Investigation on fluorescein derivatives with thermally activated delayed fluorescence and their applications in imaging

Fluorescein derivatives with thermally activated delayed fluorescence (TADF) show much stronger competition ability and vaster prospects than traditional fluorescein dyes due to their prominent long lifetime. It will be of great significance to synthesize more fluorescein derivatives with TADF. In this work, compounds DCF-MPYA and FL with TADF properties were obtained by fine tuning the substituents' structure on the basis of fluorescein derivative DCF-MPYM. Their long-lived triplet excited states (21.78 μs, 32.0 μs) were proved by nanosecond time-resolved transient difference absorption spectra. The steady-state and time-resolved fluorescence spectra showed that DCF-MPYA and FL exhibited red fluorescence around 645 nm and 651 nm, respectively. The results of sensitivity to oxygen and heavy atoms further demonstrated that the time-resolved fluorescence spectra originate from the delayed fluorescence. The time correlated single-photon counting (TCSPC) data indicated that DCF-MPYA and FL showed long-lived lifetimes of 13.16 μs and 23.72 μs, respectively. The energy gap (ΔEST) between the singlet (S1) and triplet (T1) states of DCF-MPYA and FL was calculated to be 3.32 meV and 9.98 meV from the decay rate of DF as a function of temperature. The small energy gap is conducive to the occurrence of efficient TADF at room temperature. Meanwhile, Gaussian calculation was employed to observe the electron density of DCF-MPYA and FL in the ground and excited states. The calculation results indicate that the shapes and energy levels of the highest occupied molecular orbitals (HOMOs), lowest unoccupied molecular orbitals (LUMOs), and LUMOs+1 for the monoanion and dianion forms are similar and thus DCF-MPYA and FL exhibit almost the same luminescence properties. Finally, DCF-MPYA and FL with low toxicity were used in confocal and time-resolved fluorescence imaging. Our construction strategy will be beneficial for developing more fluorescein derivatives with TADF in the future.


Introduction
Fluorescein dyes were rst obtained from resorcinol and phthalic anhydride by von Bayer under the catalysis of zinc chloride in 1871. They have the advantages of large molar extinction coefficient, 1 high uorescence quantum yield, 2 easy synthesis and purication, 3 and low environmental and biological toxicity. 4 Therefore, uorescein is one of the uorescent dyes which is widely used in the development of uorescent probes. 5 However, the traditional uorescein dyes still have some disadvantages, such as: (1) the absorption and emission wavelengths for the designed uorescent probes based on uorescein dyes are mainly concentrated in the visible band, which limits the application of these uorescent probes in the elds of clinical laboratory diagnosis and chemical biology; 6 (2) the uorescein dyes have small Stokes shi (<25 nm), which causes uorescence self-quenching and errors because of the Rayleigh scattering; 7 (3) the uorescence lifetime (nanosecond scale) of uorescein dyes is short, which brings about the interfere of background uorescence. 8 Therefore, it is of great signicance to develop uorescein dyes with long emission wavelength, large Stokes shi and long-lived uorescence lifetime.
In 1961, Parker and Hatchard discovered that eosin has the TADF property with long uorescence lifetime. 9 Compared with traditional rare earth and transition metal complexes, eosin not only has long uorescence lifetime but also has better biocompatibility. Thus, this excellent property has greatly attracted the scientic researchers' attention. Following the dye eosin, Song group rstly introduced substituents at the position 4 0 and 5 0 of 2 0 , 7 0 -dichlorouorescein (DCF) matrix through Duff and Knoevenagel reactions. 6 And they obtained the dye DCF-MPYM with long wavelength emission (l em > 590 nm), large Stokes shi (>150 nm) and long-lived TADF lifetime (22.11 ms in deoxy ethanol). 8 According to the characteristics of long-lived delayed uorescence of DCF-MPYM, it was used in timeresolved uorescence imaging to achieve the effect of no background uorescent interference. Based on this derivation, this group has done a lot of application researches on the performance of TADF. [10][11][12] For example, DCF-MPYM-N1 and DCF-MPYM-N2 with the integration of nitro-reductase activated diagnosis and treatment were designed and synthesized on the basis of TADF uorescein derivative DCF-MPYM. 10 They can be used for specic uorescence diagnosis and photodynamic therapy of hypoxic tumours with overexpression of nitroreductase. In 2018, 11 based on the uorescence resonance energy transfer (FRET) mechanism, they developed the ratio hypochlorous acid uorescent probe FL-CyN by utilizing DCF-MPYM as energy donor and cyanine as energy acceptor. In 2019, 12 they constructed dual targeted diagnosis and treatment integration photosensitizer FL-RGD through covalently coupling TADF uorescein derivative FL with cyclic tripeptide RGD, which was successfully applied to the integration of tumour diagnosis and treatment. Therefore, the above results show that developing more uorescein derivatives with TADF is signicant for the future research of uorescent diagnosis and photodynamic therapy.
It is assumed that it will be possible to obtain uorescein derivatives with TADF property by ne tuning the substituents' structure on the basis of DCF-MPYM. In this study, 2,6-dimethyl-4H-pyran-4-ylidene malononitrile used for constructing DCF-MPYM was transformed to 2-cyano-2-(2,6-dimethyl-4h-pyran-4ylidene) ethyl acetate, DCF derivative DCF-MPYA with long delayed uorescence lifetime was synthesized (Scheme 1). Meanwhile, another uorescein derivative FL was obtained by adjusting the ortho-carboxyl group on the benzene ring to the para-position. Their structures were characterized by 1 H, 13 C NMR and high-resolution mass spectrometry. The luminescence properties of DCF-MPYA and FL were analysed by the steady-state uorescence spectroscopy, time-resolved uorescence spectroscopy, nanosecond transient absorption technology and uorescence lifetime. All the results indicated that both DCF-MPYA and FL showed long-lived lifetimes and TADF. Besides, the density functional theory (DFT) was used to observe the electron density of DCF-MPYA and FL at the ground and excited state. The calculation results show that the shapes and energy levels of HOMOs, LUMOs, and LUMOs+1 for monoanion and dianion forms are similar. Thus, it explains the reason that DCF-MPYA and FL exhibit almost same luminescent properties through the analysis of electron density. The aforementioned results show that ne tuning the structures of dyes with TADF property is helpful to construct new TADF dyes. Our constructing strategy of uorescent dyes and the research methods will be conducive to the development of more organic dyes with TADF in the future.

Synthesis
Compound DCF-MPYA. To a solution of DCF dialdyhyde 6 (0.159 g, 0.348 mmol) in dry EtOH (50 mL)/CH 3 OH (10 mL), ethyl 2-cyano-2-(2,6-dimethyl-4h-pyran-4-ylidene) ethyl acetate (0.147 g, 0.670 mmol) and piperidine (0.172 g, 2.00 mmol) were added under N 2 atmosphere. The mixture was reuxed for 24 h under N 2 atmosphere at 85 C. Aer nishing reaction, the reaction mixture was allowed to cool to 25 C, and then the solvent was evaporated in vacuum. Aer being puried by column chromatography on silica gel (elution : methanol/ dichloroethane from 0.1 : 250 to 1 : 250), compound DCF-MPYA was obtained as black red powder (0.130 g, 0.152 mmol, 22.7% yield). R f ¼ 0.38 (5% CH 3 OH in dichloromethane), 1   summarized in Tables S1 and S2. † DCF-MPYA and FL show two absorption peaks, both of them have large Stokes shis ($144 nm) in different organic solvents and exhibit slightly shorter in water (#95 nm) (Tables S1 and S2 †). However, the Stokes shis of both dyes are much larger than those of conventional Rhodamine, Fluorescein and Rhodol dyes. Thus, it can be observed that the photophysical properties were similar with the reported compound DCF-MPYM in different solvents. Since the structures of DCF-MPYA and FL are analogous to DCF-MPYM, it is inferred that the luminescent characteristics of DCF-MPYA and FL may be consistent with those of DCF-MPYM. Thus, the spectra of two dyes will be studied in detail.

Results and discussion
It was reported that the uorescein derivative DCF-MPYM emits TADF and the most essential reason is the production of long-lived triplet state. 8 Therefore, it was expected that the DCF-MPYA and FL also can exhibit the same characteristic with DCF-MPYM. First, nanosecond time-resolved transient difference absorption spectra of DCF-MPYA and FL were carried out to prove whether the T 1 state of the uorescein derivatives existed. As seen from Fig. 1, upon pulsed laser excitation at 532 nm, signicant bleaching of the transient absorption bands was observed at about 400, 466, 561, and 650 nm in the range of 300-700 nm. The bleaching peaks at 466 nm and 561 nm were attributed to the depletion of the ground state of DCF-MPYA. Transient absorption bands at about 400 nm and 650 nm were due to the transient absorption of T 1 for DCF-MPYA. The T 1 lifetime for the transients of DCF-MPYA was determined as 21.78 ms in deaerated ethanol (Fig. S3A †). Similar transient spectra were also observed for FL in Fig. S4 † and the T 1 lifetime of FL was estimated as 32.0 ms (Fig. S3B †). These results indicate that both the compounds DCF-MPYA and FL exhibit long-lived triplet excited state upon visible light excitation, therefore, the existence of long lifetime triplet state will provide prerequisite for the TADF property of DCF-MPYA and FL.
Luminescence spectra of DCF-MPYA and FL Next, to investigate whether the luminescence spectra of DCF-MPYA and FL were related to the long-lived triplet lifetime, the steady-state and time-resolved uorescence spectra were studied and recorded by using uorescence spectrophotometer. As displayed in Fig. 2, a strong peak at 645 nm was observed in time-resolved uorescence spectrum (delayed uorescence, DF), which closely agreed with the prompt uorescence (PF, 634 nm) in steady-state uorescence spectrum at room temperature. It can be found that the delayed spectrum of DCF-MPYA showed a little red-shi in comparison with the steady-state uorescence spectrum, and similar results were also observed on FL (Fig. S5 †). The maximum steady-state and time-resolved emission wavelengths of FL peaked at about 649 nm and 651 nm, respectively. The red-shi phenomenon was observed in the previous report. 8 It can be attributed to the difference of nuclear conguration between the singlet and triplet excited states of DCF-MPYA and FL. According to the above results, it may be inferred that the prompt and delayed uorescence spectra simultaneously contribute to the long wavelength region of DCF-MPYA and FL. Meanwhile, the time-resolved uorescence spectra were relevant with their triplet state lifetimes.
As we all know, the triplet state is sensitive to oxygen 13 and heavy atom. 14 Thus, the time-resolved spectra will be affected by the deoxygenation and addition of substances containing heavy atoms. To clarify the origination from the delayed component, the time-resolved spectra of DCF-MPYA and FL were also measured with and without N 2 bubbling. It was found that the luminescence intensity of DCF-MPYA at 645 nm enhanced rapidly aer deoxygenation (Fig. 3), and the measured photoluminescence intensity of DCF-MPYA in N 2 rich environment has 20 times enhancement than that in air atmosphere. Meanwhile, FL also shows the similar result, the luminescence intensity at 651 nm has 4 times enhancement aer deoxygenation (Fig. S6 †). In addition, the steady-state uorescence spectra of DCF-MPYA and FL were also recorded aer deoxygenation (Fig. S7 †). It was observed that the steady-state uorescence spectra had no signicant changes whether in nondeaerated or deaerated atmosphere. Furthermore, the delayed uorescence spectra were also obtained aer the addition of ethyl iodide into the DCF-MPYA and FL nitrogen-saturated solution ( Fig. 4 and S8 †). As we expected, it was noted that the time-resolved uorescence spectra showed much enhancement  at about 600-700 nm, but the steady-state uorescence spectra exhibited no obvious enhancement (Fig. S7 †). Thus, the above results further proved that the delayed emission at 645 nm and 651 nm results from the T 1 state of the derivatives and the timeresolved uorescence can be attributed to TADF.

Luminescence lifetime measurement of DCF-MPYA and FL
It is known that the lifetime of delayed uorescence can extend microsecond scale. [15][16][17] Here, the photoluminescent lifetime (PL) of DCF-MPYA and FL for nanosecond and microsecond time scale were measured by time correlated single-photon counting (TCSPC). As shown in Fig. S9-S12, † both the nanosecond and microsecond scale lifetimes of DCF-MPYA exhibit second-order exponential decay modal in N 2 atmosphere (Tables S3 † and 1 (Table 1). Aer deaeration, the uorescent lifetimes of FL for nanosecond and microsecond scale were 3.67 ns and 23.72 ms (Tables 1 and S4 †), respectively. According to the results of lifetime, it can be concluded that the delayed lifetimes of DCF-MPYA and FL enhanced at least 1000 times in comparison with traditional uorescent dyes. Thus, the long wavelength emission can be attributed to the spectral overlap of PF and DF. In other words, the DF at long wavelength region exhibits almost same spectrum as the PF based on the time-resolved and steady-state uorescence. 18 Therefore, there is a uorescence intensity ratio (I DF /I PF ) between PF and DF. According to the reported calculation strategy, 8,19 the intensity ratio I DF /I PF of DCF-MPYA and FL can be approximately calculated to be 3.07 at 645 nm and 6.97 at 651 nm, respectively. This result suggests that the long wavelength region predominantly originates from the slow decay of long-lived DF component.

The energy difference estimation between S 1 and T 1 of DCF-MPYA and FL
Literature reported that the small energy difference between the S 1 and T 1 plays a signicant role in the realization of reverse intersystem conversion (RISC) in pure organic uorescent dyes. 8 It is the prerequisite to realize efficient TADF. The energy difference can be quantitatively calculated by the equation: where k B is Boltzmann's constant, T is temperature, and k T/S ISC is the average rate constant for ISC. The lifetime of DF is mainly determined by RISC ðk T/S ISC Þ and thus the decay rate of DF can be used to represent k T/S ISC for DE ST estimation. Here, the temperature-dependent long lifetimes for DCF-MPYA and FL were measured at 640 nm from 77 K to 300 K (Tables S5 and  S6 †). Then the logarithm of the uorescence decay rate vs. the reciprocal of temperature for DCF-MPYA and FL was obtained in Fig. 5 and S13, † the red tting curves were obtained on the basis of eqn (1). The energy gap between the S 1 and T 1 (DE ST ) was calculated to be 3.32 meV for DCF-MPYA and 9.98 meV for FL, respectively. The small energy gap is protable for efficient TADF. 20 The results were consistent with our previously reported results of DCF-MPYM, which fully proves that the luminescence mechanism of DCF-MPYA and FL is the same as the reported DCF-MPYM. 8    All the electron density of HOMO for them mainly locates on the xanthene unit, the LUMO+1 electron density is positioned at the partial xanthene moiety as well as appendant part.  (Tables S7-S10 †). This also fully explains the reason that the two dyes actually have the same luminescence characteristics.
The confocal and time-resolved uorescence imaging of DCF-MPYA and FL Based on the excellent photophysical properties of DCF-MPYA and FL, they can be expanded to the biological application. First, low cytotoxicity of organic uorophores is one of the important factors for live cells imaging. 24 MTT assays were performed in the MCF-7 cell lines by monitoring the absorbance of formazan at 570 nm to conrm the low cytotoxicity of DCF-MPYA and FL. As shown in Fig. 7, the low cytotoxicity of them in vitro was observed aer different incubation time. Even the incubation time was extended to10 h, the cytotoxicity was moderately low for cells. Then the uorescent images of DCF-MPYA and FL were performed on confocal uorescence microscope. The results showed that both of them have good biocompatibility and they can well stain the MCF-7 cell lines (Fig. 8), thus they exhibit promising potential in biological application. To utilize the advantages of long-lived lifetime of DCF-MPYA and FL, the time-resolved uorescence images of DCF-MPYA and FL were also taken from the laboratory-used time-domain luminescence microscope. As shown in Fig. 9, DCF-MPYA and FL-loaded MCF-7 cells emitted clearly red luminescence aer 15 ms delay time. Thus, the aforementioned results manifested that DCF-MPYA and FL with TADF characteristic will be good candidates for uorescent imaging.

Conclusions
In summary, two red-emission uorescein derivatives (DCF-MPYA and FL) with TADF property were synthesized on the basis of ne tuning the substituents' structure of DCF-MPYM. Luminescence characteristics of them were investigated by the nanosecond time-resolved transient difference absorption spectra, steady-state and time-resolved uorescence spectra. The results indicated that DCF-MPYA and FL exhibited longlived T 1 lifetime and delayed uorescence. Then the lifetimes of delayed uorescence for them were measured to be microsecond scale by TCSPC. Besides, the low energy gap between S 1 and T 1 was estimated to prove the reason for the occurrence of TADF.  2À are similar, which explains the reason that DCF-MPYA and FL exhibit almost same luminescent properties. DCF-MPYA and FL were nally used in the confocal and time-resolved uorescence imaging of MCF-7 cells, which proves that DCF-MPYA and FL will have great potentials in biological applications. This work manifests that ne tuning the structures of dyes with TADF property is helpful and provides a good platform for the development of more organic dyes with TADF in the future.

Conflicts of interest
The authors declare no competing nancial interest.