and D3GlcA by a simple “dilute and shoot” approach without the need for any cleanup. Limit of detection and apparent recovery of D3GlcA was 3 μg l⁻¹ and 88%, respectively. The identity of D3GlcA in human urine was confirmed by comparison with LC-MS/MS measurements of the synthetically produced D3GlcA standard which was also used for external calibration. The applicability of the method was demonstrated through the analysis of urine samples obtained from a volunteer during regular and cereal-restricted diet, respectively. In regular-diet urine samples, D3GlcA was quantified in concentrations >30 μg l⁻¹ by this approach.     

Ultrasonic-Assisted Aqueous Two-Phase Extraction and Properties of Water-Soluble Polysaccharides from Malus hupehensis

Malus hupehensis (M. hupehensis), an edible and medicinal plant with significant antioxidant and hypoglycemic activity, has been applied to new resource foods. However, the structural characterization and biological effects of its polysaccharides (MHP) are less known. The optimum extraction parameters to achieve the highest extraction efficiency (47.63%), the yield (1.68%) and purity of MHP (89.6%) by ultrasonic-assisted aqueous two-phase system (ATPS) were obtained under the liquid-to-solid ratio of 23 g/mL, ultrasonic power of 65 W, and ultrasonic time of 33 min. According to the analysis results, MHP was composed of Man, GlcA, Rha, GalA, Glc, Gal, Xyl, Ara, and Fuc, in which Ara and Gal were the main components, and the content of GlcA was the lowest. In in vitro activity analysis, MHP showed a significant antioxidant capacity, and an inhibition activity of α-glucosidase and the advanced glycation end products (AGEs) formation in the BSA/Glc reaction model. MHP interacted with α-glucosidase and changed the internal microenvironment of the enzyme, and inhibited the AGEs formation, which provides more evidence for the antihyperglycemic mechanism of MHP. The results suggest that ATPS is an efficient and environmentally friendly solvent system, and M. hupehensis has broad application prospects in functional foods, healthcare products, and pharmaceuticals.     

Glucuronidase-assisted Transglycosylation for the Synthesis of Highly Functional Disaccharides: β-D-Glucuronyl 6-O-Sulfo-β-D-Gluco- and -β-D-Galactopyranosides

The substrate specificity of snail (Helix pomatia and Helix aspersa), limpet (Patella vulgata), and bovine glucuronidases was examined by using p-nitrophenyl glucuronide (GlcA-O-pNP) and p-nitrophenyl 6-O-sulfo-β-D-glycopyranosides as the glycosyl donor and acceptors, respectively. When the donor was treated with these enzymes in the absence of the acceptors, β (1 → 3) glucuronyl disaccharides were obtained as the major products together with β (1 → 2) isomers as the result of an enzymatic “self-transglycosylation” reaction. When p-nitrophenyl 6-O-sulfo-β-D-glucopyranosides (6-O-sulfo-Glc-O-pNP and 6-O-sulfo-Glc-S-pNP) were applied as acceptor substrates, every glucuronidase transferred the GlcA residue to either the O-3 or O-2 position in 6-O-sulfo-Glc to yield a mixture of GlcAβ (1 → 3)- and GlcAβ (1 → 2)-linked disaccharides in a ratio of 12:1 ～ 1:1. On the other hand, when p-nitrophenyl 6-O-sulfo-β-D-galactopyranosides (6-O-sulfo-Gal-O-pNP and 6-O-sulfo-Gal-S-pNP) were applied, limpet and bovine glucuronidases gave a GlcAβ (1 → 3)-linked disaccharide regioselectively, while the snail enzymes showed no reactivity.     

Semi-synthesis of unusual chondroitin sulfate polysaccharides containing GlcA(3-O-sulfate) or GlcA(2,3-di-O-sulfate) units

The extraction from natural sources of Chondroitin sulfate (CS), a polysaccharide used for management of osteoarthritis, leads to very complex mixtures. The synthesis of CS by chemical modification of other polysaccharides has seldom been reported due to the intrinsic complexity that arises from fine chemical modifications of the polysaccharide structure. In view of the growing interest in expanding the application of CS to pharmacological fields other than osteoarthritis treatment, we launched a program to find new sources of known or even unprecedented CS polysaccharides. As part of this program, we report herein on an investigation of the use of a cyclic orthoester group to selectively protect the 4,6-diol of N-acetyl-galactosamine residues in chondroitin (obtained from a microbial source), thereby facilitating its transformation into CSs. In particular, three CS polysaccharides were obtained and demonstrated to possess rare or hitherto unprecedented sulfation patterns by 2D NMR spectroscopy characterization. Two of them contained disaccharide subunits characterized by glucuronic acid residues selectively sulfated at position 3 (GlcA(3S)), the biological functions of which are known but have yet to be fully investigated. This first semi-synthetic access to GlcA(3S)-containing CS could greatly expedite such studies, since it can easily furnish considerable amounts of these polysaccharides, which are usually isolated with difficulty and in very low quantity from natural sources.     

Degeneracy of the Antithrombin Binding Sequence in Heparin: 2-O-Sulfated Iduronic Acid Can Replace the Critical Glucuronic Acid

Heparin binds to and activates antithrombin (AT) through a specific pentasaccharide sequence, in which a trisaccharide subsite, containing glucuronic acid (GlcA), has been considered as the initiator in the recognition of the polysaccharide by the protein. Recently it was suggested that sulfated iduronic acid (IdoA2S) could replace this “canonical” GlcA. Indeed, a heparin octasaccharidic sequence obtained by chemoenzymatic synthesis, in which GlcA is replaced with IdoA2S, has been found to similarly bind to and activate antithrombin. By using saturation-transfer-difference (STD) NMR, NOEs, transferred NOEs (tr-NOEs) NMR and molecular dynamics, we show that, upon binding to AT, this IdoA2S unit develops comparable interactions with AT as GlcA. Interestingly, two IdoA2S units, both present in a ¹C₄-²S₀ equilibrium in the unbound saccharide, shift to full ²S₀ and full ¹C₄ upon binding to antithrombin, providing the best illustration of the critical role of iduronic acid conformational flexibility in biological systems.     

Kinetics of Iterative Carbohydrate Transfer to Polysaccharide Catalyzed by Chondroitin Polymerase on a Highly Sensitive Flow‐Type 27 MHz Quartz‐Crystal Microbalance

Using a highly sensitive flow‐type 27 MHz quartz crystal microbalance, we could detect a small mass change during stepwise and alternating one‐sugar transfer of glucuronic acid (GlcA) and N‐acetylgalactosamine (GalNAc) to an acceptor, catalyzed by chondroitin polymerase from Escherichia coli strain K4 (K4CP), and analyze the elongation mechanism of K4CP. K4CP was found to bind strongly to a chondroitin acceptor (K_d=0.97 μM ). Although the binding affinity and the catalytic rate constant for each monomer were considerably different, the apparent catalytic efficiency (k_cat/K_m) was similar (6.3×10⁴ M ^?1 s^?1 for GlcA transfer and 3.4×10⁴ M ^?1 s^?1 for the GalNAc transfer). This is reasonable for the smooth alternating elongation of GlcA and GalNAc on the acceptor. This is the first study to report the determination of kinetic parameters for enzymatic, alternated, sugar elongation.     

Characterisation of oligosaccharides from Baizhu by HILIC-MS

Polysaccharides from Baizhu were separated by preparative hydrophilic interaction liquid chromatography on an XAmide column and its components were characterised as inulin-type polysaccharides with structures of α-D-glucopyranosyl-[-(1 → 2) -β-D-fructofuranosyl-]_n ? 1-(1 → 2) -β-D-fructofuranoside (n = 3–20) by a combinatory application of electrospray-ionisation mass spectrometry, nuclear magnetic resonance and IR, as well as the chemical analysis of monosaccharide composition. In addition, the contents of nystose and 1F-fructofranosylnystose in the crude and purified Baizhu polysaccharides were determined to be 5.81%, 4.92% and 0.70%, 0.84% (w/w), respectively. In addition, MTT assay indicated that the Baizhu polysaccharides could effectively promote spleen lymphocyte transformation for the enhancement of organism immunity. It is for the first time that inulin-type polysaccharides were discovered in Baizhu and its immuno-enhancing activity was reported, which is a vigorous evidence to explain the efficacy of Baizhu.     

Molecularly Imprinted Polymer Coated Quantum Dots for Multiplexed Cell Targeting and Imaging

Advanced tools for cell imaging are of great interest for the detection, localization, and quantification of molecular biomarkers of cancer or infection. We describe a novel photopolymerization method to coat quantum dots (QDs) with polymer shells, in particular, molecularly imprinted polymers (MIPs), by using the visible light emitted from QDs excited by UV light. Fluorescent core–shell particles specifically recognizing glucuronic acid (GlcA) or N‐acetylneuraminic acid (NANA) were prepared. Simultaneous multiplexed labeling of human keratinocytes with green QDs conjugated with MIP‐GlcA and red QDs conjugated with MIP‐NANA was demonstrated by fluorescence imaging. The specificity of binding was verified with a non‐imprinted control polymer and by enzymatic cleavage of the terminal GlcA and NANA moieties. The coating strategy is potentially a generic method for the functionalization of QDs to address a much wider range of biocompatibility and biorecognition issues.     

Evaluation of anticoagulant activity of two algal polysaccharides

Marine algae are important sources of phycocolloids like agar, carrageenans and alginates used in industrial applications. Algal polysaccharides have emerged as an important class of bioactive products showing interesting properties. The aim of our study was to evaluate the potential uses as anticoagulant drugs of algal sulphate polysaccharides extracted from Ulva fasciata (Chlorophyta) and Agardhiella subulata (Rhodophyta) collected in Ganzirri Lake (Cape Peloro Lagoon, north-eastern Sicily, Italy). Toxicity of algal extracts through trypan blue test and anticoagulant action measured by activated partial thromboplastin time (APTT), prothrombin time (PT) test has been evaluated. Algal extracts showed to prolong the PT and APTT during the coagulation cascade and to avoid the blood coagulation of samples. Furthermore, the algal extracts lack toxic effects towards cellular metabolism and their productions are relatively at low cost. This permits to consider the algae as the biological source of the future.     

Comparison and characterization of polysaccharides from natural and cultured <Emphasis Type="Italic">Cordyceps</Emphasis> using saccharide mapping

Comparison and characterization of polysaccharides from natural and cultured Cordyceps on the basis of their chemical characteristics such as glycosidic linkages were performed for the first time using saccharide mapping. The results showed that polysaccharides from most of the natural and cultured Cordyceps had similar responses to enzymatic digestion. These polysaccharides mainly contained (1→4)-β-D-glucosidic linkages, and (1→4)-α-glucosidic, (1→6)-α-glucosidic, 1,4-β-D-mannosidic, as well as (1→4)-α-D-galactosiduronic linkages also existed in some polysaccharides. Especially, natural and cultured Cordyceps polysaccharides could be discriminated on the basis of high performance liquid chromatography profiles of pectinase hydrolysates, which is helpful to control the quality of polysaccharides from Cordyceps.     

二色补血草多糖的结构表征及其对Hela细胞的抑制作用

从中药二色补血草中分离提取到一种水溶性多糖(LP).¹HNMR,¹³CNMR,IR,HPLC和GC-MS等方法分析表明,LP是以α-D-[GlcA(1→6)Glc]二糖为结构单位,每个重复的二糖单位彼此以α-(1→4)糖苷键连接成直链多糖.荧光光谱显示LP的荧光谱峰出现在460nm附近,Zn²⁺,Ca²⁺通过与LP络合,其荧光强度增加.MTT实验表明,LP具有抑制肿瘤细胞活性,对Hela细胞抑制的IC₅₀为62.2μg/mL.     

Synthesis and Cytotoxic Activity of Oxidized Galactomannan/ADR Conjugate

Abstract

Many polysaccharides are expected to apply as biomaterials because they generally show good biocompatibilities and biodegradabilities. It has recently been reported that the saccharides play important roles in biological recognition and the transmission of biological information on a cellar surface. Galactomannan (GalM) is a polysaccharide whose main chain is composed of β-1,4-linked mannose units only. It has some branching α-galactose residues at the C-6 position of mannose units. Therefore, it was of interest of us to use GalM as a drug carrier which was targeted to hepatocyte having a galactose receptor on its cellar surface. Dicarboxy-galactomannan (DC-GalM), which has reactive functional groups and is a carboxylic acid derivative of galactomannan, was prepared by IO^4-/CIO^2- oxidation of GalM. The obtained DC-GalM showed specific binding with maclura pomifera (MPA) [1] which has a specificity to α-galactose. Moreover, DC-GalM showed selective incorporation into hepatocyte. Adriamycine (ADR), which is one of the most prominent anticancer agents, was immobilized to DC-GalM. The DC-GalM/ADR conjugate showed specific cytotoxic activity against HepG2 human hepatoma cells which have a galactose receptor on the cell surface, compared with Hela utrocervical carcinoma cells which have no galactose receptor.     

Molecular characterization of branched polysaccharides from Tremella fuciformis by asymmetrical flow field‐flow fractionation and size exclusion chromatography

To accurately characterize branched polysaccharides with high molecular weights from medicinal and edible mushrooms and identify the limitations of size exclusion chromatography, molecular characteristics of polysaccharides from Tremella fuciformis were determined and compared by asymmetrical flow field‐flow fractionation coupled with multiangle laser light scattering and refractive index detection, and size exclusion chromatography coupled with multiangle laser light scattering and refractive index detection, respectively. Results showed that molecular weights of three batches of T. fuciformis polysaccharides were determined as 2.167 × 10⁶ (TF1), 2.334 × 10⁶ (TF2), and 2.435 × 10⁶ Da (TF3) by size exclusion chromatography, and 3.432 × 10⁶ (TF1), 3.739 × 10⁶ (TF2), and 3.742 × 10⁶ Da (TF3) by asymmetrical flow field‐flow fractionation, as well as 3.469 × 10⁶ Da (TF1) by off‐line multiangle laser light scattering, respectively. Results suggested that size exclusion chromatography was unable to accurately characterize T. fuciformis polysaccharides, which may be due to its limitations such as shear degradation and abnormal coelution. Compared to size exclusion chromatography, asymmetrical flow field‐flow fractionation could be a better technique for the molecular characterization of branched polysaccharides with high molecular weights from medicinal and edible mushrooms, as well as from other natural resources.     

Monosaccharide composition analysis of immunomodulatory polysaccharides by on‐line hollow fiber microextraction with high‐performance liquid chromatography

The monosaccharide compositions of functional polysaccharides are essential for structure elucidation and biological activity determination. A sensitive method based on on‐line hollow‐fiber liquid‐phase microextraction with high‐performance liquid chromatography has been established for the analysis of ten monosaccharide compositions (two uronic acids, two amino sugars and six neutral sugars) of the immunomodulatory polysaccharides. After derivatization , the sample was injected into the lumen of a hollow fiber immersed in butyl ether and separated by liquid chromatography. Under optimized conditions, the calibration curves were linear (r ≥ 0.9996) in the range of 10–2000 μmol L^?1. The limits of detection were in the range of 0.04–1.58 μmol L^?1, and the recoveries were in the range of 92.1–99.6%, which shows that the method is applicable to the analysis of the monosaccharide composition of various polysaccharides.     

Effect of vinegar steaming on the composition and structure of Schisandra chinensis polysaccharide and its anti-colitis activity

In this study, infrared spectroscopy, high-performance liquid chromatography, and matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) technology were applied to systematically explain the Schisandra chinensis’s polysaccharide transformation in configuration, molecular weight, monosaccharide composition, and anti-ulcerative colitis (UC) activity after vinegar processing. Scanning electron microscopic results showed that the appearance of S. chinensis polysaccharide changed significantly after steaming with vinegar. The MALDI-TOF-MS results showed that the mass spectra of raw S. chinensis polysaccharides (RSCP) were slightly lower than those of vinegar-processed S. chinensis polysaccharides (VSCP). The RSCP showed higher peaks at m/z 1350.790, 2016.796, and 2665.985, all with left-skewed distribution, and the molecular weights were concentrated in the range of 1300–3100, with no higher peak above m/z 5000. The VSCPs showed a whole band below m/z 3000, with m/z 1021.096 being the highest peak, and the intensity decreased with the increase of m/z. In addition, compared to RSCPs, VSCPs can significantly increase the content of intestinal short-chain fatty acids (SCFAs). This study showed that the apparent morphology and molecular weight of S. chinensis’s polysaccharides significantly changed after steaming with vinegar. These changes directly affect its anti-UC effect significantly, and its mechanism is closely related to improving the structure and diversity of gut microbiota and SCFA metabolism.     

Configurational statistics of polysaccharides. VI. Linear (1→4)-linked galactan

The conformational energies for (1→4)-linked α-D- and β-D-galactans have been computed by considering nonbonded, torsional, and electrostatic interactions. The electrostatic interactions are estimated by assigning the charges to various atoms in the molecule by the method of Del Re. The characteristic ratios C_N = 〈r²〉₀/Nl_v² are computed for α-D- and β-D-galactans as a function of the degree of polymerization N and the angle τ at the bridge oxygen atom. These values of characteristic ratios obtained for α-D-galactan are very much higher than for β-D-galactan, indicating that the former assumes a highly extended conformation compared to the latter. The values of characteristic ratios of both these polysaccharides show a decrease with increase in τ similar to that observed for other (1→4)-linked polysaccharides. The calculated values of C_∞ of (1→4)-linked polysaccharides show no correlation with the number of allowed conformations but are affected both by the orientation of the interunit glycosidic bonds and the hindered potential associated with chain units. It has also been shown that the magnitude of the steric factor σ may not be used as an index of flexibility for polysaccharides which differ in type of linkage.     

Optimization of the Hydrolysis of Freshwater Polysaccharides

Abstract

Three techniques of acid hydrolysis are verified for their ability to efficiently hydrolyse freshwater polysaccharides. Polysaccharides isolated from two eutrophic lakes were hydrolysed rapidly as compared to model or marine polysaccharides. No single technique is 100% satisfactory for all polysaccharides (i.e. complete hydrolysis with no degradation). Hydrolysis by 0.1 M HCl appeared to give the best results while acceptable results were also obtained for 1.2 M H₂SO₄. The results suggest that it is prudent to hydrolyse freshwater polysaccharides under the least drastic conditions and for as short of a time as possible in order to minimize decomposition of the sample, (esp. uronic acids). Interferences due to humic acid; did not appear to increase as a function of hydrolysis time.     

Mass‐Spectrometric Studies of Providencia SR‐Form Lipopolysaccharides and Elucidation of the Biological Repeating Unit Structure of Providencia rustigianii O14‐Polysaccharide

Enterobacteria Providencia are opportunistic human pathogens causing multiple types of infections. Earlier we have studied the S‐ and R‐form lipopolysaccharides (LPSs) of Providencia strains of various O‐serogroups and established the structures of the O‐polysaccharides (O‐antigens) and core‐region oligosaccharides, respectively. Now we report on mass spectrometric studies of oligosaccharides consisting of the core moiety with one O‐polysaccharide repeating unit attached, which were derived from the SR‐form LPSs of Providencia strains. The site of attachment of the O‐polysaccharide to the core and the structure of the O‐polysaccharide biological repeating unit were elucidated in Providencia rustigianii O14 using NMR spectroscopy.     

Synthesis of L-Rhamnose and N -Acetyl-D-Glucosamine Derivatives Entering in the Composition of Bacterial Polysaccharides by Use of Glucansucrases

Transglucosylation reactions using sucrose as glucosyl donor and either N-acetyl-D-glucosamine, L-rhamnose, or methyl α -L-rhamnopyranoside as acceptors were carried out with recombinant glucansucrases from families 70 and 13 of glycoside-hydrolases. Depending on the enzyme specificity, various carbohydrate structures were synthesized and characterized including α -D-glucopyranosyl-(1 → 6)-N-acetyl-D-glucosamine, α -D-glucopyranosyl-(1 → 4)-N-acetyl-D-glucosamine, α -D-glucopyranosyl-(1 → 1)-β -L-rhamnopyranoside, α -D-glucopyranosyl-(1 → 4)-α -D-glucopyranosyl-(1 → 1)-β -L-rhamnopyranoside, methyl α -D-glucopyranosyl-(1 → 4)-α -L-rhamnopyranoside, and methyl α -D-glucopyranosyl-(1 → 3)-α -L-rhamnopyranoside. Disaccharides were obtained with yields going up to 64%. The structural diversity generated as well as the obtained yields appear to be related to enzyme active site architecture, which can be modulated and improved by enzyme engineering. Several of the obtained disaccharides enter in the composition of surface polysaccharides of pathogenic bacteria, among which is Shigella flexneri. Our results outline the potential of glucansucrases in the chemo-enzymatic synthesis of complex carbohydrates of biological interest whose chemical synthesis may be seen as a limitation.