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Reducing End (reducing + end)
Selected AbstractsCapillary electrophoresis analysis of glucooligosaccharide regioisomersELECTROPHORESIS, Issue 6 2004Gilles Joucla Abstract Complex gluco-oligosaccharide mixtures of two regioisomer series were successfully separated by CE. The gluco-oligosaccharide series were synthesized, employing a dextransucrase from Leuconostoc mesenteroides NRRL B-512F, by successive glucopyranosyl transfers from sucrose to the acceptor glucose or maltose. The glucosyl transfer to both acceptors, occurring through the formation of ,1,6 linkages, differed for the two series only in the glucosidic bond to the reducing end namely ,1,6 or ,1,4 bond for glucose or maltose acceptor, respectively. Thus, the combination of the two series results in mixed pairs of gluco-oligosaccharide regioisomers with different degrees of polymerization (DP). These regioisomer series were first derivatized by reductive amination with 9-aminopyrene-1,4,6-trisulfonate (APTS). Under acidic conditions using triethyl ammonium acetate as electrolyte, the APTS-gluco-oligosaccharides of each series were separated enabling unambiguous size determination by coupling CE to electrospray-mass spectrometry. However, neither these acidic conditions nor alkaline buffer systems could be adapted for the separation of the gluco-oligosaccharide regioisomers arising from the two combined series. By contrast, increased resolution was observed in an alkaline borate buffer, using differential complexation of the regioisomers with the borate anions. Such conditions were also successfully applied to the separation of glucodisaccharide regioisomers composed of ,1,2, ,1,3, ,1,4, and ,1,6 linkages commonly synthesized by glucansucrase enzymes. [source] Crystal structures of isomaltase from Saccharomyces cerevisiae and in complex with its competitive inhibitor maltoseFEBS JOURNAL, Issue 20 2010Keizo Yamamoto The structures of isomaltase from Saccharomyces cerevisiae and in complex with maltose were determined at resolutions of 1.30 and 1.60 Å, respectively. Isomaltase contains three domains, namely, A, B, and C. Domain A consists of the (,/,)8 -barrel common to glycoside hydrolase family 13. However, the folding of domain C is rarely seen in other glycoside hydrolase family 13 enzymes. An electron density corresponding to a nonreducing end glucose residue was observed in the active site of isomaltase in complex with maltose; however, only incomplete density was observed for the reducing end. The active site pocket contains two water chains. One water chain is a water path from the bottom of the pocket to the surface of the protein, and may act as a water drain during substrate binding. The other water chain, which consists of six water molecules, is located near the catalytic residues Glu277 and Asp352. These water molecules may act as a reservoir that provides water for subsequent hydrolytic events. The best substrate for oligo-1,6-glucosidase is isomaltotriose; other, longer-chain, oligosaccharides are also good substrates. However, isomaltase shows the highest activity towards isomaltose and very little activity towards longer oligosaccharides. This is because the entrance to the active site pocket of isomaltose is severely narrowed by Tyr158, His280, and loop 310,315, and because the isomaltase pocket is shallower than that of other oligo-1,6-glucosidases. These features of the isomaltase active site pocket prevent isomalto-oligosaccharides from binding to the active site effectively. [source] The crystal structure of a xyloglucan-specific endo-,-1,4-glucanase from Geotrichum sp.FEBS JOURNAL, Issue 18 2009M128 xyloglucanase reveals a key amino acid residue for substrate specificity Geotrichum sp. M128 possesses two xyloglucan-specific glycoside hydrolases belonging to family 74, xyloglucan-specific endo-,-1,4-glucanase (XEG) and oligoxyloglucan reducing-end-specific cellobiohydrolase (OXG-RCBH). Despite their similar amino acid sequences (48% identity), their modes of action and substrate specificities are distinct. XEG catalyzes the hydrolysis of xyloglucan polysaccharides in endo mode, while OXG-RCBH acts on xyloglucan oligosaccharides at the reducing end in exo mode. Here, we determined the crystal structure of XEG at 2.5 Å resolution, and compared it to a previously determined structure of OXG-RCBH. For the most part, the amino acid residues that interact with substrate are conserved between the two enzymes. However, there are notable differences at subsite positions ,1 and +2. OXG-RCBH has a loop around the +2 site that blocks one end of the active site cleft, which accounts for its exo mode of action. In contrast, XEG lacks a corresponding loop at this site, thereby allowing binding to the middle of the main chain of the substrate. At the ,1 site in OXG-RCBH, Asn488 interacts with the xylose side chain of the substrate, whereas the ,1 site is occupied by Tyr457 in XEG. To confirm the contribution of this residue to substrate specificity, Tyr457 was substituted by Gly in XEG. The wild-type XEG cleaved the oligoxyloglucan at a specific site; the Y457G variant cleaved the same substrate, but at various sites. Together, the absence of a loop in the cleft and the presence of bulky Tyr457 determine the substrate specificity of XEG. [source] Synthesis of a Potential 10E4 Tetrasaccharide Antigen Involved in Scrapie PathogenesisHELVETICA CHIMICA ACTA, Issue 11 2006Pascal Bindschädler Abstract To test the hypothesis that tetrasaccharide 3 is involved in scrapie pathogenesis, tetrasaccharide derivative 32 functionalized with an amine linker at the reducing end was synthesized. A (2,+,2) glycosylation approach was chosen to furnish the target compound in fully protected form. To investigate its biological role, tetrasaccharide 32 was further functionalized to the corresponding thiol 33 using Traut's reagent. During the course of the synthesis, the N,N -diacetyl protecting group proved surprisingly labile to radical and acidic conditions. [source] 27 ps DFT molecular dynamics simulation of ,-maltose: A reduced basis set study,JOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 11 2010Udo Schnupf Abstract DFT molecular dynamics simulations are time intensive when carried out on carbohydrates such as ,-maltose. In a recent publication (Momany et al., J. Mol. Struct. THEOCHEM, submitted) forces for dynamics were generated from B3LYP/6-31+G* electronic structure calculations. The implicit solvent method COSMO was applied to simulate the solution environment. Here we present a modification of the DFT method that keeps the critical aspects of the larger basis set (B3LYP/6-31+G*) while allowing the less-essential atom interactions to be calculated using a smaller basis set, thus allowing for faster completion without sacrificing the interactions dictating the hydrogen bonding networks in ,-maltose. In previous studies, the gg,-gg-c solvated form quickly converged to the "r" form during a 5 ps dynamics run. This important conformational transition is tested by carrying out a long 27 ps simulation. The trend for the "r" conformer to be most stable during dynamics when fully solvated, is confirmed, resulting in ,20/80% c/r population. Further, the study shows that considerable molecular end effects are important, the reducing end being fairly stable, the O6H pointing at the O5, while the nonreducing end moves freely to take on different conformations. Some "kink" and transition state forms are populated during the simulation. The average H1,···H4 distance of 2.28 Å confirms that the syn form is the primary glycosidic conformation, while the average C1,O1,C4 bond angle was 118.8°, in excellent agreement with experimental values. The length of this simulation allowed the evaluation of vibrational frequencies by Fourier transform of the velocity correlation function, taken from different time segments along the simulation path. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2010 [source] Synthesis of an oligosaccharide,polylysine dendrimer with reducing sugar terminals leading to acquired immunodeficiency syndrome vaccine preparationJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 11 2005Huricha Baigude Abstract A novel cellobiose,polylysine dendrimer with reducing sugar terminals was synthesized in which the reactive reducing end of a disaccharide cellobiose was directing outward. Hexa- O -benzyl-4,-(1-carboxyethyl)-cellobioside (HBCEC) was synthesized through the reaction of a 4,-hydroxyl group of benzyl hexa- O -benzyl-cellobioside with methyl 2-chloropropionate, followed by the removal of the methyl ester group. HBCEC was reacted with polylysine dendrimer generation 3 (G3) to produce benzylated cellobiose,polylysine dendrimer G3. After debenzylation, a cellobiose,polylysine dendrimer G3 was obtained in which the reducing end of the cellobiose was the terminal group of the dendrimer. For the preparation of a dendrimer-type acquired immunodeficiency syndrome vaccine, the cellobiose,polylysine dendrimer was reacted with a tripeptide (glycyl,prolyl,leucine) and a cyclic oligopeptide from the human immunodeficiency virus by reductive amination; this produced a tripeptide-bound cellobiose,polylysine dendrimer and an insoluble compound, respectively. The structure analysis was carried out with NMR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 2195,2206, 2005 [source] Elucidation of the molecular structure of lipid A isolated from both a rough mutant and a wild strain of Aeromonas salmonicida lipopolysaccharides using electrospray ionization quadrupole time-of-flight tandem mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 12 2005Anas El-Aneed The chemical structure of lipid A, isolated by mild acid hydrolysis from a rough mutant and a wild strain of Aeromonas salmonicida lipopolysaccharide, was investigated using electrospray ionization quadrupole time-of-flight (QqToF) hybrid tandem mass spectrometry and showed a great degree of microheterogeneity. The chemical structure of the main constituent of this heterogeneous mixture was identified as a , -D-(1,,,6) linked D-glucosamine disaccharide substituted by two phosphate groups, one being bound to the non-reducing end at position O-4, and the other to the position O-1 of the reducing end of the D-glucosamine disaccharide. The location of the fatty acids linked to the disaccharide backbone was established by identifying diagnostic ions in the conventional QqToF-MS scan. Low-energy collision tandem mass spectrometry analysis of the selected precursor diagnostic ions confirmed, unambiguously, their proposed molecular structures. We have established that myristyloxylauric (C14:0(3- O(12:0))) acid residues were both N-2, and O-3, linked to the non-reducing end of the D-GlcN residue, and that two 3-hydroxymyristic (C14:0(3-OH)) acid chains acylated the remaining positions of the reducing end. The MS and MS/MS data obtained allowed us to determine the complex molecular structure of lipid A. The QqToF-MS/MS instrument has shown excellent superiority over a conventional quadrupole-hexapole-quadrupole tandem instrument which failed to fragment the selected precursor ion. Copyright © 2005 John Wiley & Sons, Ltd. [source] Application of the StrOligo algorithm for the automated structure assignment of complex N-linked glycans from glycoproteins using tandem mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 24 2003Martin Ethier Oligosaccharides associated with proteins are known to give these molecules specific conformations and functions. Analysis of proteins would not be complete without studying the glycans. However, high-throughput techniques in proteomics will soon overwhelm the current capacity of methods if no automation is incorporated into glycomics. New capabilities of the StrOligo algorithm introduced for this purpose (Ethier et al., Rapid Commun. Mass Spectrom., 2002; 16: 1743) will be discussed here. Experimental tandem mass spectra were acquired to test the algorithm using a hybrid quadrupole-time-of-flight (QqTOF) instrument with a matrix-assisted laser desorption/ionization (MALDI) source. The samples were N-linked oligosaccharides from monoclonal antibody IgG, beta interferon and fetuin, detached by enzymatic deglycosylation and labeled at the reducing end. Improvements to the program were made in order to reduce the need for user intervention. StrOligo strips the spectra down to monoisotopic peaks only. The algorithm first builds a relationship tree, accounting for each observed loss of a monosaccharide moiety, and then analyzes the tree and proposes possible structures from combinations of adducts and fragment ion types. A score, which reflects agreement with experimental results, is then given to each proposed structure. The program then decides which combination is the best one and labels relevant peaks in the experimental mass spectrum using a modified nomenclature. The usefulness of the algorithm has been demonstrated by assigning structures to several glycans released from glycoproteins. The analysis was completed in less than 2 minutes for any glycan, which is a substantial improvement over manual interpretation. Copyright © 2003 John Wiley & Sons, Ltd. [source] Crystallization and preliminary X-ray crystallographic study on a xyloglucan-specific exo-,-glycosidase, oligoxyloglucan reducing-end specific cellobiohydrolaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2003Katsuro Yaoi A novel xyloglucan-specific exo-,-glycosidase, oligoxyloglucan reducing-end specific cellobiohydrolase (OXG-RCBH), recognizes the reducing end of oligoxyloglucan and releases two glucosyl residue segments from the main chain. OXG-RCBH was crystallized by the hanging-drop vapour-diffusion method with polyethylene glycol 3000 and polyethylene glycol 400,as precipitants. The crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 61.0, b = 146.9, c = 211.9,Å. The crystals diffract to a resolution of 2.2,Å and are suitable for X-ray structure analysis. [source] Preparation and Use of Microarrays Containing Synthetic Heparin Oligosaccharides for the Rapid Analysis of Heparin,Protein InteractionsCHEMISTRY - A EUROPEAN JOURNAL, Issue 34 2006Christian Noti Abstract Heparin is a highly sulfated, linear polymer that participates in a plethora of biological processes by interaction with many proteins. The chemical complexity and heterogeneity of this polysaccharide can explain the fact that, despite its widespread medical use as an anticoagulant drug, the structure,function relationship of defined heparin sequences is still poorly understood. Here, we present the chemical synthesis of a library containing heparin oligosaccharides ranging from di- to hexamers of different sequences and sulfation patterns. An amine-terminated linker was placed at the reducing end of the synthetic structures to allow for immobilization onto N -hydroxysuccinimide activated glass slides and creation of heparin microarrays. Key features of this modular synthesis, such as the influence of the amine linker on the glycosidation efficiency, the use of 2-azidoglucose as glycosylating agents for oligosaccharide assembly, and the compatibility of the protecting group strategy with the sulfation-deprotection steps, are discussed. Heparin microarrays containing this oligosaccharide library were constructed using a robotic printer and employed to characterize the carbohydrate binding affinities of three heparin-binding growth factors. FGF-1, FGF-2 and FGF-4 that are implicated in angiogenesis, cell growth and differentiation were studied. These heparin chips aided in the discovery of novel, sulfated sequences that bind FGF, and in the determination of the structural requirements needed for recognition by using picomoles of protein on a single slide. The results presented here highlight the potential of combining oligosaccharide synthesis and carbohydrate microarray technology to establish a structure,activity relationship in biological processes. [source] An Expedient Synthesis of the Repeating Unit of the Acidic Polysaccharide of the Bacteriolytic Complex of Lysoamidase,CHEMISTRY - A EUROPEAN JOURNAL, Issue 3 2005Remy E. J. N. Litjens Abstract The first synthesis of the trisaccharide repeating unit of the acidic polysaccharide of the bacteriolytic complex of lysoamidase is presented. The construction is based on a linear glycosylation strategy that starts from the reducing end and employs thio- and selenoglycosides in a highly stereoselective manner by a single set of activation conditions. The thus-formed trisaccharide is selectively deprotected and oxidised, after which a final deprotection step furnishes the desired repeating unit. [source] An Improvement in the Bending Ability of a Hinged Trisaccharide with the Assistance of a Sugar,,Sugar InteractionCHEMISTRY - A EUROPEAN JOURNAL, Issue 22 2005Hideya Yuasa Prof. Abstract Hinged di- and trisaccharides incorporating 2,4-diamino-,- D -xylopyranoside as a hinge unit (Hin) were synthesized. Bridging of the diamino group of Hin by carbonylation or chelation to a metal ion results in a conformational change from 4C1 to 1C4, which in turn causes a bending of the oligosaccharides. In this study, the bending abilities of the hinged oligosaccharides were compared, in terms of the reactivities toward carbonylation and chelation. Di- or trisaccharides containing a 6- O -glycosylated mannopyranoside or galactopyranoside at their reducing ends had bending abilities similar to that of the Hin monosaccharide, probably because there were neither attractive nor repulsive interactions between the reducing and nonreducing ends. However, when Hin was attached at O2 of methyl mannopyranoside (Man,Me), the bending ability was dependent on the nonreducing sugar and the reaction conditions. Typically, a disaccharide,Hin,(1,2)Man,Me,was difficult to bend under all the tested reaction conditions, and the bent population in the presence of ZnII was only 4,%. On the other hand, a trisaccharide,Man,(1,3)Hin,(1,2)Man,Me,was bent immediately after the addition of ZnII or HgII, and the bent population reached 75,%, much larger than those of all the other hinged trisaccharides ever tested (<40,%). This excellent bending ability suggests an attractive interaction between the reducing and nonreducing ends. The extended conformation was recovered by the addition of triethylenetetramine, a metal ion chelator. Reversible, quick, and efficient bending of the hinged trisaccharide was thus achieved. [source] | ||||||||||