High Photostability in Nonconventional Coumarins with Far-Red/NIR Emission through Azetidinyl Substitution

Abstract

Replacement of electron-donating N,N-dialkyl groups with three- or four-membered cyclic amines (e.g., aziridine and azetidine, respectively) has been described as a promising approach to improve some of the drawbacks of conventional fluorophores, including low fluorescent quantum yields (Φ) in polar solvents. In this work, we have explored the influence of azetidinyl substitution on nonconventional coumarin-based COUPY dyes. Two azetidine-containing scaffolds were first synthesized in four linear synthetic steps and easily transformed into far-red/NIR-emitting fluorophores through N-alkylation of the pyridine moiety. Azetidine introduction in COUPY dyes resulted in enlarged Stokes' shifts with respect to the N,N-dialkylamino-containing parent dyes, but the Φ were not significantly modified in aqueous media, which is in contrast with previously reported observations in other fluorophores. However, azetidinyl substitution led to an unprecedented improvement in the photostability of COUPY dyes, and high cell permeability was retained since the fluorophores accumulated selectively in mitochondria and nucleoli of HeLa cells. Overall, our results provide valuable insights for the design and optimization of novel fluorophores operating in the far-red/NIR region, since we have demonstrated that three important parameters (Stokes' shifts, Φ, and photostability) cannot be always simultaneously addressed by simply replacing a N,N-dialkylamino group with azetidine, at least in nonconventional coumarin-based fluorophores.

Modulating Photostability and Mitochondria Selectivity in Far-Red/NIR Emitting Coumarin Fluorophores through Replacement of Pyridinium by Pyrimidinium

“…This red-shift was considerably larger in the 4-CF3 analogue (e.g., compare abs = 600 nm for 13 and abs = 568 nm for in PBS). As previously found with the first generation of COUPY dyes, 11,12 10-13 showed negative solvatochromism since the absorption maxima was blue-shifted with increasing polarity of the solvent (e.g., compare the abs of 10-13 in DCM with the corresponding values in non-protic (ACN) and protic (EtOH) polar solvents). Moreover, the molar absorption coefficients of the two derivatives containing the ortho,para-pyrimidine moiety were similar even higher than to those of their respective parent dyes, especially in the 4-CF3 analogue (e.g., compare  = 58 mM -1 cm -1 for 13 and  = 20 mM -1 cm -1 for 5 in EtOH).…”

“…It is worth noting that in all of the COUPY scaffolds the chemical shift of H3 appears at higher δ in the Z isomer (e.g., 8.18 ppm in 7 and 8.26 ppm in 8) than in the E isomer (e.g., 6.70 ppm in 7 and 6.75 ppm in 8). Next, we synthesized the corresponding N-methylated pyridinium (10) and pyrimidinium (11)(12)(13) dyes by reaction with methyl trifluoromethanesulfonate in DCM at room temperature. 11 The four new coumarin derivatives were isolated after silica column chromatography as reddish-orange (10-11), purple (12) and dark blue ( 13) solids (yields 64-80 %), and their purity was assessed by reversed-phase HPLC (Figure S1).…”

“…Next, we synthesized the corresponding N-methylated pyridinium (10) and pyrimidinium (11)(12)(13) dyes by reaction with methyl trifluoromethanesulfonate in DCM at room temperature. 11 The four new coumarin derivatives were isolated after silica column chromatography as reddish-orange (10-11), purple (12) and dark blue ( 13) solids (yields 64-80 %), and their purity was assessed by reversed-phase HPLC (Figure S1). Characterization was carried out by HR ESI-MS and 1D and 2D NMR spectroscopy.…”

“…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 dyes and a higher selectivity for mitochondria of HeLa cells was achieved.…”

Regio- and Diastereoselective Synthesis of 2-Arylazetidines: Quantum Chemical Explanation of Baldwin’s Rules for the Ring-Formation Reactions of Oxiranes

“… 13 , 16 − 21 They have a significant role as synthetic building blocks of foldamers, 22 , 23 N -heterocycles, 24 − 28 and polymers. 29 It has also been demonstrated, that the introduction of these strained rings improves enormously the fluorescent properties of rhodamines 30 − 32 and coumarins 33 and the efficacy of homogenous catalysts as ligands, 34 , 35 which makes them an attractive and challenging synthetic topic for chemists nowadays. Compared to their widespread uses, there are relatively few synthetic methods available, although they cover a wide range of structural variability.…”