THE DEVELOPMENT OF FLUORESCENT PROBES FOR LYSOSOMES

Exciting opportunities exist for the development fluorescent probes for intracellular targets, and there is a significant need for the development of new fluorescent probes for live cell imaging studies. Many fluorescent dyes possess ionic charges to enhance their water solubility, but these charges inhibit the membrane permeability of the dyes. We have discovered a new class of fluorescent 2-hydrazinylpyridine (HPY) dyes that are neutral and have many advantageous features for use as receptor- and organelle-targeted probes in cellular imaging studies. We recently developed a new Red-Orange dye designated HPY(530) that is based on 9-fluorenone, a compound that exhibits only very weak fluorescence. We hypothesized that the introduction of a basic amine group would result in accumulation of the dye in acidic intracellular compartments, and could provide useful new probes for staining lysosomes, the acidic organelles that break down waste materials and debris inside the cells. We synthesized the desired HPY(530) compounds in excellent yields starting from the hydrazone formation of 2-hydrazinylpyridines and 9-fluorenone, followed by cyclization with boron trifluoride. We measured the important photophysical properties of HPY(530)-dioxyamine and compared these with the simple HPY(530) dye and the parent compound 9-fluorenone. The HPY dyes exhibited high quantum yields and large separation of absorption and emission bands. Live cell imaging studies of HPY(530)-dioxyamine were conducted through collaboration with Dr. Shuster’s cellular biology laboratory. These studies demonstrated that HPY(530)-dioxyamine localizes in lysosomes. These results suggest that HPY dyes are a promising new class of fluorescent probe for live cell imaging.