Automated Segmented-Flow Analysis - NMR with a Novel Fluoropolymer Flow Cell for High-Throughput Screening.

High-throughput analysis in fields such as industrial biotechnology, combinatorial chemistry, and life sciences is becoming increasingly important. Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique providing exhaustive molecular information on complex samples. Flow NMR in particular is a cost- and time-efficient method for large screenings.

Positional preferences of acetyl esterases from different CE families towards acetylated 4-O-methyl glucuronic acid-substituted xylo-oligosaccharides.

Background: Acetylation of the xylan backbone restricts the hydrolysis of plant poly- and oligosaccharides by hemicellulolytic enzyme preparations to constituent monosaccharides. The positional preferences and deacetylation efficiencies of acetyl esterases from seven different carbohydrate esterase (CE) families towards different acetylated xylopyranosyl units (Xylp) - as present in 4-O-methyl-glucuronic acid (MeGlcA)-substituted xylo-oligosaccharides (AcUXOS) derived from Eucalyptus globulus - were monitored by (1)H NMR, using common conditions for biofuel production (pH 5.0, 50°C).

Fast and robust method to determine phenoyl and acetyl esters of polysaccharides by quantitative ¹H NMR.

The acetyl (AcE), feruloyl (FE), and p-coumaroyl (pCE) ester contents of different cereal and grass polysaccharides were determined by a quantitative ¹H NMR-based method. The repeatability and the robustness of the method were demonstrated by analyzing different plant polysaccharide preparations. Good sensitivity and selectivity for AcE, FE, and pCE were observed.

A robust and universal NMR method for the compositional analysis of polysaccharides.

A method is presented for the detailed and accurate quantitative determination of the monomeric composition of polysaccharides. The method is a modification of the well-known Saeman hydrolysis in combination with 600 MHz (1)H NMR quantification. Experimental conditions for this two-step hydrolysis have been optimized for cellulose and hemicelluloses, and the method has been applied to several other polysaccharides as well.

Assessing carbohydrate-carbohydrate interactions by NMR spectroscopy: the trisaccharide epitope from the marine sponge Microciona prolifera.

WEAK RECOGNITION PROCESSES: Weak calcium-mediated carbohydrate-carbohydrate interactions have been detected by DOSY and TRNOESY NMR methods by employing a gold glyconanoparticle as a multivalent system. In addition, 3D models of trisaccharide-Ca(II)-trisaccharide complexes based on results from molecular dynamics simulations are proposed. Diffusion-ordered NMR spectroscopy (DOSY-NMR) and TR-NOESY-NMR experiments are used to detect ligand binding to macromolecular receptors.

Adhesion forces in the self-recognition of oligosaccharide epitopes of the proteoglycan aggregation factor of the marine sponge Microciona prolifera.

Cell aggregation in the marine sponge Microciona prolifera is mediated by a multimillion molecular-mass aggregation factor, termed MAF. Earlier investigations revealed that the cell aggregation activity of MAF depends on two functional domains: (i) a Ca(2+)-independent cell-binding domain and (ii) a Ca(2+)-dependent proteoglycan self-interaction domain. Structural analysis of involved carbohydrate fragments of the proteoglycan in the self-association established a sulfated disaccharide beta-D: -GlcpNAc3S-(1-->3)-alpha-L: -Fucp and a pyruvated trisaccharide beta-D: -Galp4,6(R)Pyr-(1-->4)-beta-D: -GlcpNAc-(1-->3)-alpha-L: -Fucp.

Identification of the smallest structure capable of evoking opsonophagocytic antibodies against Streptococcus pneumoniae type 14.

Synthetic overlapping oligosaccharide fragments of Streptococcus pneumoniae serotype 14 capsular polysaccharide (Pn14PS), [6)-[beta-D-Galp-(1-->4)-]beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->](n), were conjugated to CRM(197) protein and injected into mice to determine the smallest immunogenic structure. The resulting antibodies were then tested for Pn14PS specificity and for their capacity to promote the phagocytosis of S. pneumoniae type 14 bacteria.

Gold nanoparticles coated with a pyruvated trisaccharide epitope of the extracellular proteoglycan of Microciona prolifera as potential tools to explore carbohydrate-mediated cell recognition.

The species-specific cell adhesion in the marine sponge Microciona prolifera involves the interaction of an extracellular proteoglycan-like macromolecular complex, otherwise known as aggregation factor. In the interaction, two highly polyvalent functional domains play a role: a cell-binding and a self-interaction domain. The self-recognition has been characterized as a Ca(2+)-dependent carbohydrate-carbohydrate interaction of repetitive low affinity carbohydrate epitopes.

The application of neoglycopeptides in the development of sensitive surface plasmon resonance-based biosensors.

The development of a biosensor based on surface plasmon resonance is described for the detection of carbohydrate-binding proteins in solution on a Biacore 2000 instrument, using immobilized glycopeptides as ligands. Their selection was based on previous screenings of solid-phase glycopeptide libraries with Ricinus communis agglutinin (RCA(120)) and human adhesion/growth-regulatory galectin-1 (h-Gal-1). Glycopeptides were immobilized on Au sensor chips functionalized with mixed self-assembled monolayers of different ratios of 11-mercapto-1-undecanol and 11-mercaptoundecanoic acid, and of 3-mercapto-1-propanol and 11-mercaptoundecanoic acid.

Analysis of carbohydrate-carbohydrate interactions using gold glyconanoparticles and oligosaccharide self-assembling monolayers.

Carbohydrates are the most extended structures exposed at the surface of most cells. These carbohydrate chains, when arranged in polyvalent clusters, offer a rich supply of low-affinity binding sites, making them a reliable and flexible system to regulate cell adhesion and recognition. The very first model system for cell-cell recognition by means of carbohydrate-carbohydrate interactions in the animal kingdom came from a primitive invertebrate animal: the marine sponge.

Determination of the specificity of monoclonal antibodies against Schistosoma mansoni CAA glycoprotein antigen using neoglycoconjugate variants.

The immunogenic O-glycan of circulating anodic antigen (CAA) is a high-molecular-mass polysaccharide with the unique -->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GalpNAc-(1--> repeating unit. To obtain information at the molecular level about the specificity of monoclonal antibodies against CAA, the immunoreactivity of two series of bovine serum albumin-coupled synthetic oligosaccharides related to the CAA O-glycan was monitored using ELISA and surface plasmon resonance spectroscopy. The importance of the axial hydroxyl group of beta-D-GalpNAc for antibody binding was investigated using the following series of analogues: beta-D-GlcpA-(1-->3)-beta-D-GlcpNAc-(1-->O); beta-D-GlcpNAc-(1-->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GlcpNAc-(1-->O); and beta-D-GlcpA-(1-->3)-beta-D-GlcpNAc-(1-->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GlcpNAc-(1-->O).

SPR studies of carbohydrate-protein interactions: signal enhancement of low-molecular-mass analytes by organoplatinum(II)-labeling.

The relatively insensitive surface plasmon resonance (SPR) signal detection of low-molecular-mass analytes that bind with weak affinity to a protein--for example, carbohydrate-lectin binding--is hampering the use of biosensors in interaction studies. In this investigation, low-molecular-mass carbohydrates have been labeled with an organoplatinum(II) complex of the type [PtCl(NCN-R)]. The attachment of this complex increased the SPR response tremendously and allowed the detection of binding events between monosaccharides and lectins at very low analyte concentrations.

Synthesis and conjugation of oligosaccharide analogues of fragments of the immunoreactive glycan part of the circulating anodic antigen of the parasite Schistosoma mansoni.

The gut-associated circulating anodic antigen (CAA) is one of the major excretory antigens produced by the parasite Schistosoma mansoni. The immunoreactive part of CAA is a threonine-linked polysaccharide composed of long stretches of the unique repeating disaccharide-->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GalpNAc-(1-->. Previously, using surface plasmon resonance and ELISA techniques, it has been shown that some anti-CAA IgM monoclonal antibodies (MAbs) also recognize members of a series of bovine serum albumin (BSA)-coupled synthetic di- to penta-saccharide fragments of the CAA glycan.