Third Generation Photo-Cross-Linked Small-Molecule Affinity Matrix: A Photoactivatable and Photocleavable System Enabling Quantitative Analysis of the Photo-Cross-Linked Small Molecules and Their Target Purification

The third generation of photoactivatable beads designed to capture bioactive small molecules in a chemo- and site-nonselective manner upon irradiation at 365 nm of UV light and release them as coumarin conjugates after exposure to UV light of 302 nm is described. These photoactivatable and photocleavable beads enable quantification of the amount and distribution of immobilized small molecules prior to the pull-down experiments to identify target protein(s) for the immobilized small molecules. The newly developed system was then used to analyze the functional group compatibility of the photo-cross-linking technology as well as the preferable nature of small molecules to be immobilized. As a result, compounds having a hydroxyl group, carboxylic acid, or aromatic ring were shown to give multiple conjugates, indicating that these compounds are well compatible with the photoactivatable beads system. (Chemical Equation Presented).