New Caged Coumarin Fluorophores with Extraordinary Uncaging Cross Sections Suitable for Biological Imaging Applications

• Published in: Journal of the American Chemical Society 2004-04, Vol.126 (14), p.4653-4663
• Main Authors: Zhao, YuRui, Zheng, Quan, Dakin, Kenneth, Xu, Ke, Martinez, Manuel L, Li, Wen-Hong
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Coumarins - chemical synthesis; Coumarins - chemistry; Fluorescence; Fluorescent Dyes - chemical synthesis; Fluorescent Dyes - chemistry; Fundamental and applied biological sciences. Psychology; HeLa Cells; Humans; Image Processing, Computer-Assisted; Molecular biophysics; Photochemistry. Photosynthesis. Bioluminescence; Photolysis; Radiation-biomolecule interaction
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja036958m

Photocaged fluorescent molecules are important research tools for tracking molecular dynamics with high spatiotemporal resolution in biological systems. We have designed and synthesized a new class of caged coumarin fluorophores. These coumarin cages displayed more than 200-fold fluorescence enhancement after UV photolysis. Remarkably, the uncaging cross section of a 1-(2-nitrophenyl)ethyl (NPE)-caged coumarin is 6600 at wavelength of 365 nm, about 2 orders of magnitude higher than previously described caged fluorophores. Product analysis of the photolytic reaction showed clean conversion of NPE-caged coumarin to 2-nitrosoacetophenone and the parent coumarin, suggesting that the mechanism of the photolysis follows the known photochemical reaction pathway of the 2-nitrobenzyl group. We have also measured the two-photon uncaging cross sections of NPE-caged coumarins 2a and 5 at 740 nm to be near 1 Goeppert-Mayer (GM). The mechanistic study, together with the two-photon uncaging data, suggested that the coumarin moiety serves as an antenna to enhance the light harvesting efficiency of the coumarin cage and that the photonic energy absorbed by coumarin was utilized efficiently to photolyze the NPE group. Future explorations of this type of “substrate-assisted photolysis” may yield other cages of high uncaging cross sections. For cellular imaging applications, we prepared a cell permeable and caged coumarin fluorophore, NPE-HCCC2/AM (10), which can be loaded into fully intact cells to high concentrations. Initial tests of this probe in a number of cultured mammalian cells showed desired properties for the in vivo imaging applications. The combined advantages of robust fluorescence contrast enhancement, remarkably high uncaging cross sections, noninvasive cellular delivery, and flexible chemistry for bioconjugations should generate broad applications of these caged coumarins in biochemical and biological research.