Amino-isocyanoacridines: Novel, Tunable Solvatochromic Fluorophores as Physiological pH Probes

Amino-isocyanoacridines (ICAAcs), as first members of their class, turned out to be a novel, multifunctional acridine orange (AO) type dye family with a number of additional favorable properties. They have enhanced solvatochromic emission range, low quantum yields (ΦF = 2.9–0.4%) in water, reduced basicity (pKa = 7.05–7.58), and their optical behavior could be fine-tuned by complexation with Ag(I) ions, too. Based on both their vibronic absorption and the charge transfer bands, ICAAcs can be applied as stable pH-probes with great precision (2–3% error) in the physiological pH range of 6–8 using UV-vis and fluorescence detection. The dyes are also able to sense pH change in different microenvironments, such as the Stern layer, as it was demonstrated on sodium lauryl sulfate micelles. The optical behavior of the ICAAc derivatives is discussed based on high-level quantum chemical calculations. All three dyes are well-applicable with conventional epifluorescence imaging. Furthermore, at the blue excitation, diMICAAc is optimally suited as a whole-cell probe for both the conventional microscopic and the laser-illumination studies, like flow- and imaging cytometric, or confocal laser-scanning microscopic examinations.


Results and Discussion
Steady-state fluorescence properties. All the quantum chemical (DFT) calculations, steady-state emission, absorption and excitation spectra in various solvents are presented in the Supporting Information (SI) for all the substances as separate chapters.
The UV-vis and fluorescence properties of ICAAc, monoMICAAc, and diMICAAc for solvents of different polarity are summarized in Fig. 2a and Table 1. Independently of solvent polarity, similarly for all three compounds, a broad long wavelength absorption band is seen in the range of ~370-540 nm (Fig. 2a inset), which can be attributed to the internal charge transfer transition (ICT) between the donor amino and the acceptor isocyano groups. This ICT band is accompanied by overlapping structured absorption bands belonging to the locally excited state (LE) characteristic for the aromatic acridine ring in the region of ~300-370 nm. These structured bands are also present in the UV-vis spectrum of 2-aminoacridine 35 , however they are completely missing from the spectrum of both proflavine and acridine orange. The reappearance of the structured bands may be explained by the presence of the rigid electron withdrawing isocyanide group, which breaks the symmetry of the molecule compared to AO. While the position of the LE bands remains unaffected by changing the solvent polarity, the longer wavelength (ICT) absorption bands show a considerable redshift with increasing solvent polarity. Increasing the electron donating ability of the amino group by methylation also results in a bathochromic shift www.nature.com/scientificreports www.nature.com/scientificreports/ of the higher wavelength absorption bands. These shifts are greater than those observed when changing solvent polarity. That is, the higher the electron donating ability of the amino group the higher wavelength the ICT absorption peak is located at. As it is evident from Fig. 2 and Table 1, all three dyes have a considerable emission range spanning from green to orange. The emission maximum of diMICAAc in water is the highest of the three dyes with λ em = 576 nm, which is 50 nm higher than that of AO (λ em = 526 nm). In addition, AO has a solvatochromic range of approximately 30 nm, while in the case of monoMICAAc and diMICAAc this value is doubled to more than 60 nm.
To get a better understanding of the solvatochromic behavior of the ICAAc derivatives Lippert-Mataga plots (Figs S14, 15) were constructed based on Eqs S1, S2 in the SI. As solvent polarity increases the emission maximum shifts bathochromically (Fig. S14b), due to the solvent reorientation around the excited dye, reducing the energy gap between the ground and excited state and as a result the energy of the emitted photon also decreases. Additionally, the quantum yields (Φ F ) show a linear decrease with increasing solvent polarity parameter E T (30) values, which is in line with the data of Fig. 2 and Table 1. All our dyes have very low quantum yields (Φ F = 2.9-0.4%) in water, that of diMICAAc being the lowest. The low Φ F may be advantageously used in cell staining applications where the suppression of background fluorescence is essential. The extended solvatochromic range of the ICAAc derivatives can be explained by their increased dipole moment difference between the ground (μ g ) and excited state (μ e ) due to the the electron withdrawing isocyano group, which makes the dyes more polar. The dipole moment difference was determined experimentally from the slope of the Lippert-Mataga plot (Fig. S14a) and by DFT calculations. As it is evident from the data of Table 1, the μ e -μ g values of the ICAAc derivatives are at least 3 times higher than that of AO and are increasing with the methylation of the amino group.
The absorption spectrum of all three compounds contains a broad CT band with a maximum of λ abs,max = 470 nm for ICAAc, 475 nm for monoMICAAc and 502 nm for diMICAAc at pH = 3. This maximum suffers a hypsochromic shift upon increasing the pH of the medium to λ abs,max = 428 nm for ICAAc, 446 nm for monoM-ICAAc and 470 nm for diMICAAc at pH = 11.
The base (DB) and protonated (DBH + ) forms ( Fig. 4) can be distinguished by the shape of the minor acridine-like absorption bands located between λ abs = 320 nm and 400 nm. It should be noted that these bands are completely absent from the absorption spectrum of AO, making our dyes more suitable for pH sensing. The UV-vis spectrum of the free base in the same region exhibits a more structured absorption pattern consisting of three moderate bands of different intensity, while the spectrum of the protonated species contains only two broader and higher intensity peaks. This low intensity three-band structure is characteristic for all solvents investigated except water. Therefore, to further support our assignation, UV-vis spectra of diMICAAc were recorded in acetonitrile in the presence of water, ammonia and acetic acid (Fig. 5).   Table 1. Comparison of the optical properties of the dyes in solvents of different polarity. Emission (λ em,max ), excitation (λ ex,max ) maxima, Stokes shift (Δν), molar absorbance (ε) and quantum yield (Φ F ), as well as theoretically calculated and experimentally estimated dipole moment difference data. a Taken from ref. 38 . * The optical data of AO was obtained from ref. 39 . www.nature.com/scientificreports www.nature.com/scientificreports/ According to Fig. 5 the absorption spectra do not change either upon the addition of small amounts of water or base (NH 3 ). After the addition of acetic acid in ~7 molar excess, a new band starts to appear at ~375 nm and a broad double band structure only appears after the addition of a large excess (~35x molar) of acetic acid. After recording the absorption spectra in the concentration range of 10 −6 -10 −4 M in water and in buffers too, no deviation from the Lambert-Beer law was observed, which excludes the presence of dimers.
Assuming a simple equilibrium between DB and DBH + (Fig. 4) and denoting the pH-dependent property (absorbance or PL intensity) by Y(λ,pH), the acid dissociation constant (K a ) can be determined by fitting Eq. 1. to the Abs(502)-pH experimental plots as presented in the inset of Fig. 6.
( ) 10 The obtained pK a values show only a slight variation between pK a = 7.05 and 7.58 (UV-vis) and pK a = 6.96 and 7.71 (Fluorescence). These pK a values make the ICAAc derivatives three orders of magnitude weaker bases than AO (pK a = 10.5) 22 . The fluorescence intensity also shows strong pH dependence, it increases in the more acidic medium in the case of ICAAc, and monoMICAAc. The PL intensity ratio I 3 /I 11 calculated from the intensities  Table 2. Absorption (λ abs,max ) and emission (λ em,max ) maxima of the ICAAc derivatives at pH = 3 and pH = 11, respectively, in aqueous buffer. I 3 /I 11 is the fluorescence intensity ratio measured at pH = 3 and pH = 11. pK a is the acid dissociation constant at a logarithmic scale.  www.nature.com/scientificreports www.nature.com/scientificreports/ measured at pH = 3 and pH = 11 is 2.25 and 1.69 for ICAAc, and monoMICAAc, respectively. The situation is completely different for diMICAAc, where PL intensity increases at higher pH and I 3 /I 11 is only 0.58.

Investigation of the applicability of diMICAAc for pH determination.
Favorably, the pK a value of diMICAAc is 7.5, which falls in the physiological pH range making it a possible candidate for a fluorescent biosensor. For the precise determination of the pH, calibration curves were constructed based on Eq. 2.
where r is the ratio of the absorbances (PL intensities) measured at different wavelengths, b 1 , b 2, and b 3 are parameters used for fitting. The UV-vis titration curves are presented in Fig. 6 and the fluorescence titration curves in Fig. S18.
As it is evident from Fig. 6 both curves have a very similar run. The A 502 /A 470 ratio varies between 1.43-0.73, while the A 375 /A 359 between 1.05-0.34 in the pH range of 5.5-10.0. The majority of the change happens between pH = 6.5-8.5, making it the most useful region for pH determination. In addition, it should be noted that there is a perfect correlation between the two absorbance ratios as is presented in Fig. 6 inset. Unlike UV-vis, the fluorescence (excitation) titration curve is shifted approximately 1 pH unit to the left, that is the drop in the curve is located between pH = 5.5-8.0, extending the useful section more in the acidic region. To test the practical applicability of our dye and the reproducibility of the method, that is its dependence on the dissolved chemical species and ionic strength, buffer media of different pH and concentration were prepared and their absorption and fluorescence spectra were recorded after the addition of diMICAAc. The results are summarized in Table 3.   Table 3. Reproducibility of the spectroscopic pH sensing method using different buffers. NaH 2 PO 4 /Na 2 HPO 4 (normal text) NaH 2 BO 3 /Na 2 HBO 3 (italic text). pH Set , pH Abs, and pH PL are the pH(s) determined using a pH meter, from the UV-vis and the fluorescence spectrum, respectively. c buffer = 0.05 M in both cases.
www.nature.com/scientificreports www.nature.com/scientificreports/ The spectroscopically measured pH values for both the phosphate and borate buffers agree well with the previously set values in the 6.00-9.00 pH region. No selectivity for different chemical species could be detected. The largest errors 2-4% can be observed on the edges of the range, whereas between pH = 7.00-8.50 the errors are well below 1% for both the UV-vis and the fluorescence methods, which is important for biological applications. The method was also tested on a 0.10 M NaHCO 3 solution with a pH of 8.50 and the measured values were pH = 8.53 ± 0.07 (UV-vis) and pH = 8.59 ± 0.01 (PL). The stability of the method was also tested in phosphate buffer at pH = 6 and 8 respectively for an hour. No significant change (the relative errors were 0.6% at pH = 6 and 0.4% at pH = 8) could be observed during this period as can be seen in Figs S19, 20. To investigate the salt effect, samples in BRB buffer at pH = 6 and 8, respectively were titrated with saturated KCl solution. As Fig. S21 shows the ionic strength has no significant effect on the method, the errors are below 1-2% even at 0.5 M KCl concentrations. Hence, we can conclude that the method is stable and can be used as soon as 5 minutes after mixing the dye with the medium.
Critical micelle concentration (CMC) studies. It has been shown previously that the pH in the Stern layer of micelles formed from anionic surfactants such as sodium lauryl sulfate (SLS) can significantly differ from that of the bulk 28 . It is known that AO interacts with SLS micelles through both hydrophobic and electrostatic interactions. Since our dyes are highly dipolar and the intracyclic imino group gets easily charged through protonation, it is more likely that the dyes will reside in the Stern layer instead of penetrating into the nonpolar core. In order to investigate the behavior of our dyes in the micellar medium, we have studied diMICAAc in SLS solutions prepared in 50 mM BRB buffer at pH = 7 (to keep the bulk pH constant). The pH values were calculated from the excitation spectra using the ratio of I 376 /I 362 and the fluorescence intensities were read at 575 nm (peak maximum). The results are summarized in Fig. 7. As it is evident from Fig. 7 the pH of the microenvironment of diMICAAc starts to decrease in the first four steps by increasing the SLS concentration. This linear decrease is followed by a sharp drop to pH = 6 and then fluctuates around this value. Since Φ F = 0.4% for diMICAAc, the interaction with micelles results in a sharp increase in fluorescence intensity at 575 nm. The change in pH and PL intensity vs concentration curves correlate perfectly, the sharp drop (jump) happens at approximately 4 mM which can be considered the critical micelle concentration (CMC) in this medium. However, this value is a bit lower than the one reported in pure water, it is known that the use of a buffer solution, therefore the elevated ionic strength results in a lower CMC 36 .
The experiments were repeated using deionized water and diMICAAc and the results are presented in Fig.  S22. Similarly to the buffered solution, below CMC the PL intensity decreases until a minimum is reached followed by a sudden increase after which the PL intensity runs into saturation above CMC. It is evident from Fig.  S22b. that the run of the intensity vs SLS concentration curves are identical and no matter which wavelength we use the same CMC value of approximately 7 mM is obtained, which is in good agreement with the values found in the literature for SLS. When looking at Fig. S22a. we can see the variation of the excitation spectrum as a function of SLS concentration. It should be emphasized that independently from the wavelengths used, the CMC of SLS can be very well guessed from the excitation spectra too. At 5.74 and 6.54 mM SLS concentrations (indicated by thick lines) we can see the shape of the spectra alter, that is a shoulder at 530 nm appears on the ICT band and it almost becomes a double band. This change can be assigned to the start of the increasing part of the intensity curve in the premicellar region of SLS concentration. Above CMC the 530 nm shoulder remains, but the 503 nm www.nature.com/scientificreports www.nature.com/scientificreports/ band becomes dominant. It should be noted that λ em,max does not change above or below CMC with respect to the one recorded in pure water. Therefore the dye is not likely to penetrate to the nonpolar core of the micelles and it most likely resides in the Stern layer of the micelles where the interaction is of electrostatic character. For comparison, the CMC was also determined using the more sophisticated method of Pineiro et al. 37 , where the CMC is defined as the total surfactant concentration [S] 0 at which the change in the gradient of the monomer concentration [S 1 ] with respect to [S] 0 is maximum, that is where the third derivative of [S 1 ] is zero. Consequently, we describe the second derivative of [S 1 ] with respect to [S] 0 by a Gaussian function centered at the CMC as is presented in Fig. S23. The CMC obtained by this method is 7.5 mM which is very close to the literature value of 8.2 mM. In summary, diMICAAc is well applicable for both CMC determination and microenvironment pH mapping.

Complexation of diMICAAc with Ag(I) ions.
We have shown earlier 34 that aromatic amino-isocyano compounds tend to form complexes in the presence of Ag(I) ions and the process is accompanied by significant spectral changes. Steady-state fluorescence titration of diMICAAc in dioxane revealed a sharp decrease of PL intensity (to one-third of the original) with increasing silver(I) concentration as is presented in Fig. 8. In addition, a 20 nm bathochromic shift of the emission maximum occurs from λ em,max = 548 nm to λ em,max = 568 nm, which manifests in a color change from greenish-yellow to red in visible light and from neon green to orange when excited with λ ex = 365 nm UV light (Fig. 8 insets a and b respectively). The shape of the inset graph in Fig. 8 indicates the formation of a 1:1 complex, which was also detected under ESI-MS conditions. The first isotopic mass of the complex with a formula of [Ag(C 16 H 13 N 3 )] + is 354.015 Da as can be seen in Fig. S24 and no additional mass corresponding to other Ag:diMICAAc composition could be observed. The equilibrium constant of complexation was determined using non-linear regression analysis described in ref. 34 and turned out to be K = (4.5 ± 0.9) × 10 5 M −1 . However, this equilibrium constant is lower than those obtained for ICAN derivatives (K = 10 6 M −1 and 10 7 M −1 ) it still indicates high binding affinity of silver(I) towards diMICAAc. The formation of a 1:1 complex is further backed by quantum chemical (DFT) calculations since the calculated PL spectrum agrees very well with the measured one as can be seen in Fig. S25. DFT calculations also revealed that Ag + is attached to the isocyano moiety instead of the imino nitrogen in the ring (Fig. S26).
Computational results. The calculated absorption and emission spectra of the three amino-isocyanoacridine molecules are shown in Figs S27-32. In nonpolar solvents, the calculated and experimental spectra show very good agreement. Considering polar solvents, however, it seems that additional effects, other than simple dielectric interactions play an important role. First, we investigated whether the observed fluorescence quenching is due to the decoupling of either the amino or the isocyano group from the acridine ring by simply replacing the two functional groups with a hydrogen atom. As Fig. S33 shows, both spectra are blue-shifted compared to the unmodified ICAAc but no dark states or lower oscillator strengths are calculated. The TICT (Twisted Intramolecular Charge Transfer) mechanism is unlikely because the planarization of the amino group in the excited state is not hindered by nearby hydrogens according to geometry optimization in the excited state. Another commonly reported quenching mechanism, the dimerization is also not expected to play a role because dimer formation of ICAAc is endergonic by 3.3-5.0 kcal mol −1 . Since these molecules can be used as pH sensors, we also explored the possible sites of protonation, as well as the optical properties of these protonated species using DFT. We considered three www.nature.com/scientificreports www.nature.com/scientificreports/ protonation modes, as shown in Fig. 9a,b. It can be seen, that in both the ground and S1 states, the protonation of the acridine nitrogen is by far the most favored case. Protonation of the amino group is also interesting because it gives rise to the observed double peak structure below 400 nm in the absorption spectrum but the low energy band disappears. The change in the experimental fluorescence spectrum of ICAAc upon protonation is reproduced by the calculations well, however, the emission behavior of diMICAAc remains an open question. As Fig. 9c shows, the frontier orbitals barely change upon protonation which is reflected by the very small change in the calculated emission spectra. We also considered another approach where an excited state proton transfer was modeled using an explicit water molecule. The spectral changes following the gradual decrease of the N-H distance are shown in Fig. 9d. This shows the experimentally observed behavior of a minimal red shift together with a nearly twofold increase in intensity. Correlation between the protonation coordinate and change in emission wavelength for the protonation and deprotonation of the amino group is shown in Figs S34, 35. The calculated wavelength shift is in the opposite direction which further strengthens the view that protonation takes place on the acridine nitrogen.  www.nature.com/scientificreports www.nature.com/scientificreports/

Conclusions
Novel, multifunctional, amino-isocyanoacridines (ICAAc, monoMICAAc and diMICAAc) were prepared by the reaction of 3,6-diaminoacridine with dichlorocarbene. The resulting dyes showed tunable solvatochromic behavior (Δλ em = 53-63 nm) owing to the dipolar nature (μ e -μ g varies between 5.6-8.9 Debye) of the molecules. The introduction of the electron withdrawing isonitrile group, resulted in a reduced basicity (pK a = 7.05-7.58), which in combination with their unique pH-sensitive vibronic absorption bands make them promising pH-probes in the physiological pH range. It was demonstrated using UV-vis and fluorescence spectroscopy that the pH of different buffer solutions could be recovered with great precision (2-3% error) between pH = 6-8 and the method is stable for at least an hour. The dyes were shown to be able to sense pH change in different microenvironments such as the lowered pH in the Stern layer as it was demonstrated on SLS micelles. Furthermore, the isocyano group is an excellent ligand for complexation with Ag(I), this way the optical behavior of our dyes can be fine-tuned by ions too. 24 hour MTT assays on HeLa cells revealed LD 50 values of 7.27, 5.78, and 7.50 μM for ICAAc, monoMICAAc and diMICAAc dyes, respectively. The applicability of ICAAc, monoMICAAc, and diMICAAc dyes for live cell imaging was demonstrated on HeLa cells at 0.3 μg/ml dye concentration. diMICAAc stained cells demonstrated the best-preserved morphology. DiMICAAc probably binds cell membranes, some unknown intracellular vesicular structures, probably the DNA slightly, and possible binds to some extent to the RNA. All the three dyes are well-applicable with conventional epifluorescence imaging at the UV excitation. Furthermore, at the blue excitation, diMICAAc is optimally suited as a whole-cell probe for both the conventional microscopic and the laser-illumination studies, like flow-and imaging cytometric, or confocal laser-scanning microscopic examinations.
Synthesis. The preparation and characterization of the ICAAc derivatives are detailed in the SI.

Methods. UV-vis.
The UV-vis spectra were recorded on an Agilent Cary 60 spectrophotometer (Agilent, Santa Clara, CA, USA) in a quartz cuvette of 1.00 cm optical length. 3.00 cm 3 solution was prepared from the sample.
Fluorescence measurements. Steady-state fluorescence measurements were carried out using a Jasco FP-8200 fluorescence spectrophotometer equipped with a Xe lamp light source. The excitation and emission spectra were recorded at 20 °C, using 2.5 nm excitation, 5.0 nm emission bandwith, and 200 nm/min scanning speed. Fluorescence quantum yields (Φ) were calculated by using quinine-sulfate as the reference, using the following equation: where Φ r is the quantum yield of the reference compound (quinine-sulfate in 0.1 mol/L sulfuric acid, absolute quantum efficiency (Φ r = 55%)), n is the refractive index of the solvent, I is the integrated fluorescence intensity and A is the absorbance at the excitation wavelength. The absorbances at the wavelength of excitation were kept below A = 0.1 in order to avoid inner filter effects. For UV-vis and fluorescence measurements the investigated compounds were dissolved in acetonitrile at a concentration of 1.2 mM and was diluted to 2.4 × 10 −5 M and 4.8 × 10 −6 M in the solvents in interest.
Measurements on the pH-dependence of the optical properties. Britton-Robinson buffer (BRB) was prepared by mixing 100-100 cm 3 0.04 M H 3 BO 3 , 0.04 M H 3 PO 4 and 0.04 M acetic acid solution, then titrated to the desired pH with 2.0 M NaOH solution. The cuvette was charged with 3.00 cm 3 buffer solution, and 30 μl stock solution of the dye in acetonitrile was added. NaH 2 PO 4 /Na 2 HPO 4 and NaH 2 BO 3 /Na 2 HBO 3 buffers were prepared by the titration of 0.05 M Na 2 HPO 4 or Na 2 B 4 O 7 solution with 1.0 M NaOH or HCl to set the desired pH.
CMC studies. A stock solution of sodium lauryl sulfate (SLS) was prepared in water or 0.004 M pH = 7.01 BRB buffer at a concentration of 0.082 M, and was diluted to the given concentration. The cuvette was charged with 3.00 cm 3 SLS solution, and 12 μl stock solution of the dye in acetonitrile was added. Optical properties were measured after 1 hour equilibration.
Complexation of diMICAAc with Ag(I) ions. The cuvette was charged with 3.00 cm 3 dioxane and 120 μl stock solution of the dye in acetonitrile, then silver trifluoroacetate stock solution (3.4 mM in dioxane) was added in 10 μl portions. Laser-Scanning Cytometry and microscopy and MTT cell viability assay are presented in the Supporting Information.