Strategies for the Synthesis and Screening of Glycoconjugates. 2. Covalent Immobilization for Flow Cytometry

Vetter, Dirk; Tate, Emily M.; Gallop, Mark A.

doi:10.1021/bc00033a014

“…We hoped this would allow us to directly screen carbohydratederivatized beads for binding to protein receptors (32). To evaluate the potential of the TentaGel resin for on-bead screening, we treated separate samples of the Gal␤1,3GalNAc-derivatized beads as well as underivatized beads with various concentrations of Arachis hypogaea (peanut) lectin (23), a protein known to recognize Gal␤1,3GalNAc.…”

Section: Resultsmentioning

confidence: 99%

Polyvalent binding to carbohydrates immobilized on an insoluble resin

Liang

¹

,

Loebach

²

,

Horan

³

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Numerous studies have established that polyvalency is a critical feature of cell surface carbohydrate recognition. Nevertheless, carbohydrate-protein interactions are typically evaluated by using assays that focus on the behavior of monovalent carbohydrate ligands in solution. It has generally been assumed that the relative affinities of monovalent carbohydrate ligands in solution correlate with their polyvalent avidities. In this paper we show that carbohydrate ligands synthesized directly on TentaGel beads interact with carbohydrate-binding proteins in a polyvalent manner. The carbohydrate-derivatized beads can, therefore, be used as model systems for cell surfaces to evaluate polyvalent carbohydrate-protein interactions. By using a combinatorial approach to synthesize solid-phase libraries of polyvalent carbohydrates, one can rapidly address key issues in the area of cell surface carbohydrate recognition. For example, studies reported herein demonstrate that there is an unanticipated degree of specificity in recognition processes involving polyvalent carbohydrates. However, the correlation between polyvalent avidities and solution affinities is poor. Apparently, the presentation of carbohydrates on the polymer surface has a profound inf luence on the interaction of the ligand with the protein receptor. These findings have implications for how carbohydrates function as recognition signals in nature, as well as for how polyvalent carbohydrate-protein interactions should be studied.Interactions between carbohydrates on the surface of one cell type and proteins on the surface of another cell type play critical roles in a wide variety of biochemical recognition processes (1, 2). However, the details of these interactions are poorly understood. Typically, receptor-ligand binding events are studied by making derivatives of the ligand and directly quantitating the affinity. Applying this approach to cell surface carbohydrates has been problematic because carbohydrates are notoriously difficult to synthesize; it is usually not feasible to make more than a small number of derivatives, and even that can take years (3). Moreover, it is difficult to measure the binding affinities by using direct methods because individual carbohydrates bind weakly (K d Ϸ 10 Ϫ3 M) to their protein receptors. Therefore, the relative affinities of carbohydrates are obtained from the concentrations of ligand required to inhibit some event or process-e.g., cell agglutination-that is caused by interactions between the protein receptor and carbohydrates presented on the cell surface. These inhibition assays have shown that many carbohydrate-binding proteins can bind a variety of different structures with similar affinities (4). The broad specificity makes it hard to evaluate which structural features are critical for recognition.Despite the low affinity and broad specificity of individual carbohydrate-protein interactions, carbohydrates function as very specific signals in a wide variety of cell-cell recognition events. Proteins involved...

show abstract

“…For example, carbohydrate ligands bound to TentaGel resin were screened in parallel arrays using digoxigenin-labelled Maackia amurensis agglutinin (a sialic acid specific lectin). [146] Beads binding fluorescent-labeled lectin were detected using flow cytometry. [146] The solid-phase screening of the 1300-compound carbohydrate library (Section 6.3) was carried out on the entire library utilizing a colorimetric assay.…”

Section: Screening Of Oligosaccharide and Glycopeptide Librariesmentioning

confidence: 99%

“…[146] Beads binding fluorescent-labeled lectin were detected using flow cytometry. [146] The solid-phase screening of the 1300-compound carbohydrate library (Section 6.3) was carried out on the entire library utilizing a colorimetric assay. [59] Beads were incubated with biotinlabeled Burhinia purpurea lectin and bound lectin was detected by secondary incubation with alkaline phosphatase conjugated streptavidin.…”

Section: Screening Of Oligosaccharide and Glycopeptide Librariesmentioning

confidence: 99%

Glycopeptide and Oligosaccharide Libraries

Hilaire

¹

,

Meldal

²

2000

Angew. Chem. Int. Ed.

View full text Add to dashboard Cite

Despite the burgeoning interest in the various biological functions and consequent therapeutic potential of the vast number of oligosaccharides found in nature on glycoproteins and cell surfaces, the development of combinatorial carbohydrate chemistry has not progressed as rapidly as expected. The reason for this imbalance is rooted in the difficulty of oligosaccharide assembly and analysis that renders synthesis a rather cumbersome endeavor. Parallel approaches that generate series of analogous compounds rather than real libraries have therefore typically been used. Since generally low affinity is obtained for interactions between carbohydrate receptors and modified oligosaccharides designed as mimetics of natural carbohydrate ligands, glycopeptides have been explored as alternative mimics. Glycopeptides have been proven in many cases to be superior ligands with higher affinity for a receptor than the natural carbohydrate ligand. High-affinity glycopeptide ligands have been found for several types of receptors including the E-, P-, and L-selectins, toxins, glycohydrolases, bacterial adhesins, and the mannose-6-phosphate receptor. Furthermore, the assembly of glycopeptides is considerably more facile than that of oligosaccharides and the process can be adapted to combinatorial synthesis with either glycosylated amino acid building blocks or by direct glycosylation of peptide templates. The application of the split and combine approach using ladder synthesis has allowed the generation of very large numbers of compounds which could be analyzed and screened for binding of receptors on solid phase. This powerful technique can be used generally for the identification and analysis of the complex interaction between the carbohydrates and their receptors.

show abstract

“…Here, the exact identity of the compounds has been lost during the synthesis. Combinatorial libraries can be screened, while the compounds are still attached to the resin beads or onto a surface [17], [18], [83][84][85]. Thus, the solid support (often TentaGel resin) and its linkers must be soluble in water and for quantitative results (e.g., structure -activity relationships) the beads must be uniform in both size and loading capacity.…”

Section: Hit Identification In Combinatorial Libraries By High-througmentioning

confidence: 99%

“…The identity of the bioactive substance can be limited to a few alternative structures by mass spectrometric determination of the molecular mass. Very efficient, automated methods have been developed to isolate the labeled beads, for example, by use of a fluorescence-activated cell sorting instrument [84].…”

Section: Hit Identification In Combinatorial Libraries By High-througmentioning

confidence: 99%

Combinatorial Chemistry

Tiebes

¹

,

Peters

²

2000

Ullmann's Encyclopedia of Industrial Chemistry

0

View full text Add to dashboard Cite

The article contains sections titled: 1. Introduction 2. Concept of Combinatorial Chemistry 3. Methods and Techniques of Combinatorial Synthesis 3.1. Synthetic Strategies Towards Combinatorial Libraries 3.1.1. Split ‐ Pool Synthesis Towards Combinatorial Libraries 3.1.1.1. Techniques using Resin Beads 3.1.1.2. Tea‐Bag Method 3.1.2. Parallel Synthesis Towards Combinatorial Libraries 3.1.2.1. Reaction Apparatus using Resin Beads 3.1.2.2. Multipin Technique 3.1.2.3. Spatially Addressable Parallel Synthesis on Silica Wafer 3.1.3. Reagent Mixture Synthesis Towards Combinatorial Libraries 3.2. Synthetic Methodology for Organic Library Construction 3.2.1. Solid‐Phase Organic Synthesis 3.2.1.1. Solid Support, Linker, and Cleavage Strategies 3.2.1.2. Reaction Portfolio 3.2.2. Synthesis in Solution and Liquid‐Phase Synthesis 4. Characterization of Combinatorial Libraries 4.1. Analytical Characterization 4.2. Hit Identification in Combinatorial Libraries by High‐Throughput Screening 4.2.1. Strategies for Libraries of Compound Mixtures 4.2.1.1. On‐Bead Screening 4.2.1.2. Deconvolution 4.2.1.2.1. Iterative Deconvolution 4.2.1.2.2. Deconvolution by Positional Scanning 4.2.1.2.3. Deconvolution by Orthogonal Libraries 4.2.1.3. Encoding 4.2.1.4. Multiple Cleavable Linkers 4.2.2. Strategies for Libraries of Separate Single Compounds 4.2.3. Conclusion 5. Automation and Data Processing 5.1. Synthesis Automation and Data Processing 5.2. Automated Purification 6. Library Design and Diversity Assessment 6.1. Diversity Assessment for Selection of Building Blocks or Compound 6.2. Iterative Optimization Methods 7. Economic Aspects and Industrial Case Studies 8. Further Developments 8.1. Combinatorial Chemistry and Catalysis 8.2. Combinatorial Chemistry and Material Science

show abstract

Strategies for the Synthesis and Screening of Glycoconjugates. 2. Covalent Immobilization for Flow Cytometry

Cited by 27 publications

References 15 publications

Polyvalent binding to carbohydrates immobilized on an insoluble resin

Polyvalent binding to carbohydrates immobilized on an insoluble resin

Glycopeptide and Oligosaccharide Libraries

Combinatorial Chemistry

Contact Info

Product

Resources

About