TOC ABSTRACT GRAPHIC 2 ABSTRACT Mitochondrial dysfunction has been associated with several human pathologies, including cancer, aging and neurodegenerative diseases. Thus, the availability of selective fluorescent probes for mitochondria could play an important role in the future for monitoring cellular function and disease progression. In this work, we have studied how the photophysical properties and subcellular accumulation of nonconventional coumarin-based COUPY fluorophores can be

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... d through replacement of the para-pyridinium moiety with several heterocycles. Among them, ortho,para-pyrimidinium substitution provided novel fluorophores with suitable photophysical properties for bioimaging applications, including emission in the far-red to NIR region, large Stokes' shifts and high photostability. Furthermore, the compounds exhibited excellent cell membrane permeability in living cells and a higher selectivity for mitochondria compared with the parent COUPY fluorophores. Overall, these results provide useful insights for the development of novel mitochondriatargeted fluorescent probes based on small organic molecules, since higher selectivity for this organelle can be achieved through the replacement of conventional N-alkylated pyridinium moieties by the corresponding N-alkylated-ortho,para-pyrimidinium counterparts. 4 was replaced by cyano(4-pyridine)methylene moiety (e.g., compounds 2 and 3; see Scheme 1) with the aim of increasing the push-pull character of the -delocalized system. 11 N-Alkylation of the pyridine heterocycle provides low molecular weight fluorophores, nicknamed COUPYs, with several attractive characteristics, including emission in the far-red/NIR region, large Stokes' shifts and good brightness. 11 Moreover, COUPY dyes (e.g., see compounds 4 and 5 in Scheme 1 as representative examples) exhibited moderate to good aqueous solubility and excellent cell membrane permeability, and accumulate preferentially in two specific cellular compartments, mitochondria and nucleoli. After the discovery of this promising fluorescent platform, we have initiated a systematic study to unveil the principles governing the structure-photophysical property relationships (SPPR) of COUPY dyes, which are necessary to design new fluorescent probes according to need. Compared with conventional coumarins (e.g., 1), the molecular framework of COUPY scaffolds offers several advantages for carrying out a systematic SPPR study. Indeed, we have found that absorption and emission maxima can be red-shifted through the incorporation of strong electron-withdrawing groups like CF3 either at the 4-position or via N-alkylation of the pyridine heterocycle, 11 while photostability can be increased through replacement of the electron-donating N,N-dialkyl groups (e.g., NMe2 or NEt2) at position 7 with azetidine. 12 In addition, conjugatable versions of COUPY dyes can be easily obtained by incorporation of suitable functional groups (e.g., carboxylic acid, amino, azide or alkyne) via N-alkylation of the pyridine moiety. Such COUPY derivatives allowed us to label receptor-binding peptides on solid-phase by using efficient click chemistry methodologies, 13 and to develop novel photosensitizers for photodynamic anticancer therapy via conjugation to cyclometalated Ir(III) complexes. 14 On the basis of all these precedents, in this work we focused on investigating how the modification of the pyridine moiety in COUPY chromophores could influence the photophysical properties and subcellular accumulation of the compounds. With this idea in mind, herein we describe the synthesis and characterization of four analogues (compounds 10-13, Scheme 1) of our original COUPY dyes (4) (5). Surprisingly, the replacement of para pyridine heterocycle by ortho pyridine (10) or ortho,ortho pyrimidine (11) had a negative effect on the spectroscopic properties of the compounds since both absorption and emission were blue-shifted. By contrast, the incorporation of ortho,para pyrimidine (12-13) led to highly photostable fluorophores with improved photophysical properties compared with the parent compounds, including emission in the far-red to NIR region and large Stokes's shifts. Furthermore, high cell permeability was retained in both ortho,para-pyrimidine-containing 5 dyes and a higher selectivity for mitochondria of HeLa cells was achieved. Overall, these results provide new insights for the design and optimization of mitochondria-targeted fluorescent probes. Scheme 1. Rational design of novel COUPY fluorophores.