Frontal Affinity Chromatography for the Screening of Mixtures

Author(s): N. W.C. Chan, D. F. Lewis, S. Hewko, O. Hindsgaul, D. C. Schriemer

Journal Name: Combinatorial Chemistry & High Throughput Screening
Accelerated Technologies for Biotechnology, Bioassays, Medicinal Chemistry and Natural Products Research

Volume 5 , Issue 5 , 2002

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A protein stationary phase for frontal affinity chromatography was prepared, containing biotinylated β-galactosidase immobilized to controlled pore glass beads via covalently bonded streptavidin. Single microaffinity columns of approximately 30 pmol of active β-galactosidase were prepared from this material and characterized with a known ligand by frontal analysis. These columns were used to measure the specific interactions between the bound β-galactosidase and a library of modified β-galactopyranosides using electrospray mass spectrometry as the means of detection. The library contained 89 entries, each representing 4 diastereomers for a total of 356 library members. A single entry was analysed revealing differential activity among the 4 isomers. The library was grouped into 10 mixtures of 24-40 members each with each mixture infused under frontal chromatographic conditions. This deconvolution procedure led to the identification of 34 entries containing isomers with Kd values better than 10 μM. A method based on a displacement principle was implemented as a rapid prescreen which served as the basis for a parallel column high throughput screening assay.

Keywords: frontal affinity chromatography

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Article Details

Year: 2002
Page: [395 - 406]
Pages: 12
DOI: 10.2174/1386207023330192
Price: $65

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