Shozo Onishi1 Naoya Asamura1 Hitomi Seki2 Yosuke Niko2 Seiji Tani1 Jun Kawamata1 Yasutaka Suzuki1

1, Yamaguchi University, Yamaguchi, , Japan
2, Kochi University, Kochi, , Japan

Monitoring mitochondrial membrane potential (MMP) by luminescence microscope is important for understanding activity of a living cell. Our research group has created a novel type of MMP sensitive two-photon excitable green fluorescent probe [1]. When MMP is high, the compound accumulates on mitochondria and transfers to nucleus as the mitochondrial membrane potential decreases. This translocation is reversible. A key parameter of this ability has been found to be polarizability of the compound. In this study, we have undertaken to extend the color variation of this type of MMP sensitive probe. Namely, we have designed red or blue fluorescent probes that exhibit MMP sensitivity.
In order to obtain a red fluorescent probe, a compound possessing a low polarizability but a rather longer π-electron system must be designed. To satisfy this dilemma, we designed pyrene derivatives possessing substituents at 1 and 3 or 1 and 8 positions, 4,4’-[1,3-pyrenediyldi-(1E)-2,1-ethenediyl]bis(1-methylpyridinium) diiodide (1,3-PY), and 4,4’-[1,8-pyrenediyldi-(1E)-2,1-ethenediyl]bis(1-methylpyridinium) diiodide (1,8-PY). As for a blue fluorescent MMP sensitive probe, 1,1’-dimethyl-4,4’-(2,6-naphthalenediyl)dipyridinium diiodide (NPJ) has been designed. This compound possessing rather smaller π-electron system compared to that of the previous biphenyl derivative. The optical properties and MMP sensitivities of these compounds were compared with those of the previously reported MMP sensitive fluorescent probe, 4,4’-[(1,1’-biphenyl)-4,4’-diyldi-(1E)-2,1-ethenediyl]bis(1-methylpyridinium) diiodide (BP).
The wavelengths of absorption maxima of 1,3-PY, 1,8-PY, BP and NPJ were 451, 470, 384 and 338 nm, while the fluorescence maxima were 630, 618, 527 and 455 nm, respectively. As we expected, red, green and blue fluorescent probes were obtained. The maximum value of the two-photon absorption cross-section of these compounds was 952, 1664, 250 and 42 GM (1 GM=10-50 cm4 s photon-1 molecule-1), respectively. These values were sufficient for applying to a two-photon microscope. MMP sensitivity of these compounds were confirmed by a multi-photon fluorescence microscope. Before decreasing MMP, the probes were localized at mitochondria of HEK293 cells. After decreasing MMP by addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP), these compounds transferred from mitochondria to the nucleus. When recovering the MMP by washing out the CCCP, the probes were transferred from nuclei to mitochondria.

[1] H. Moritomo, et al. Cell. Struct. Funct., 2014, 39, 125-133