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Rational design, synthesis, and verification of affinity ligands to a protein surface cleft

¹ Amersham Biosciences, Björkgatan 30, S-751 84 Uppsala, Sweden
² Amersham Biosciences, 3-25-1, Hyakunincho, Shinjuku-ku, Tokyo 169-0073, Japan
³ Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala, Sweden

Reprint requests to: Andreas Axén, Ulf Tedebark, or Enrique Carredano, Amersham Biosciences R&D, Björkgatan 30, S-751 84 Uppsala, Sweden; e-mail: andreas.axen{at}eu.amershambiosciences.com; ulf.tedebark{at}eu.amershambiosciences.com; or enrique.carredano{at}eu.amershambiosciences.com; fax: +46 18 612 18 44.

The structure-based design, synthesis, and screening of a glucuronic acid scaffold library of affinity ligands directed toward the catalytic cleft on porcine pancreas -amylase are presented. The design was based on the simulated docking to the enzyme active site of 53 aryl glycosides from the Available Chemicals Directory (ACD) selected by in silico screening. Twenty-three compounds were selected for synthesis and screened in solution for binding toward -amylase using nuclear magnetic resonance techniques. The designed molecules include a handle outside of the binding site to allow their attachment to various surfaces with minimal loss of binding activity. After initial screening in solution, one affinity ligand was selected, immobilized to Sepharose (Amersham Biosciences), and evaluated as a chromatographic probe. A column packed with ligand-coupled Sepharose specifically retained the enzyme, which could be eluted by a known inhibitor.

Keywords: Affinity; -amylase; chromatography; glucuronic acid; ligand; NMR; separation; Sepharose; structure-based design; synthesis

Abbreviations: ACD, Available Chemicals Directory • DMSO_d6, perdeuterated dimethyl sulfoxide • DTT, dithiothreitol • MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight • NMR, nuclear magnetic resonance • PPA, porcine pancreas -amylase • RP-HPLC, reversed phase high pressure liquid chromatography • Sepharose HP, Sepharose high performance • STD, saturation transfer difference • TFA, trifluoroacetic acid

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