A small-structure two-photon platform equipped with weak electron donor: Development of permeable two-photon probes for lysosomes and mitochondria in living cells

Small-structure two-photon dyes displaying intense and long-wavelength emission in aqueous solutions are extremely attractive, because they are powerful platforms for design of fluorescent probes targeting various targets in cytoplasma. However, design of such dyes is difficult, because the photo-induced twisted intramolecular charge transfer (TICT) process often leads to low quantum yields. An efficient strategy is to introduce covalent bonds to constrain the intramolecular rotation, such as fluorecein and rhodamine dyes. But this strategy would bring great difficulty in molecular design and synthesis, and therefore cannot be always used. In this work, we provide a novel strategy by linking weak electron donor and strong electron acceptor to a simple conjugated system, and a small-molecule two-photon dye 1-(2-hydroxyethyl)-4-(4-(methylthio)styryl) pyridin-1-ium iodide (HMTP) was synthesized. Experimental results demonstrated that HMTP exhibited suppressed TICT process and strong green fluorescence in buffer solutions with modest two-photon performances (δ = 320 GM). In consequence, HMTP has been modified as two probes, 4-(4-(methylthio)styryl)-1-propylpyridin-1-ium iodide (HMTP-MT) and 4-(4-(methylthio)styryl)pyridine (HMTP-LY), to bioimage mitochondria and lysosomes with intense one- and two-photon fluorescence. These results demonstrate the ability of HMTP as novel platform for the design of various one- and two-photon fluorescent probes, and also the validity of the strategy to construct fluorophores with weak electron donor and strong electron acceptor.