Carbohydrate Arrays for the Evaluation of Protein Binding and Enzymatic Modification

Abstract

This paper reports a chemical strategy for preparing carbohydrate arrays and utilizes these arrays for the characterization of carbohydrate-protein interactions. Carbohydrate chips were prepared by the Diels-Alder-mediated immobilization of carbohydrate-cyclopentadiene conjugates to self-assembled monolayers that present benzoquinone and penta(ethylene glycol) groups. Surface plasmon resonance spectroscopy showed that lectins bound specifically to immobilized carbohydrates and that the glycol groups prevented nonspecific protein adsorption. Carbohydrate arrays presenting ten monosaccharides were then evaluated by profiling the binding specificities of several lectins. These arrays were also used to determine the inhibitory concentrations of soluble carbohydrates for lectins and to characterize the substrate specificity of beta-1,4-galactosyltransferase. Finally, a strategy for preparing arrays with carbohydrates generated on solid phase is shown. This surface engineering strategy will permit the preparation and evaluation of carbohydrate arrays that present diverse and complex structures.

The molecular level modification of surfaces: from self-assembled monolayers to complex molecular assemblies

“…No protection schemes are therefore required, and the reaction is also fast and efficient in aqueous media. Diels-Alder reactions have been used for the immobilization of diene-and maleimide-functionalized oligonucleotides, 160 carbohydrates, 161 and peptides 162 on gold and glass surfaces. In a very elegant work, Chaikof and co-workers have prepared complex interfaces using sequential Diels-Alder cycloaddition and CuAAC reactions (Fig.…”

Diels–Alder Ligation of Peptides and Proteins

“…[86][87] Pozsgay et al also utilized the Diels-Alder reaction for the synthesis of a glyconjugate vaccine against gram-negative bacterium Neisseria meningitidis A. [88][89] Furthermore, Mrksich et al have recently described in successive reports the development of biochips microarrays prepared by the Diels-Alder immobilization of monosaccharides [90] and small peptides [91][92][93] to self-assembled monolayers on gold-coated glass surfaces.…”

“…So far only maleimide and benzoquinone have been utilized as dienophiles for Diels-Alder assisted biomolecule modifications. [85][86][87][88][89][90][91][92][93] In order to determine the influence of dienophile double bond activation on the rate of cycloaddition, the N-terminal acryloyl peptide 31 was prepared by solid-phase synthesis following the same procedure as described for the synthesis of 5, but replacing the maleoyl-glycine part for Nacryloylglycine 32 (Scheme 14). This acrylamide was obtained from reaction of glycine methyl ester with acryloyl chloride and subsequent C-terminal deprotection by saponification.…”

“…Whereas the Diels-Alder reaction has been successfully employed for the surface attachment of small biomolecules in microarrays, [86,87,[90][91][92][93] the challenge of immobilizing complex macromolecules such as proteins by this method still Three different types of diene conjugates were designed to assist the Diels-Alder protein immobilization (Scheme 31). One encloses the 2,4-hexadienyl group, the diene functionality that has been extensively studied in previous investigations.…”

Glycosylation Characterization of Human and Porcine Fibrinogen Proteins by Lectin-Binding Biophotonic Microarray Imaging

“…In addition, aggregation of the lectins has been observed at higher concentration observing multi-lectin binding to the tethered-sugar surfaces 39 which is consistent with the Figure 3B for WGA binding to GlcNAc. The relevant dissociation constants K D for tethered-lectin-(mannose containing saccharide) binding reported in thermodynamic studies are in the range of 50-770 M for mono and disaccharides [44][45][46] , and up to 750 nM for more complex longer and branched oligosaccharides 45 . The lectins specificity, Figure 5, indicates binding to the expected tethered-sugars with some notable exceptions and binding of a series of lectins to glycoprotein, pig FBR, Table 2 and Table 3.…”