Rational Engineering of Bioinspired Anthocyanidin Fluorophores with

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Rational Engineering of Bioinspired Anthocyanidin Fluorophores with Excellent Two Photon Properties for Sensing and Imaging Tianbing Ren, Wang Xu, Fangping Jin, Dan Cheng, Lili Zhang, Lin Yuan, and Xiaobing Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02538 • Publication Date (Web): 09 Oct 2017 Downloaded from http://pubs.acs.org on October 9, 2017

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Analytical Chemistry

Rational

Engineering

of

Bioinspired

Anthocyanidin

Fluorophores with Excellent Two Photon Properties for Sensing and Imaging Tianbing Ren,†,§Wang Xu,†,‖ § Fangping Jin,† Dan Cheng,† Lili Zhang,† Lin Yuan,*† Xiaobing Zhang† †

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical

Engineering, Hunan University, Changsha 410082 (PR China) ‖

Current address: Department of Chemistry, Stanford University

Abstract: Fluorescent materials are widely employed in biological analysis owing to their biorthogonal chemistries for the imaging and sensing purposes. However, it is always a challenge to design fluorophores with desired photophysical and biological properties, due to their complicated molecular and optical nature. Inspired by anthocyanidin, a class of flower pigments, we designed a new fluorescent molecular framework, AC-Fluor. The new fluorescent materials can be rationally engineered to produce a broad range of fluorescent scaffolds with flexibly tunable emission spectra covering the whole visible light, from 467 nm to 707 nm. Furthermore, they exhibit unprecedented environment-insensitive two-photon properties with a substantial cross section as large as 1100 GM in aqueous solution. AC-Fluors demonstrate their biological values through two-photon deep tissue imaging, with the penetration depth as much as 300 μm, while exhibiting minimal cytotoxicity. These features engender a rational engineering strategy for the design and optimization of new fluorescent materials for biological imaging. Introduction Fluorescence techniques, including fluorescence spectroscopy and fluorescence imaging, have emerged as one of the most important approaches to interrogate the biological systems.1-4 1

ACS Paragon Plus Environment


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Importantly, small-molecule fluorescent probes and labels have seen widespread applications owing to their non-invasive nature, superior sensitivity, fine spatiotemporal resolution and emission/target tenability, etc.5-9 To fulfill the growing needs for fluorescent probes, various small-molecule fluorophores with distinct photophysical properties have been developed, such as Tokyo Green,10 CS-Fluors,11 Si-rhodamine,12-14 P-rhodamine,15 Keio Fluors,16 Janelia,17 Fluor PPCys,18 GFP-Fluor,19-22 and Seoul-Fluor,23 etc. However, only a few of these fluorophores have been subjected to systematic structure photophysical property relationship (SPPR) studies, which is an indispensible step to develop fluorophores with desired optical and biological characteristics.24-28 Alongside the chemical development of new fluorophores, optical instrumentation has also seen remarkable advances.6, 29-31 Among them, two-photon excitation (TPE) imaging has emerged to investigate deep tissue levels, owing to its unique advantages of near-infrared (700-1000 nm) excitation, reduced photo-bleaching, suppressed photo-damage/photo-toxicity, etc.26,

30, 32-38

However, most of the developed TPE probes emit between 420 nm and 550 nm, which is below yellow fluorescence region. Within such regime, there is serious TPE auto-fluorescence in tissues, leading to significantly compromised signal-to-noise ratio.29, 32 Therefore, it is in great demand to search for TPE fluorophores with bathochromic emission and large cross section for high TPE efficiency. Inspired by nature once again, we adapted the fluorophore design from a flower pigment anthocyanidin and developed a new class of small-molecule fluorophores (AC-Fluor). Through the fixation of the rotational linker, AC-Fluor shows substantially higher quantum yields than anthocyanidin. Furthermore, the xanthene-like structure of AC-Fluors can be easily synthesized 2


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with one-step ring formation. A systematic study on the substituent effects yields further understanding of SPPR in the AC-Fluor, which further expands the emission coverage of AC-Fluor over the full visible light spectra by tuning the electron donating ability of substituent. Bio-imaging studies of AC-Fluor under both one photon and two-photon excitation have indicated its powerful applications in chemical biology with negligible effects on the cell viability, mainly due to the natural origin of AC-Fluor. To the best of our knowledge, AC-Fluor is the first single fluorophore core that can cover the emission wavelengths from ultraviolet to near-infrared, with broad two-photon absorption cross-sections and tunable quantum yields. All of these data lay a firm foundation that AC-Fluor can function as a versatile platform for developing desirable fluorescent probes. Our new fluorophore provides a colorful future with broad applications in chemical biology. Experimental Section Materials and instruments. For details, see Supporting Information. Calculation of fluorescence quantum yield. Fluorescence quantum yield was determined using optically matching solutions of rhodamine B (Φf = 0.65 in ethanol39) as the standard and the quantum yield was calculated using the following equation 1: Φs = Φr (ArFs/AsFr) (ns2/nr2)2

(1)

where, s and r denote sample and reference, respectively, A is the absorbance, F is the relative integrated fluorescence intensity, and n is the refractive index of the solvent. Measurement of two-photon cross section. The two-photon cross section (σ) was determined by using a femtosecond (fs) fluorescence measurement technique. AC-Fluors were dissolved in pH 7.4, 25 mM PBS buffer, respectively, at a concentration of 5.0 × 10-6 M and then the two-photon 3



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fluorescence intensity was measured at 750-860 nm by using Rhodamine B in MeOH as the standard, whose two-photon property has been well characterized in the literature.40 The two-photon cross-section was calculated by using the following equation 2: σ = σr(Ftnt2ФrCr) /(Frnr2ФtCs)

(2)

where the subscripts t and r stand for the sample and reference molecules, F is the average fluorescence intensity, n is the refractive index of the solvent, C is the concentration, Ф is the quantum yield, and σr is the two-photon cross-section of the reference molecule. Cell

incubation.

The

cytotoxic

effects

of

AC-Fluors

were determined

by MTT

(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl- 2-H-tetrazolium bromide) assays. HeLa cells (1 × 104 cells/well) were placed in a flatbottom 96-well plate in 100 μL culture medium and incubated in 5% CO2 at 37 ˚C for 24 h incubation. The cells were treated with different concentrations (0-20 μM) of AC-Fluors. After 24 h incubation, MTT solution (5.0 mg/mL) was added into each well (10 μL/well, 0.5 mg/mL) and the residual MTT solution was removed after 4 h, and then DMSO (100 μL) was added to each well to dissolve the formazan crystals. After shaking for 10 min, the absorbance values of the wells were recorded using a microplate reader at 490 nm. The cytotoxic effects (VR) of AC-Fluors were assessed using the following equation: VR = A/A0 × 100%, where A and A0 are the absorbance of the experimental group and control group, respectively. The assays were performed in six sets for each concentration. Cell culture and fluorescence imaging. HeLa cells were grown in MEM (modified Eagle’s medium) supplemented with 10% FBS (fetal bovine serum) in an atmosphere of 5% CO2 and 95% air at 37 ˚C. The cells were plated in 35 mm glass-bottom culture dishes and allowed to adhere for 24 h. Before the experiments, the cells were washed with PBS buffer. The HeLa cells were then 4


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incubated with Mito-tag (1 μM) and Mito-Tracker Green (1 μM) for 30 min at 37 ˚C. After washing with PBS three times to remove the remaining Mito-tag and Mito-Tracker Green, the cells were imaged with an Olympus FV1000 equipped with a CCD camera. Preparation of fresh mouse slices and two-photon fluorescence imaging. The slices were prepared from 20-day-old mice. The slices were cut to 400 μm thickness by using a vibrating-blade microtome in 25 mM PBS (pH 7.4). For the control experiments, the slices were incubated with 5 μM ACF27-Cu2+ in PBS buffer bubbled with 95% O2 and 5% CO2 for 30 minutes at 37 ˚C. The slices were then washed three times with PBS, transferred to glass-bottomed dishes, and observed under a twophoton confocal microscope (Olympus FV1000). To obtain the two-photon fluorescence images of the tissues incubated with both the probe and Cu2+, the slices were pretreated with ACF27-Cu2+ for 30 minutes, and then incubated with various concentrations of Cu2+ (2-20 μM). Following this incubation for 20 minutes at 37 ˚C, the slices were washed three times and imaged. Images were acquired using 820 nm excitation and fluorescent emission windows of 605-680 nm. Results and Discussion Design. Anthocyanidin is a natural pigment found in many genus of flower pedals. As demonstrated by nature, slight tuning of the anthocyanidin scaffold could result in a diverse spectra of absorbance colors. However, there is very limited report on its emission profile, mainly due to its instability and poor quantum yield. We reason that: 1) the nucleophilic reaction on the 4 position of ring B leads to the instability of anthocyanidin, as a result of electron migration to the positively charged oxygen; 2) the free rotation through the single bond between ring B and ring C could dissipate away the excitation energy (Figure 1). Since 2015, our and Wang group 5



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independently discovered redshifted fluorescence properties of fused anthocyanidin scaffolds.41-43 However, there lacks a clear understanding of the structure-optical properties relationship of anthocyanidin scaffold. Therefore, it is practically impossible to optimize molecular properties of anthocyanidin without a thorough study. Prior to the systematic synthesis of anthocyanidin analogues, we performed the density functional theory (DFT) calculations with anthocyanidin.37, 44

DFT calculations indicate that position 7 on ring A and position 4’ on ring C are the junction

points of electron distribution (Figure S1). Therefore, altering the substituents at these two positions should significantly affect the emission profile of anthocyanidin. Hence, we propose the rational design and engineering of AC-Fluor, which caps the electron-deficient 4 position, while restraining the rotation between ring B and ring C to minimize energy dissipation. Substituent effect is also systematically studied at position 7 and position 4’ for the optical optimization of AC-Fluor.

Figure 1. The rational engineering principle of anthocyanidin fluorophore, A, B and C represent the three rings for

the chemical modification.

Firstly, to increase the quantum yield, we restricted the rotation between ring B and ring C by linking them with a five-membered ring (Figure 2a). Remarkably, compounds 1a and 1b, although sharing the same substituents, exhibit 7-fold difference of quantum yields (Figure S5 and Table S2). These two dyes display very similar absorbance and emission wavelength; hence the low quantum yield of 1a is mainly attributed to the energy dissipation induced by the single bond rotation. Furthermore, in the process of spectra measurement, we discovered that both compounds 6


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1a and 1b showed poor photostability as their emission maxima decreased significantly over time under ambient condition (Figure 2c). Further analysis reveals that the poor photostability is existing regardless of the substituents on ring A and ring C, as compounds 1a, 1b, 1c and 1e all exhibit substantially decreasing emission intensities with time (Figure 2b, 2c and Figure S6-8). It prompts us to study the decomposition mechanism, which leads to the assumption that the electron deficiency of C-4 in anthocyanidin makes it labile to even very weak nucleophilic reagents under physiological condition, such as H2O molecules. As confirmed by liquid chromatogram and mass data, we observe the formation of 1c-H2O and 1d-H2O adducts (Figure S9-10). The Effects of R1 Substituents at Ring B on the Photo-physical Properties of AC-Fluor. To further test this assumption, compounds ACF1-4 were synthesized with their 4-position capped by either methyl (trifluoromethyl) or o-benzoic acid groups (Table S3) and their stabilities were also measured (Figure 2b & 2c and Figure S11-14). Clearly, the time-dependent intensity curves and spectra demonstrate the stability effects brought by capping the labile 4-position of anthocyanidin fluorophores. Interestingly, the pendent aryl ring at 4-position brings electronic modulations to the spectral properties (Figure 2d-2f). The introduction of phenyl group (ACF5) poses minimal alterations to the absorbance/emission wavelengths, hence indicating weak ground-state interactions between the phenyl moiety and the anthocyanidin core. In fact, due to the rotation between the phenyl ring and the anthocyanidin core, the quantum yield of ACF5 is even weaker than 1e. Introduction of steric hundrance to the appended phenyl ring, such as introducing an ortho carboxylic acid group (ACF4), can increase the quantum yield of anthocyanidin. On the other hand, appending an electron donating group to the phenyl ring causes significant photo-induced

7



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electron transfer (PeT)23 effect (ACF6) that readily quenches the fluorescence. This phenomenon can be utilized to develop PeT based fluorescence turn-on probes.45

Figure 2. a) Limiting the rotation could significantly increase the quantum yield. b) Anthocyanidin structures with capped or upcapped 4-position. c) time-dependent emission profile of the anthocyanidin derivatives. d) anthocyanidin structures with various 4-position capping moieties. e) Absorbance and f) emission spectra of structures in d.

The Effects of R2 Substituents at Ring A on the Photo-physical Properties of AC-Fluor. After the investigation of quantum yield and photo-stability, we proceed to further examine the substituent effects. Firstly, we aim at studying the electronic effect on ring A. A group of new AC-Fluors were synthesized, with restricted rotation and methyl capped 4-position, while ring A are substituted with electron donors at C-5 to C-7 positions (Figure 3a). Our initial DFT calculations indicate that the calculated energy gap between the S0 and S1 states decrease dramatically with intensifying electron donating group (EDG) at the C-7 position (Table S1). Indeed, as shown in Figure 3b-3c and Table S4, increment of the electron donating effects of the substituent results in remarkably longer emission maxima (ACF7 < ACF8 < ACF1), while modifications at 5 or 6 positions hardly affect the emission wavelengths (ACF9 & ACF10). 8


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Furthermore, in contrast to the substantial ratiometric pH-dependence of ACF8 (Figure S15), ACF9 and ACF10 only display fluorescence quenching response when the pH increase from 4 to 10, without clear emission shifts (Figure S16-17). Hence the electron-donating character of C-7 has a more pronounced effect on the emission wavelengths than C-5 or C-6, implying that derivatization on C-7 imposes a more drastic perturbation of the excited-state dipole moment (Figure S1). When an electron donor occupies the C-7 position, effective internal charge transfer states form between the donor and the benzopyranium core. The other derivatization positions (C-5 and C-6) do not elect such a marked effect. As a result, the C-7 electron donor lowers the HOMO–LUMO gap (HLG), thus inducing redshifted emission.44 In addition, we synthesized a group of AC-Fluors with C-4 capped by ortho-benzoic acid, only varying the 7-position on ring A. The absorption/emission maxima shift from 478/548 nm to 490/550 nm, 522/582 nm and 557/608 nm and then to 565/619 nm and 582/632 nm, when the substituents on C-7 position change from hydrogen (ACF11) to methoxyl (ACF12), hydroxyl (ACF4) and dimethyl-amino (ACF13) and then to diethyl-amino (ACF3) and fusion amino (ACF14) (Figure 3d-3f and Table S5). These observations are caused by the electron resonance from the R2 substituent at C-7 position to anthocyanidin.46 On the other hand, a small bathochromic shift was also observed by introducing chlorine to the C-7 position (ACF15), while still keeping its high quantum yield. This is ascribed to the dualism of halogen substituent, i.e. competing inductive electron withdrawing group (EWG)/resonant electron donating group (EDG) interactions.47 These observations are also verified by DFT calculation results (Figure S2). Such findings have strengthened our knowledge of the fluorescent scaffold and allowed us to facilely design fluorophores with distinct emission wavelengths. 9



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Figure 3. a) Anthocyanidin derivatives with fixed 4-position. b) Absorbance and c) emission of the derivatives with R1 = methyl. d) Anthocyanidin derivatives with varied ring A substituents. e) Absorbance and f) emission of the derivatives with R1 = o-benzoic acid.

The Effects of R3 Substituents at Ring C on the Photo-physical Properties of AC-Fluor. Since the electron donating ability of the substituents at C-7 of ring A directly correlates with the absorption/emission wavelengths of AC-Fluors, we then synthesized a new batch offluorophores to further investigate the electronic effects of ring C. Firstly, regioisomeric hydroxyl 1-indanone derivatives are incorporated to investigate the positional effect of R3, while fixing R1 and R2 groups as diethyl amino and benzoic groups (Figure 4a). Among ACF16-19, the 4’-position hydroxyl-substituted ACF17 exhibits the best quantum yield of 0.22, whereas the other substituents are poorly emitting (Figure 4b-4c and Table S6). This phenomenon is in good accordance with the DFT calculation results (Table S1), which implies the formation of stable resonance structure when the electron-donating substituent is at the C-4’ position, para to ring B (Figure S3). After evidencing that C-4’ position interacts best with the fluorescent scaffold, we then focused on the electronic effects of this position. We introduced a series of groups, such as chlorine, methoxyl, hydroxyl, amino, ethyl-amino and diethyl-amino, onto the C-4’ positions of the AC-Fluor and construct a group of fluorescent compounds with the same ortho-benzoic acid 10


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capping at 4-position and diethyl amino substituent on ring A, while only varying the groups on ring C (Figure 4d). The spectral data turns out to be more complicated. For instance, ACF20 bearing a weak electron withdrawing group, chlorine, at the 4’-position displays weak fluorescence intensity; changing chlorine to hydrogen (ACF21) and to moderate EDG (OCH3, ACF22), and then to strong EDGs, such as hydroxyl anion O- (ACF17) and amino groups (ACF3), causes a slight hypsochromic shift accompanying the improvement of fluorescence quantum yields (Figure 4e-4f and Table S7). These results indicate that on C-4’, the inductive effect plays a more significant role than the resonance effect on the photo-physical properties of AC-Fluor.

Figure 4. a) Anthocyanidin derivatives with varied ring C substituents. b) Absorbance and c) emission of the derivatives with regioisomeric hydroxyl group on ring C. d) Anthocyanidin derivatives with varied R3 substituents fixed at 4’-position. e) Absorbance and f) emission of structures in d.

In addition, when the electron donating ability on C-4’ increases from amino (ACF3) to ethyl-amino (ACF23) and diethyl-amino (ACF24), a slight fluorescence intensity decrease and bathochromic shift are observed (Figure 4e-4f and Table S7). Such a phenomenon may attribute to extended molecular orbital and narrowed HOMO–LUMO gaps (HLG) by the hyper-conjugation effect between the electrons in the σ bonds of the substituent on the amino group with the adjacent p and π orbitals, which is also verified by DFT calculation results (Figure S4).48, 49 These results 11



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prove that the positional effect of R3 substituent on the photo-physical properties of AC-Fluor is through both inductive effect and hyper-conjugative effect. Combining all the above observations, we conclude that strong electron donors at the C-4’ position of AC-Fluor benefit high quantum yield and long emission wavelength. NIR-excited Red to Far-red Emission AC-Fluors with Excellent Two-photon Properties. The structure-optical property studies have enabled us deeper understanding of the fluorophore. Since the AC-Fluors have been engineered to afford push-pull pairs and great one-photon absorption coefficients, we next proceed to examine their two-photon properties. We first selected a few compounds with apparent push-pull pairing (ACF3, ACF13, ACF17 and ACF24) and measured their cross sections using two-photon induced fluorescence instrument in PBS buffer containing 1% DMF. Excitingly, all the compounds exhibit grand cross sections (σ > 600 GM), with the best dye (ACF3, σ = 1139 GM) absorbing at 810 nm more than 4 times of rhodamine B (σ = 260 GM) measured in MeOH (Figure 5d and Table S8). We then synthesized several more fluorescent compounds with even stronger electron donors (Figure 5a-5c, ACF25-ACF27), hoping to reach longer emission wavelengths upon two-photon excitation. The results correspond well with our previous structural studies, as we successfully push the emission to more than 640 nm while keeping the two-photon cross sections above 360 GM (ACF26, Figure 5d-5e and Table S8). Notably, to the best of our knowledge, AC-Fluors are by far the best two-photon fluorophore measured in aqueous solution (Table S9).26, 35 Such unprecedented two-photon properties have allowed researchers to readily apply these fluorophores to various biological and medical investigations. Furthermore, it is of particular interest that in contrast to most known two-photon dyes such as acedans analogues, which are in general sensitive to the environment and display 12


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polarity-dependent emission wavelengths, the fluorescence properties of AC-Fluor display comparatively smaller dependence on the solvent polarity (Δλ < 10 nm) (Figure S18). This is highly beneficial for accurate two-photon bio-imaging on the basis of circumventing the problems of classic two-photon dyes.

ACF13 ACF3 ACF17 ACF24 ACF25 ACF26 ACF27

25000 20000 15000 10000 5000 0

550

1200 1000 800 600 400 200 0

σ /GM

30000

σ /GM

Fl. Intensity (a. u.)

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600 650 700 Wavelength, nm

ACF13 ACF3 ACF17 ACF24 ACF25 ACF26 ACF27

760 780 800 820 840 860 Wavelength, nm

Figure 5. a), b) & c) Structures of ACF25-27. d) & e)Two photon emission spectra and two-photon cross sections.

Stability and bioimaging Studies. Ideally, fluorophores that could be used as bioimaging probes should meet the following requirements: (1) Far red to NIR excitation and emission to avoid auto-fluorescence from biosample and high penetration depth; (2) tunable quantum yields by simple structural modification or interactions with the target; (3) good stability (including chemical stability, thermal stability and photostability) for practical application and (4) suitable lipid and water solubility and low bio-toxicity. Previously studies have indicated that AC-Fluors derivatives can satisfy the requirements 1 to 2 and have the remarkable chemical stability in PBS solution. However, anthocyanidins were reported to be susceptable to thermal degradation in aqueous solutions.50 Then their thermal stability of AC-Fluor was assessed by monitoring their fluorescence intensity with time. As shown in Figure S19, the fluorescence intensity of all the representative dyes ACF3, ACF13, ACF14, ACF26, ACF27 remained constant for up to 3 hours when it is incubated at 37 oC in PBS solution (25 mM, pH 7.4, containing 1% DMF). In addition, they also exhibited stable fluorescence intensity when the temperature of the PBS solution changed (Figure S20). All 13



the results demonstrate the excellent thermal stability of AC-Fluor. These observations may be caused by capping the labile 4-position of anthocyanidin, which enhanced the stablity of anthocyanidin moiety and restrained their themml degradation to produce the chalcone forms.51 Finally, to test the photo-stability of AC-Fluor, ACF17, ACF24 and Cy5 in PBS (25 mM, pH 7.4, containing 1% DMF) were irradiated continuously by a Xe-lamp. It is observed that ACF17 and ACF24 remain highly emissive after continuous irradiation for up to one hour (99% and 95% initial values), whereas the fluorescence intensity of commercially available Cy5 decreases to approximate 78% of its initial value under the same irradiation condition (Figure S21). Futhermore, MTT assay confirms that even a concentration as high as 10 μM of randomly selected dyes would not affect cell viability (Figure S22). All the experiments indicate that AC-Fluor possess great potential as an excellent organic fluorophore platform for the imaging applications. d) Signal loss / %

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0% 20%

Mito-Tracker Green Mito-tag

40% 60% 80%

100%

0

5

10

15

20

25

30

Time/min

Figure 6. (a) The design and synthesis of Mito-tag. (b) Images of HeLa cells pretreated with Mito-tag (5 μM) for 10min, followed by 1 μM Mito-Tracker Green for 20 min. (Ⅰ) The fluorescence image of Mito Tracker Green (irradiation HV: 35%), λex = 488 nm, λem = 495-520 nm; (Ⅱ) The fluorescence image of dye Mito-tag (irradiation HV: 35%), λex = 559 nm, λem = 580-620 nm; (Ⅲ) Merged images ofⅠand Ⅱ; (Ⅳ) Merged images of bright field and Ⅲ. (c) TPM images of live cells. (Ⅰ) The fluorescence image of dye Mito-tag, λex = 820 nm, λem = 500-660 nm; (Ⅱ) merged images of bright field and Ⅰ. (d) Signal loss (%) of fluorescent emission of Mito-tag (1.0 μM) and Mito-Tracker Green (1.0 μM) in living tissues with continuous irradiation for 30 min using the confocal microscope (irradiation HV: 95%).

We next investigate the viability of AC-Fluor as bio-imaging reagents. Owing to the positive charge of AC-Fluors, we designed a mitochondria tagger (ACF28, Mito-tag, Figure 6a) and compared its performance to commercially available Mito-tracker green. Figure 6 and Figure S23 14


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show that Mito-tag not only can penetrate the plasma membrane of living cells very well and localize selectively in mitochondria (Figure 6b I-IV & Figure 6c I-II), but also shows much higher photostability than Mito-tracker green under the same excitation condition (Figure 6d and Figure S23). These results corroborate the usage of AC-Fluor in biological applications. The above investigations have indicated that AC-Fluor possessed an excellent two-photon properties in PBS solution and may be highly beneficial for two-photon bio-imaging, dyes ACF3 and ACF27 were chosen for tissue imaging of rat tissue slices by confocal microscopy (Olympus, FV1000) in z-scan mode. As is shown in Figure S24-25, with the concentration of ACF3 increased from 1 to 5 and then to 10 μM, the two-photon fluorescence intensity of ACF3 in tissues were gradually enhanced, simultaneously its tissue imaging capability at depths increased from 80 μm to 220 μm and then to more than 300 μm upon excitation at 810 nm. These data indicated that AC-Fluor have a good tissue penetration even in a very low concentration. This result was further confirmed by ACF27. It can be clearly seen that, with 5 μM ACF27 incubated, the living rat tissue can image from 40 to 360 μm at depth upon excitation at 820 nm (Figure S26a), while the widely used dye rhodamine B is only capable of tissue imaging at depths of 80–200 μm (Figure S26b). This results indicated the tissue penetration capability of ACF27 was much more superior to rhodamine B. Moreover, under the same excitation condition, ACF27 showed much brighter two-photon signals compared to rhodamine B for imaging rat tissue slices. All these results demonstrated the potential of AC-Fluor in two-photon tissue imaging applications. Advantages of TPE with AC-Fluor Compared to the Traditional imaging method. In order to exhibit the uniqueness of TPE for bioimaging especially compared with conventional imaging method with AC-Fluor, ACF3 and ACF27 were chosen for further imaging analysis. The 15



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imaging assay was carried out in living rat tissues by confocal microscopy (Olympus, FV1000). As shown in Figure S27-28, under the same parameters, the tissues incubated with AC-Fluor excitated by two-photon can image brightly at 300 μm depth, while the one-photon excitation (OPE) showed negligble signals even at at depths less than 120 μm. In addition, TPE also created much less tissue autofluorescence than that of OPE (Figure S29). These data indicates that TPE for tissue imaging is much more superior than OPE, whereas AC-Fluor materials (λem > 600 nm) have a great potential to deep tissue imaging with miniaml autofluorescence by TPE. In addition, the heating effect of TPE imaging with AC-Fluor in living tissues was also investigated. As shown in figures S30a & b, within 30 minutes of irradiation at 810 nm, no observable heating effect was identified on the rat liver tissues. When ACF3, one of the best two-photon fluorophores, was incubated with the tissue and subjected to near-infrared excitation, no heating effect was identified within 30 minutes. On the other hand, the fluorescence intensity of ACF3-stained tissues remained constant, as exhibited by figures S30c & d. Figure S31 quantifies the results from figure S30. In total, the results demonstrate that our experimental conditions and our AC-Fluor materials are exempted from the potential heating effects. They are well suitable for two-photon fluorescence imaging. Development of a Novel NIR excitation, Far-red Emission Two-photon Fluorescent Turn-on Copper ions Sensor and Its Application for Imaging in Tissues. To validate the possibility of AC-Fluor for in vivo detection and imaging, we designed and synthesized ACF27-Cu2+ as an NIR TPE fluorescent turn-on probe for Cu2+ (Figure 7a). As designed, ACF27-Cu2+ is essentially non-fluorescent because it formed an intramolecular spirocyclic ring. Upon addition of Cu2+ in HEPES (25 mM, pH 7.0, 20% CH3CN) at room temperature, the peak fluorescence intensity at 627 16


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nm and absorption intensity at 578 nm were observed (Figures 7b and S32a). Remarkably, ACF27-Cu2+ showed large fluorescence enhancement upon binding with Cu2+ aggregates (229-fold enhancement, Figure 7b). Additionally, the off/on sensor ACF27-Cu2+ exhibits a high selectivity for Cu2+ over other metal ions represented by Hg2+, Co2+, Na+, Mg2+, Cd2+, Zn2+, Mn2+, Ni2+, Ca2+, Fe3+ (Figure 7c) and shows a linear response to Cu2+ with a detection limit of 24 nM (3s/slope)43 (Figure S32b). Encouraged by the above results, the probe was then applied for imaging in live tissues by TPE. In the control experiment, tissue incubated with only probe ACF27-Cu2+ (5 μM) for 30 min shows non-fluorescence at the emission window of 500–660 nm (Figure 7d). When the tissues are pretreated with ACF27-Cu2+ for 30 minutes, and then incubated with various 200 eq.

9000

Cu 6000

0

3000 0 600

12000

5 10 20

200 100 0

Fe 3+

2

750

4000

bla

0

650 700 Wavelength, nm

8000

nk Hg 2+

80 60 40 20 0

2+

Co 2+ Na + Mg 2 + Cu 2+ Cd 2+ Zn 2+ Mn 2 + Ni 2 + Ca 2+


12000


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Mean Fl. intensity

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Figure 7. (a) The design and synthesis of the probe ACF27-Cu2+. (b) Fluorescence spectra of probe ACF27-Cu2+ (5 μM) in the presence of various concentrations of (0-200 eq.) in HEPES (25 mM, pH 7.0, 20% CH3CN) buffer with excitation at 574 nm. (c) The fluorescence intensity of probe ACF27-Cu2+ (5 μM) at 627 nm excited at 574 nm in the presence of various metal ions (1 mM). (d) TPM images of live tissues. Live tissues were stained with ACF27-Cu2+ (5 μM) for 30 min and then treated with 0, 2, 5, 10 and 20 μM Cu2+, respectively, for another 20 min. Then, tissues were imaged under excitation at 820 nm (irradiation HV: 35%). Scale bar represents 110 μm. Inset: relative mean fluorescence levels of tissues were quantified.

concentrations of Cu2+ (2-20 μM) for another 20 minutes, a significant fluorescence increase is observed, while the emission intensity can be used to quantify the copper concentration as well (Figure 7d). Moreover, it is worth noting that the tissue incubated with 20 μM Cu2+ can image at 17



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depth as far as 140 μm (Figure S33). All these data indicated that ACF27-Cu2+ can be employed as an efficient two-photon bioimaging probes for Cu2+ analytes. Conclusion In summary, we have rationally engineered a new class of fluorophore, AC-Fluor, from a natural flower pigment anthocyanidin and rendered these dyes with tunable optical properties. We produced a total of more than 30 fluorescent molecules, all of which through simple and scalable one-step condensation. The structural and photophysical characteristics of AC-Fluor were systematically investigated by both DFT calculation and spectroscopy measurements. We calculated the electron dispersion throughout the whole molecule and replaced the interactive joints with electron-donating or electron withdrawing groups. Through tuning the substituents, we not only cover the whole visible light spectrum with the emission of AC-Fluors, but also learn to facilely adjust their quantum yields. These studies have engendered us to better understand the structure-property relationships of AC-Fluors, as well as other classes of fluorophores. Importantly, the unique broad electronic structure and push-pull conformation endow the fluorophores grand two-photon cross section in aqueous phase unparalleled with any previous reports. To test AC-Fluor for bioimaging applications, we designed a mitochondria tag and a Cu2+ turn-on probe, both through simple one-step chemistry. The Cu2+ probe can be applied to live tissue two-photon imaging of copper species with great sensitivity and response range. We envision that our new dye scaffold AC-Fluor provides great potential for probe development and commercial labelling applications. ASSOCIATED CONTENT Supporting Information 18


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Experimental details for chemical synthesis of all compounds, supplementary photophysical characterization of probes, and imaging methods and data. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (L. Y). Fax: +86-731-88821632

Author Contributions §T.R.

and W.X. contributed equally to this work.

ACKNOWLEDGMENTS This work was financially supported by NSFC (21622504, 21302050), the Hunan Provincial Natural Science Foundation of China (14JJ2047), and the Hunan University Fund for Multidisciplinary Developing (2015JCA04). We are also grateful to Prof. Zhihong Liu in Wuhan University for use of his instrument to measure two-photon excited fluorescence data. References (1)

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Rational Engineering of Bioinspired Anthocyanidin Fluorophores with

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