Preparation of Carbohydrate Arrays by Using Diels–Alder Reactions with Inverse Electron Demand

Abstract

Carbohydrate microarrays are an emerging tool for the high-throughput screening of carbohydrate-protein interactions that represent the basis of many biologically and medicinally relevant processes. The crucial step in the preparation of carbohydrate arrays is the attachment of carbohydrate probes to the surface. We examined the Diels-Alder reaction with inverse-electron-demand (DARinv) as an irreversible, chemoselective ligation reaction for that purpose. After having shown the efficiency of the DARinv in solution, we prepared a series of carbohydrate-dienophile conjugates that were printed onto tetrazine-modified glass slides. Binding experiments with fluorescently labeled lectins proved successful and homogeneous immobilization was achieved by the DARinv. For immobilization of nonfunctionalized reducing oligosaccharides we developed a bifunctional chemoselective linker that enabled the attachment of a dienophile tag to the oligosaccharides through oxime ligation. The conjugates obtained were successfully immobilized on glass slides. The presented strategies for the immobilization of both synthetic carbohydrate derivatives and unprotected reducing oligosaccharides facilitate the preparation of high-quality carbohydrate microarrays by means of the chemoselective DARinv. This concept can be readily adapted for the preparation of other biomolecule arrays.

Inverse electron demand Diels–Alder (iEDDA)-initiated conjugation: a (high) potential click chemistry scheme

“…48 Only very recently, the iEDDA reaction has also found application in polymer science. These first macromolecular applications of iEDDA concentrate on norbornenes as the alkene reaction partner, in spite of its comparably modest reactivity, due to the easy availability and stability of norbornenes compared to trans-cyclooctenes.…”

Preparation of Small‐Molecule Microarrays by trans‐Cyclooctene Tetrazine Ligation and Their Application in the High‐Throughput Screening of Protein–Protein Interaction Inhibitors of Bromodomains

“…Our previously developed strategy of using the biotin-avidin interaction for compound immobilization met some of these requirements, [11,14] but failed to provide sufficiently intense signals for fluorescence detection (not shown), which is likely due to low compound loading of the surface. [13,16] This may lead to solvent evaporation, compound precipitation, and ultimately a decrease in immobilization efficiency. [13,16] This may lead to solvent evaporation, compound precipitation, and ultimately a decrease in immobilization efficiency.…”

“…[17] With a secondorder rate constant that exceeds 10 4 m À1 s À1 , it is the fastest bioorthogonal reaction known. The most closely related transformation that has been applied for such a process is the above-mentioned tetrazine norborene ligation for the preparation of a carbohydrate microarray, which was described by Wittmann et al, [16] and which took 24 h to reach complete compound immobilization. The most closely related transformation that has been applied for such a process is the above-mentioned tetrazine norborene ligation for the preparation of a carbohydrate microarray, which was described by Wittmann et al, [16] and which took 24 h to reach complete compound immobilization.…”

Combination of inverse electron-demand Diels–Alder reaction with highly efficient oxime ligation expands the toolbox of site-selective peptide conjugations

“…Since oxime ligation has been applied to couple exo-norbornene as a dienophile handle onto aldehydes of the reducing ends of oligosaccharides 40 and taking into consideration that this moiety is easily generated in both recombinant and synthetic peptides 41 without interference with functional side chains, we decided to use this highly efficient reaction to decorate the peptides of interest with the respective diene and dienophile partners. Our modular approach relies on the tailor-made bifunctional building blocks equipped with an aminooxy moiety for the primary incorporation into peptidic counterparts along with the DAR inv site for the successive ligation (Fig.…”