Redesigning the Coumarin Scaffold into Small Bright Fluorophores with Far-Red to Near-Infrared Emission and Large Stokes Shifts Useful for Cell Imaging

Abstract

Among the palette of previously described fluorescent organic molecules, coumarins are ideal candidates for developing cellular and molecular imaging tools due to their high cell permeability and minimal perturbation of living systems. However, blue-to-cyan fluorescence emission is usually difficult in in vivo applications due to the inherent toxicity and poor tissue penetration of short visible light wavelengths. Here, we introduce a new family of coumarin-based fluorophores, nicknamed COUPY, with promising photophysical properties, including emission in the far-red/near-infrared (NIR) region, large Stokes shifts, high photostability, and excellent brightness. COUPY fluorophores were efficiently synthesized in only three linear synthetic steps from commercially available precursors, with the N-alkylation of a pyridine moiety being the key step at the end of the synthetic route, as it allows for the tuning of the photophysical properties of the resulting dye. Owing to their low molecular weights, COUPY dyes show excellent cell permeability and accumulate selectively in nucleoli and/or mitochondria of HeLa cells, as their far-red/NIR fluorescence emission is easily detected at a concentration as low as 0.5 μM after an incubation of only 20 min. We anticipate that these coumarin scaffolds will open a way to the development of novel coumarin-based far-red to NIR emitting fluorophores with potential applications for organelle imaging and biomolecule labeling.

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

“…The photophysical properties of 4Az-7Az are summarized in Tables 1, S1 and S2 and compared with those of their parent 7-N,N-dialkylamino-COUPY dyes (4-7, respectively). 9 As shown in Figure 2, the new 7-azetidinylcoumarins reproduced the trend previously found with 4-7 and provided intense coloured solutions due to the existence of an intense absorption band in the visible spectrum, with absorption maxima ranging from 515 nm (4Az) to 592 nm (7Az) in aqueous solution (PBS buffer), and from 566 nm (4Az) to 630 nm (7Az) in DCM. Such bathochromic effects with respect conventional coumarins (e.g., compare with 7-azetidinylcoumarin 1Az:  abs = 355 nm in H 2 O) 13 are a consequence of the lengthening of the conjugated chromophore and the increased push−pull character of the conjugated system in COUPY dyes.…”

“…15 8, 155.0, 154.1, 147.2, 142.6, 136.1 (q, J = 33 Hz), 126.6, 122.1, 121.7 (q, J = 277 Hz), 116.9, 110.5 (q, J = 6 Hz), 109. 9,104.4,95.6,85.7,51.6,16.3;19 F NMR (376.5 MHz,CDCl 8, 156.4, 156.2, 155.5, 151.2, 144.5, 127.8, 122.0, 119.0, 112.7, 112.2, 111.8, 96.3, 79.9, 52.7, 46.7, 19.1, 17. 7, 150.7, 143.7, 127.1, 123.0 (q, J = 279 Hz), 117.0 (q, J = 300 Hz, TFA salt), 120. 4,117.9,111.5,111.2,110.9,95.4,78.7,56.2 (q,J = 33 Hz),51.6,18.7,15.8;19 F NMR (376.5 9,155.3,154.4,149.6,144.9,135.9 (q,J = 32 Hz), 125.9, 122.9 (q, J = 280 Hz), 122.6, 121.9 (q, J = 276 Hz), 120.7 (q, J = 323 Hz), 116.…”

“…The later coumarin scaffolds (compounds 2 and 3 in Scheme 1) allowed us to develop a new family fluorophores, nicknamed COUPY, whose photophysical properties can be easily tuned by selecting the appropriate combination of the N-alkylating group at the pyridine moiety and the substituent at position 4 of the coumarin skeleton (compounds 4-7 in Scheme 1). 9 Taking into account their low molecular weight, COUPY dyes offer many attractive features such as large Stokes' shifts, brightness and emission in the far-red/NIR region, as well as aqueous solubility and good cell permeability. Such novel fluorophores opened the way to solving some of the main drawbacks associated with conventional coumarin-based fluorophores.…”

Rationally Designed Furocarbazoles as Multifunctional Aggregation Induced Emissive Luminogens for the Sensing of Trinitrophenol (TNP) and Cell Imaging

“…Above all, the widespread use of explosive formulations poses a serious threat to the human condition, including skin irritation, anemia, and carcinogenicity, among several others . Therefore, the analysis of explosives has been of importance and several attempts have been committed to designing new materials for detecting nitroaromatics (NACs) . The high risk associated with TNT urged us to use trinitrophenol (TNP) as an analyte, which interestingly has superior explosive power when compared to TNT as well as higher water solubility which also threatens the aquatic life and biodiversity.…”

Coumarins into Polyurethanes for Smart and Functional Materials

“…Apart from biological properties and applications in pharmacology as active products, or precursors in new, effective pharmaceuticals, which are reported in different comprehensive reviews [16,17], coumarins are characterised by other interesting properties. Notable photophysical properties of coumarins have turned them into extensive dyes for probing, brightening or labelling, as a function of substitution on the core structure and the interaction with the surrounding media [18][19][20][21][22][23][24]. Besides, coumarin compounds are widely used in the laser industry as laser dyes, as well as being reported as dopants in light-emitting diodes (LEDs and OLEDs) [25][26][27][28][29][30][31][32].…”