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

Abstract

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.

Controlled two-photon photodegradation of PEG hydrogels to study and manipulate subcellular interactions on soft materials

“…30 This moiety was chosen due to its capability of undergoing two-photon photolysis 37 (at 740nm the two-photon cross section δ u Φ u ≈ 0.01 to 0.03 GM 38 ) and its previous use in biological systems. 39 Upon two-photon excitation, PEGdiPDA cleaves between the NBE and acrylate functionalities, releasing modified PEG and polyacrylate chains with pendant PEG. Previous studies have shown that the degradation products of this hydrogel formulation are cytocompatible.…”

Intercellular calcium signaling in a gap junction-coupled cell network establishes asymmetric neuronal fates in C. elegans

“…Embryonic neurons were isolated from nsy-5p::mCherry transgenic animals, in which neurons of the NSY-5 network were labeled with mCherry (Shaner et al, 2004;Shaner et al, 2005). Cultured neurons were loaded with the cell-permeable caged fluorophore NPE-HCCC2/AM (Zhao et al, 2004;Dakin et al, 2005). After entering the cell, the acetoxymethyl (AM) esters are hydrolyzed and the caged dye (NPE-HCCC2) becomes trapped inside the cell.…”

Photodegradable Macromers and Hydrogels for Live Cell Encapsulation and Release

“…Several different o-NB groups have been reported [14][15][16] , and small modifications to the structure of the o-NB linker have a significant impact on the photolysis rate. 14 The benzylic position (photodegradation site) of o-NB groups can be primary or secondary (Chart 1a, R 1 = −H or R 1 = −CH 3 ).…”