Neutral rhodol-based dyes expressing localization in mitochondria.

Neutral rhodol-based red emitters are shown to efficiently localize in mitochondria, as demonstrated by confocal microscopy and co-localization studies. A simple model is proposed to explain the localization mechanism of neutral molecules. The model takes into account the strong coupling between the molecular dipole moment and the electric field of the inner mitochondrial membrane.

Polarized, V-Shaped, and Conjoined Biscoumarins: From Lack of Dipole Moment Alignment to High Brightness.

Eleven conjoined coumarins possessing a chromeno[3,4-c]chromene-6,7-dione skeleton have been synthesized via the reaction of electron-rich phenols with esters of coumarin-3-carboxylic acids, catalyzed by either Lewis acids or 4-dimethylaminopyridine. Furthermore, Michael-type addition to angular benzo[f]coumarins is possible, leading to conjugated helical systems. Arrangement of the electron-donating amino groups at diverse positions on this heterocyclic skeleton makes it possible to obtain π-expanded coumarins with emission either sensitive to, or entirely independent of, solvent polarity with large Stokes shifts. Computational studies have provided a rationale for moderate solvatochromic effects unveiling the lack of collinearity of the dipole moments in the ground and excited states. Depending on the functional groups present, the obtained dyes are highly polarized with dipole moments of ∼14 D in the ground state and ∼20-25 D in the excited state. Strong emission in nonpolar solvents, in spite of the inclusion of a NO2 group, is rationalized by the fact that the intramolecular charge transfer introduced into these molecules is strong enough to suppress intersystem crossing yet weak enough to prevent the formation of dark twisted intramolecular charge transfer states. Photochemical transformation of the dye possessing a chromeno[3,4-c]pyridine-4,5-dione scaffold led to the formation of a spirocyclic benzo[g]coumarin.

Rhodols - synthesis, photophysical properties and applications as fluorescent probes.

The formal replacement of one dialkylamino group in rhodamines with a hydroxyl group transforms them into rhodols. This apparently minor difference is not as small as one may think; rhodamines belong to the cyanine family whereas rhodols belong to merocyanines. Discovered in the late 19th century, rhodols have only very recently begun to gain momentum in the field of advanced fluorescence imaging. This is in part due to the increased understanding of their photophysical properties, and new methods of synthesis. Rationalization of how the nature and arrangement of polar substituents around the core affect the photophysical properties of rhodols is now possible. The emergence of so-called π-expanded and heteroatom-modified rhodols has also allowed their fluorescence to be bathochromically shifted into regions applicable for biological imaging. This review serves to outline applicable synthetic strategies for the synthesis of rhodols, and to highlight important structure-property relationships. In the first part of this Review, various synthetic methods leading to rhodols are presented, followed by structural considerations and an overview of photophysical properties. The second part of this review is entirely devoted to the applications of rhodols as fluorescent reporters in biological imaging.

Modified Isoindolediones as Bright Fluorescent Probes for Cell and Tissue Imaging.

New L-shaped fluorophores possessing five conjugated rings have been synthesized through a four-step procedure involving diketopyrrolopyrrole synthesis and its double N-alkylation, followed by trimethylsilyl bromide-mediated rearrangement to thieno[2,3-f]isoindole-5,8-dione and an intramolecular Friedel-Crafts reaction. In comparison with the parent isoindolediones and π-expanded diketopyrrolopyrroles, these new dyes show red-shifted absorption and emission (up to ≈630 nm). Their structural rigidity is responsible for both the observed small Stokes shifts and large fluorescence quantum yields. Tissue imaging studies revealed that these new dyes show advantageous features including minimal autofluorescence interference and pronounced solvent-sensitive emission. Interestingly, there is a fundamental difference between a dye possessing an amino group and its analog bearing an N-alkyl substituent. The former dye under two-photon excitation at 900 nm gives bright images whereas its N-alkylated counterpart does not. A new type of membrane localization has been discovered by an N-alkylated isoindoledione possessing a benzofuryl substituent. In spite of the fact that the fluorescence quantum yield of this dye in a range of solvents is rather low, it does stain cell membranes exclusively. This new mode of cellular staining opens the door towards further development of membrane staining dyes.

Direct transformation of coumarins into orange-red emitting rhodols.

The lactone carbonyl group of coumarin derivatives has been shown to participate in intramolecular Knoevenagel condensations, enabling the unprecedented direct transformation of coumarins into rhodols. The resulting rhodols, possessing two ester groups, have very intense orange-red fluorescence.

Red emissive sulfone-rhodols as mitochondrial imaging agents.

The controlled hydrolysis of sulfone-rhodamines affords a series of core-modified red-emitting rhodols, the fluorescence of which is sensitive to solvent polarity with pronounced bathochromic shifts recorded in both DMSO and CH3CN combined with an up to 8-fold increase in the fluorescence quantum yield.