Development of Novel Dark Quenchers and Their Application to Imaging Probes

Abstract

Rhodamine dyes are among the most widely used fluorescent dyes for bioimaging due to their high fluorescence quantum yield and high photostability. Recently, novel far-red to near-infrared (NIR) fluorescent dyes have been developed: Si-rhodamines (SiRs), in which the O atom of conventional rhodamine dyes at the 10 position of the xanthene moiety is replaced with a Si atom. These SiRs are excellent long-wavelength fluorophores for bioimaging, as they retain the advantageous photophysical properties of conventional rhodamine dyes. Further, we focused on the QSY dark quenchers, which contain the rhodamine scaffold bearing aromatic rings at the N atoms at the 3,6-positions of the xanthene moiety; these show no fluorescence, irrespective of solvent polarity and pH. NIR fluorescent probes based on the Förster resonance energy transfer (FRET) mechanism have various practical advantages, and their molecular design is generally based on the use of NIR dark quenchers as cleavable FRET acceptors. However, few NIR dark quenchers can quench fluorescence in the Cy7 region (over 780 nm). We successfully developed SiR-based NIR dark quenchers (SiNQs) which show broad absorption covering this region. To demonstrate their usefulness, we designed and synthesized a NIR fluorescence probe for matrix metalloproteinase (MMP) activity using SiNQs. The developed probe was able to detect MMP activity in terms of NIR fluorescence, not only in vitro, but also in cultured cells and in a tumor-bearing mouse, in which the tumor was clearly visualized.