Synthesis and applications of heterobifunctional photocleavable cross-linking reagents

Abstract

This study designed, synthesized, and characterized a number of new heterobifunctional photocleavable cross-linking reagents that may be used to photomodulate the activity of proteins or to prepare caged fluorescent dyes. Biomolecules or fluorophores caged via a thiol group with the BNBASE reagent can be covalently linked to a second protein, ligand, or derivatized surface through the activated carboxyl group. Members of the new class of photocleavable cross-linking reagent can be used to cage amino groups in the molecule of interest, which can then be covalently linked to a second molecule through the thiol-reactive oxirane group. These crosslinking reagents may be used for the following applications: (1) to cage the activity of a protein by masking its active site with a second macromolecule, e.g., aminodextran; (2) to prepare a protein conjugate exhibiting an enhanced or new activity that is lost on irradiation with near-ultraviolet light, e.g., cross-linked actin dimer; (3) to target the caged compound to a specific site by cross linking to a specific antibody; (4) to attach the caged compound to a thiol or amino derivatized surface; and (5) to render the caged compound fluorescent in order to image or to quantify the yield of the photoactivation reaction.