Near-infrared asymmetrical heptamethine cyanines specifically imaging cancer cells by sensing their acidic lysosomal lumen

Abstract

Tumor cytoreductive surgery faces great challenges to completely remove malignant residues due to the indistinct margin between the normal and neoplastic tissues. Intra-operative delineation of a tumor invasive margin will greatly improve the prognosis of the surgery. Near-infrared (NIR) fluorescence imaging shows advantages in delineating a tumor margin due to its high sensitivity and deep tissue penetration depth. Therefore, it is important to develop NIR fluorophores with high specificity and sensitivity. Cancer cells demonstrate more acidic lysosomal lumen (pH 3.8–4.7) than that of normal cells (pH 4.5–6.0), which facilitates their invasion, migration and metastasis. In this work, we developed five asymmetrical heptamethine cyanine (Hcyanine) based NIR fluorophores, in which a primary amine as a proton sensitizing group and another functional group as an electron density modulator were labeled on the Hcyanine respectively. While these fluorophores remained silent under a neutral environment, their fluorescence quantum yields increased 18–54 times upon the acidification from pH 7.4 to 2.4. Modulating the electron density on the fluorophores could fine tune their pK_a (ΔpH = 0.1 pH units) to fit the lysosomal pH (pH_lys) in cancer cells. AsP2 with a pK_a of 4.0 visualized cancer cells, especially the invasive cancer cells with high selectivity. Even though AsP2 remained silent in the normal cells, its activation in the lysosomes after cytoplasmic acidification verified the feasibility to specifically detect tumor by distinguishing the pH_lys discrepancy between the normal and cancer cells. Considering the up-regulated lysosomal acidity is a universal characteristic of cancer cells, pH_lys responsive fluorophores are promising to accurately delineate tumor margin and guide intra-operative tumor resection with high sensitivity and universality.

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1 Introduction

Cytoreductive resection is a mainstay for most solid tumors because the extent of tumor excision directly correlates with the survival of patients.^1,2 However, due to the infiltrated nature of highly invasive cancer cells, surgeons face great challenges to completely remove tumor residues because of the indistinct margin between the normal and malignant tissues.³ Considering that tumor residues beyond the cutting edge is a primary reason leading to the high recurrence and poor prognosis of cancer patients, visualization of the extracapsular extension of a tumor during the surgery will remarkably reduce the positive surgical margins and minimize damage to adjacent functional structures such as major vasculatures and non-renewable nerves.⁴

Even though multiple imaging modalities were applied in assisting intra-operative tumor excision,⁵ near-infrared (NIR) fluorescence imaging shows great potential in the image-guided surgery due to the deep tissue penetration depth (up to centimeter) of NIR light (700–900 nm), minimized autofluorescence from endogenous molecules such as water and hemoglobin, non-ionizing radiation and handling convenience.⁶ As the only FDA and EDA approved NIR fluorescence probe, indocyanine green (ICG) has been utilized clinically in visualizing hepatocellular carcinomas (HCC)⁷ and colorectal hepatic metastases.⁸ However, the wide applications of ICG in guiding surgery are limited by its lack of tumor targeting specificity. To overcome this problem, NIR fluorescence probes labeled with tumor targeting domains including antibodies,⁹ peptides¹⁰ or small molecular ligands¹¹ were developed in assisting tumor excision. Broad translations of these receptor targeting probes are also hindered by the diversity of tumor genotypes and phenotypes. As an example, less than 25% breast cancer patients over-express Her2/neu receptor, the most widely recognized biomarker for breast tumor targeted therapy.¹² Therefore, NIR fluorescence probes visualizing invasive tumor margin regardless of their subtypes and developmental stages will be invaluable in assisting the image-guided tumor surgery.

Lysosomes are membrane-bound organelles found in all types of mammalian cells. As the “stomachs of the cells”, they digest extraneous materials from the endocytic pathway or worn-out organelles/cell debris from the autophagy.¹³ The most distinguished characteristic of lysosomes is the acidic lumen that activates more than 50 hydrolytic enzymes.¹⁴ The maximized enzymatic activity in lysosomes not only degrades the exogenously and endogenously delivered materials, but also facilitates the invasion, migration and metastasis of cancer cells by remodeling the extracellular matrix (ECM).¹⁵ While the lysosomal pH (pH_lys) in normal cells was determined in a range of 4.5–6.0, this value was measured as low as 3.8–4.7 in cancer cells, especially the cells located in invasive tumor margin,¹⁶ which was believed to maximize the enzymatic activities of the exocytosed proteases. We hypothesize that the pH_lys discrepancy between the normal and invasive cancer cells could be a useful biomarker to distinguish the positive tumor margin. Ge et al. recently reported a Darrow Red analogue based far-red fluorescence probe specifically imaging cancer cells by sensing their acidic lysosomal lumen.¹⁷ Even though this work verified the feasibility to detect cancer cells by distinguishing the pH_lys between the cancer and normal cells, the emission maximum (λ_max = 657 nm) at the edge of NIR wavelength range, impropriate pK_a (∼2.4) and far less than satisfactory pH sensitivity (11 folds quantum yield (Q.Y.) enhancement from pH 5.0 to pH 1.6) prevent this probe to image the tumor margin with high sensitivity and target to background (T/B) ratio.

To specifically visualize cancer cells by sensing their pH_lys, the aiming pH responsive probes should meet criteria including: (1) fine-tuned pK_a value close to the pH_lys of cancer cells; (2) remarkable signal variation in the pH transition point; (3) optimized absorption/emission in the NIR wavelength window; (4) high photophysical stability in acidic environment. As the most widely used NIR fluorophore in biomedical imaging, heptamethine carbocyanine (Hcyanine) derivatives show high molar extinction coefficients, high fluorescence quantum yields and favorable biocompatibility.¹⁸ We previously reported a series of Hcyanine based NIR fluorescence fluorophores that not only visualized subcutaneous tumor xenograft (cm scale)^19,20 and lung metastases (mm scale)²¹ with high T/B ratio by sensing tumor acidic extracellular fluid (pH_e 6.2–6.9), but also successfully estimated tumor metastatic potential by delineating the acidosis distribution pattern inside the tumor.²² Asymmetrical Hcyanines in which the two terminal aza-heterocyclic indoles are substituted with different functional groups attract attentions because of the convenience to optimize the photophysical parameters, strengthen the photochemical stability and fine tune the pK_a. According to above rationale, we developed five asymmetrical Hcyanines based pH responsive fluorophore AsP1–5 that possess pK_a within a narrow pH_lys range (Fig. 1). IR783 was chosen as a parent molecule due to its optimized absorption and emission wavelength, high extinction coefficient and quantum yield.²³ A primary amine as a proton sensitizing group and another functional group as an electron density modulator were labeled on the two terminal indoles respectively. Fine tuning the pK_a with interval of 0.1 pH units was achieved by adjusting the electron density of the extended p–π conjugated system. The incorporation of a cyclohexene ring in the center of the polymethine chain strengthens the molecular rigidity and enhances the photophysical properties of the aiming NIR dyes.²⁴ Additionally, the reactive allyl chlorine in the cyclohexane bridgehead can be easily substituted, which provides convenience for labeling this pH_lys responsive fluorophore into functional domain to achieve the imaging and theranostic purpose. Among these fluorophores, AsP2 with the highest photophysical stability showed specifically intracellular uptake in the lysosomes of both human normal and cancer cells. Importantly, due to its optimized pK_a (4.0), its activation in the lysosomes of cancer cells but not of normal cells offers its great potential in the image-guided tumor surgery by precisely positioning the invasive tumor margin with high sensitivity and universality.

Fig. 1 Chemical structures of AsP1–5 and their proposed mechanism of pH responsive NIR fluorescence. ICT presents intramolecular charge transfer.

2 Results and discussion

2.1. Synthesis of the pH_lys responsive NIR fluorophores

Synthesis of the asymmetrical cyanines, especially the Hcyanines is usually arduous due to the low yield and tedious purification procedure.²⁵ The shared strategy includes a condensation reaction between an unsaturated dialdehyde with two different N-alkyl quaternized indoles with a molar ratio of 1 : 1 : 1.²⁶ Though simple and straightforward, two symmetric Hcyanines as side products are inevitably generated. Due to their similar polarities, it is difficult to isolate the desirable asymmetrical product.²⁵ To reduce the side products, a hemicyanine method was introduced, in which the dialdehyde reacted with one quaternized indole first in a 1 : 1 molar ratio to offer a hemicyanine intermediate.²⁷ The following condensation between this intermediate and another indole derivative offered the aiming asymmetrical Hcyanines. Even though this strategy reduced the side-products, reverse phase column chromatography was required for purification, which prevents large-scale production. In this work, we put forward a two-step strategy to prepare the asymmetric Hcyanines (Scheme 1). The condensation between 2,3,3-trimethyl-5-tert-butoxycarbonylamino-indole sulfonate 1 and dialdehyde 2 in a solvent system composed of butanol and methylbenzene (v/v = 7 : 3) not only offered hemicyanine 3 with higher yield, but also avoided the environmentally hazardous benzene used in previous works.²⁷ Compound 3 was purified and then reacted with quaternized indole sulfonate 4a–e that substituted with nitro, chloro, proton, methyl or methoxyl group respectively to obtain the aiming asymmetrical products 5a–e. Deprotection of 5a–e in trifluoroacetic acid (TFA) offered the aiming products AsP1–5 with overall yields of 34–49%. After purification by silica gel chromatography, pH-responsive NIR fluorophores AsP1–5 were fully characterized by ¹H NMR and ¹³C NMR. Compared with conventional strategies, this two-step synthetic strategy avoided the undesirable symmetrical products and made large-scale production feasible.

Scheme 1 A two-step synthetic strategy to prepare asymmetric heptamethine cyanines. (i) Butyl alcohol, toluene; (ii) acetic anhydride, sodium acetate.

2.2. pH dependent optical properties

2.2.1. pH dependent absorption. In general, all the fluorophores displayed absorptions within the NIR wavelength range (650–900 nm) with a major peak representing fluorophore monomer and a shoulder presenting the fluorophore H-type aggregates²⁸ (Fig. 2A and S1A†). Functional groups substituted at the terminal indole changed the absorbance wavelength of both monomers (λ_mon) and aggregates (λ_agg). Compared with AsP3 without substitution of functional groups, λ_mon of AsP1 and AsP2 modified with electron-withdrawing moiety showed a slight hypsochromic shift. In contrast, bathochromic shifts were observed in AsP4 and AsP5 modified with an electron-donating moiety, which could be explained by the decreasing of energy gaps between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO).²⁹ Notably, increased absorptions with concomitant hypsochromic shifts were observed for all fluorophores upon acidification. For example, the extinction coefficient of AsP2 (ε_mon) increased four times from 0.21 × 10⁵ to 0.84 × 10⁵ cm⁻¹ M⁻¹ upon the acidification from pH 7.4 to 2.4 (Table 1). Meanwhile, a 13 nm hypsochromic shift was also observed. Above acidity induced hypsochromic shifts in absorption can be explained by the breakdown of p–π conjugation system after protonation of the terminal amine.³⁰

Fig. 2 The pH dependent absorption (A) and emission spectra (B) of AsP2 (1.0 μM, λ_ex = 765 nm). pH value changed with an interval of 0.5 units from 2.4 to 7.4. (C) NIR fluorescence image of AsP1–5 (1.0 μM) as a function of pH in a range of 3.8–7.4. (D) Substitutions at one of the terminal indole ring fine tune the pK_a of the fluorophores. pH dependent fluorescent intensities were normalized to their maximal values measured under pH 2.4.

Table 1 Yields and photophysical properties of pH responsive fluorophores

Dye	λmono^a (nm)	λagg^b (nm)	λem^c (nm)	ε^d (10^5)	Φ^g (%)	Stokes' Shift	Yield (%)
a Maximal absorption wavelength of the monomers.b Maximal absorption wavelength of the aggregates.c Maximal emission wavelength.d Extinction coefficients at λAbs.e Parameters of the deprotonated form measured under pH 7.4.f Parameters of protonated form measured under pH 2.4.g Quantum yields correlated to ICG (Q.Y.ICG = 0.12 in DMSO).
AsP1	780^e	704^e	796	0.41^e	0.01^e	12	44
	784^f	726^f		0.60^f	0.18^f
AsP2	793^e	724^e	802	0.21^e	0.01^e	22	47
	780^f	730^f		0.84^f	0.54^f
AsP3	787^e	750^e	797	0.30^e	0.02^e	21	49
	776^f	731^f		1.11^f	0.70^f
AsP4	791^e	743^e	802	0.46^e	0.10^e	25	48
	777^f	731^f		0.75^f	0.50^f
AsP5	802^e	755^e	815	0.20^e	0.05^e	36	34
	779^f	738^f		0.76^f	0.20^f

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2.2.2. pH dependent emission. The pH dependent emission of the fluorophores was measured in buffer solutions covering the whole physiological pH range (Fig. 2B and S1B†). While the signals of all the fluorophores kept quenched at neutral pH (Q.Y. < 0.05), a broad emission band with its peak centered in a range of 796–815 nm increased remarkably when the pH gradually decreased from 7.4 to 2.4. AsP2 demonstrated the highest acidity triggered fluorescence intensity enhancement with a 54 fold Q.Y. increase. Other fluorophores also demonstrated 4–35 fold Q.Y. enhancements (Table 1). NIR fluorescence images of the fluorophores (1.0 μM) in buffered solutions with selected pH values further verified the acidity correlated fluorescence enhancement (Fig. 2C). Under neutral environment, the electron transfer from the terminal amine to the Hcyanine backbone quenches the fluorescence through the formation of a nonfluorescent intramolecular charge transfer (ICT) state, in which the excited state becomes closer to the ground states as well as charge transfer excited state, resulting in a release of absorbed energy via efficient non-radiative decay.^31–33 The protonation of the bridgehead amine transfers the planar conformation (ICT state) in neutral environment to a pyramidal geometry (LE state),³⁴ which broadens the energy gap (without charge separation) between the locally excited state (LE) and the ground state, blocks the ICT process and hence activates the fluorescence (Fig. 1). The pK_a values of the fluorophores were calculated by Henderson–Hasselbalch equation via analyzing the fluorescence intensity variation as a function of pH. It is known that the basicity of the heterocyclic nuclei has an paramount influence on pK_a values, which can be tailored by introduction of either electron-withdrawing or electron-donating groups that could change the basicity of the indolenine moiety.^35,36 We hypothesize that functional groups substituted at the offside indole ring also influence the basicity of bridgehead amine and further fine tune the pK_a values of the fluorophores. As shown in Fig. 2D, the pK_a of AsP1–5 were determined to be 3.9, 4.0, 4.1, 4.2 and 4.3 respectively. Generally, the pK_a value increased with the enhanced electron donating capability of the substituted groups. For example, while AsP1 functionalized with nitro-group offered the lowest pK_a (3.9), AsP5 modified with methoxyl-group gave the highest pK_a value (4.3). Changing electron density by introducing different functional groups on the terminal indole indeed tuned the pK_a of the fluorophores. The electron-withdrawing substituents such as nitro-group decreased the electron density of the fluorophore, causing protonation in lower pH and hence lower pK_a values. Therefore, it is feasible to fine tune pK_a values of the NIR fluorophores by taking advantages of the asymmetrical Hcyanines. Time dependent fluorescence maximums of AsP1–5 were measured at pH 4.0 to evaluate their stability in acidic environments (Fig. S2A†). While AsP2 and AsP5 displayed high stability with less than 5% fluorescence intensity retreatment, more than 10% signal lost was found in AsP1, AsP3 and AsP4. Meanwhile, even though all the fluorophores demonstrated the reversible NIR fluorescence by cyclically switching the pH between 7.4 and 4.0, only AsP2 maintained its 90% fluorescence maxima after four cyclic pH switches (Fig. S2B†). Chlorine as electron-withdrawing substituent in the indole ring of cyanine dye helps to suppress oxidative photobleaching of the dye and leads to enhanced photostability.³⁷ Therefore, AsP2 with the highest stability and fine-tuned pK_a was chosen for the in vitro microscopic imaging studies. The low pH sensitivity of AsP1 may be explained by its efficient non-radiative decay upon the nitro group substituted on the indole ring.³⁸

2.2.3. Uncompromised pH-response in presence of albumin. Numerous fluorophores lost their pH sensitivity after intravenous injection due to their non-covalently binding to human serum albumin (HSA), the most abundant protein in the plasma.³⁹ The trapping of the fluorophores in the hydrophobic pocket of HSA reduces their self-aggregation and increase its monomeric absorbance subsequently.^40,41 Spectroscopic properties of AsP1–5 in the presence and absence of 5% HSA were shown in Fig. 3A, S3 and S4.† Similar to the control fluorophore, ICG, bathochromic shifts with the concomitant absorption increase of AsP1–5 were observed (Fig. S3†), which suggested an association between the fluorophore and the serum protein.⁴⁰ Impressively, all the fluorophores maintained their pH responsive fluorescence in the presence of HSA (Fig. 3A and S4†). The NIR fluorescence image of AsP2 as functions of pH in presence and absence of 5% HSA further supported above experimental results (Fig. 3B). The slight to moderate downtrend of the acidity-triggered fluorescence could be interpreted by the increase of solvent viscosity triggered by HSA.⁴² As shown in Fig. 3C, the fluorescence intensity of the protonated AsP2 increased inversely with the viscosity by adjusting the volume percentage of glycerol in the buffer solution. Previous studies demonstrated that the viscosity can significantly change the ICT efficiency by changing the transformation rate between the LE state and ICT state.^34,43 In the viscous polar solvents, the LE emission is dominant because the transformation rate between LE to ICT state (planar configuration) is low. Under such circumstances, emission is only derived from the LE state, which leads to the fluorescence attenuation of the fluorophores.³⁴

Fig. 3 AsP2 remains pH responsive in the presence of HSA. (A) Emission spectra of protonated AsP2 (1.0 μM) in the presence or absence of HSA (5%). (B) pH dependent NIR fluorescence image of protonated AsP2 (1.0 μM) in the presence or absence of HSA (5%). (C) Viscosity dependent fluorescence of protonated AsP2 (1.0 μM).

2.3. AsP2 specifically illuminating cancer cells by sensing their pH_lys

The cytotoxicity of AsP2 towards human adenocarcinoma HeLa cell line (ATCC, USA), human hepatocellular carcinoma HepG2 cell line (ATCC, USA), and normal human hepatic HL-7702 cell line (Cell Bank of the Chinese Academy of Sciences, China) were measured by using Cell Counting Kit-8 (CCK-8) assay (Fig. S5†). The viabilities of all the cell lines were over 79% even though the probe concentration was five times higher than that used for fluorescence microscopic imaging studies, which indicates the low acute cytotoxicity of AsP2 at its concentration for imaging purpose. Intracellular uptake of AsP2 distributed at peri-nuclear areas in both live human cervical HeLa cancer cells and human hepatic HepG2 cancer cells at 6 h post-incubation were shown by confocal fluorescence microscopic imaging. In contrast, only background noise was observed in normal hepatic HL-7702 cells after the similar treatment (Fig. 4A). Quantitative studies demonstrated that the average intracellular fluorescence intensities of the HepG2 and HeLa cancer cells were 5.7 and 6.0 times higher than that of HL-7702 normal cells (Fig. 4B). The colocalization coefficients (Pearson's coefficients of co-localization) between AsP2 and lysosomal marker (LysoTracker Green DND-26) were determined to be 0.84 for HepG2, 0.79 for HeLa (Fig. 4C), which indicated the lysosomal delivery of this fluorophore. In contrast, due to the low intracellular AsP2 fluorescence, the colocalization coefficient was determined as 0.13 for HL-7702 normal cells after the similar treatment. To verify that it is the pH_lys triggered fluorescence selectively illuminating cancer cells, the cytoplasmic acidification was conducted by exposing the HL-7702 cells in an acidic buffer solution (pH 4.0) for 10 min.⁴⁴ The fluorescence intensity in the lysosomes increased 5.0 times and the colocalization coefficient also increased from 0.13 to 0.67 (Fig. 4C). Above studies indicated that Asp2 specifically visualized cancer cells by sensing their acidic lysosomal lumen.

Fig. 4 AsP2 specifically imaging cancer cells by sensing their acidic lysosomal lumen. (A) Confocal fluorescence microscopic images of live human hepatic HepG2 cancer cells, human cervical HeLa cancer cells and human hepatic HL-7702 normal cells at 6 h post incubation of AsP2 (10 μM). Fluorescence of AsP2 and LysoTracker were displayed in red and green respectively. A portion of HL-7702 normal cells loaded with LysoTracker and AsP2 were cytoplasmically acidified in buffer solution (pH 4.0) for 10 min before microscopic imaging. Bar, 10 μM. (B) Mean intracellular NIR fluorescence intensities at 6 h post incubation of AsP2 (10 μM). Bars present mean ± SD, n = 12 microscopic fields from three independent tests. (C) Colocalization coefficients between AsP2 and LysoTracker at 6.0 h post probe incubation. Bars present mean ± SD, n = 5. C.A. presents cytoplasmic acidification.

Glunde et al. reported that the highly invasive/metastatic human breast cancer cells showed significant increase of the lysosomal volume, the spatial distance between the lysosomes and the nucleus after culturing in physiological acidic media (pH 6.4–6.8).⁴⁵ Microscopic fluorescence imaging studies showed that the average lysosomal diameter of HepG2 cells increased from 0.61 μm after culturing in neutral media to 0.81 μm after culturing in acidic media (pH 6.8) (Fig. 5A and B). Similarly, the average lysosomal diameter of HeLa cells increased significantly from 0.62 to 0.88 μm. Meanwhile, the lysosomal shifting toward the cell periphery was also evident after cell incubation in acidic media (Fig. 5B). While the median lysosome-to-nuclear distance of HepG2 cancer cells increased 44% to 10.7 μm after culturing in acidic media for 6.0 h, this value also increased 54% to 10.8 μm for HeLa cancer cells. Above experimental results implied the enhanced invasiveness of the cancer cells under the acidic extracellular environment. Importantly, the average lysosomal fluorescence intensities of AsP2 increased 65% and 95% respectively in HeLa and HepG2 cancer cells after incubation in acidic media (pH 6.8) for 6.0 h (Fig. 5A and C), which verified the acidification of the lysosomal lumen by up-regulating cancer cell invasiveness. The increased lysosomal diameter, relocation of lysosomes into cell periphery and the enhanced lysosomal acidity strengthen the possibility of the pH_lys responsive fluorescence probes to specifically visualize the highly invasive cancer cells in tumor invasive margin.

Fig. 5 AsP2 selectively visualized highly invasive cancer cells cultured in acidic extracellular environment. (A) Fluorescence microscopic images of live HepG2 and HeLa cancer cells cultured in neutral (pH 7.4) or acidic media (pH 6.8) for 6 h with the presence of AsP2 (10 μM). Bar: 10 μM. (B) Box-and-whisker plots of lysosomal diameter and lysosomal-to-nuclear distance (L–N distance) of cancer cells after 6.0 h culturing in neutral or acidic media. The box represents the range between the first quartile and third quartile of the respective distribution indicating the length of the interquartile range. The horizontal line in the box shows the median of the distribution. * presents P < 0.05 compared to the neutral media. (C) Mean intracellular NIR fluorescence intensities of AsP2 (10 μM) at 6.0 h post incubation in neutral (pH 7.4) or acidic media (pH 6.8). Bars present mean ± SD, n = 8.

3 Conclusions

In this work, we developed a two-step synthetic method to prepare five novel asymmetrical Hcyanine fluorophores with improved purification convenience. Modifying a primary amine and a functional moiety at each of the two terminal indole rings not only offered the pH sensitivity of these NIR fluorophores, but also showed the feasibility to fine tune their pK_a (ΔpH = 0.1 pH units) by carefully adjusting the electron density on the fluorophores. Among these fluorophores, AsP2 with a pK_a value of 4.0 specifically illuminated cancer cells, especially the highly invasive cancer cells, by sensing their higher acidic lysosomal lumen. Considering the up-regulated acidic lysosomal lumen is a common characteristic of invasive cancer cells, this pH responsive NIR fluorophore holds the promise to guide tumor resection by delineating tumor invasive margin with high sensitivity and universality.

Acknowledgements

R. Xi and J. Zhang contributed equally to this work. This work was financially supported by the National Basic Research Program of China (973 Program, 2013CB932500, 2015CB755500), the National Natural Science Foundation of China (No. 81171384, 81371624), the Nanotechnology Program of Shanghai Science and Technology Committee (13NM1400400). We appreciate the kind help of Mr Ji Tang for confocal fluorescence microscopic studies.

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Footnote

† Electronic supplementary information (ESI) available: Synthesis, characterization, experimental details and other figures and spectra. See DOI: 10.1039/c6ra12381c

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