NIR-Emitting Hemicyanines with Large Stokes’ Shifts for Live Cell Imaging: from Lysosome to Mitochondria Selectivity by Substituent Effect

Abstract

Lysosome imaging without perturbing intracellular activity remains challenging, as the current commercial lysosome probes contain weakly basic amino groups that could perturb lysosome pH. Herein, we illustrate NIR-emitting dyes 2 and 3 (λ em ≈ 700 nm) with very large Stokes' shifts (Δλ = 231−246 nm), attributing to the presence of a 2-hydroxyphenyl(benzo[d]oxazol) (HBO) unit that undergoes excited-state intramolecular proton transfer (ESIPT). The structures of 2 and 3 also contain a hemicyanine unit with benzothiazolium and indolium as a terminal group, respectively. Although the fluorescent probe 2 (Φ fl ≈ 0.28−0.35 in CH 2 Cl 2 ) does not contain any basic amino functional group, it exhibits excellent selectivity for staining intracellular lysosomes, showing the potential for long-term in vivo lysosome imaging without "alkalinizing effect." However, probe 3 (Φ fl ≈ 0.27, in CH 2 Cl 2 ) exhibits excellent selectivity toward mitochondria. The observation showed that the terminal group in the hemicyanine unit played an essential role in guiding the intracellular selectivity to different organelles. In addition, the probes also displayed a transparent optical window between 520 and 590 nm, which is useful to achieve multicolor co-staining study, without fluorescence crosstalk that is a common problem on fluorescence microscopes.

Progress in Tuning Emission of the Excited-State Intramolecular Proton Transfer (ESIPT)-Based Fluorescent Probes

“…Recently, Paul et al reported that 7a was used for hydrogen sulfide detection. 20 A later study from our group 10 suggests that the absorption peaks at ∼428 and 570 nm could be attributed to the neutral form Ar–OH and its anionic form Ar–O – , respectively. By comparison with its model compound ( 10 ), the study revealed that the absorption, λ abs , of 8 is basically determined by the cyanine fragment ( Figure 1 ).…”

“…As shown in Table 1 , the absorption λ abs values change with the type of the e-withdrawing groups (compare 11 , 12 , and 9 ), consistent with the assumption that the dye’s conjugation length is basically defined by the cyanine segment. 10 …”

“…Images D–F are 4 times digitally enhanced images of the region covered by white rectangular box in A–C, respectively. Reproduced from ref ( 10 ). Copyright 2019 American Chemical Society.…”

“…In comparison with commercial dyes that emit in the UV/vis range, NIR dyes have advantages of deep tissue penetration, less tissue damage, and minimal interference caused by tissue absorption and autofluorescence. 6 − 10 …”

“… 16 The acidity of the phenolic proton could be influenced by a suitable electron-withdrawing or electron-donating substituent, which could actively perturb the proton transfer process. 7 − 10 …”