Amphipolar, Amphiphilic 2,4-diarylpyrano[2,3-b]indoles as Turn-ON Luminophores in Acidic and Basic Media

A versatile amphiphilic pyrano[2,3-b]indole for halochromic turn-ON luminescence in acidic or basic media is accessed by an insertion-coupling-cycloisomerization and adjusting solubilizing and phenolic functionalities. While almost non-emissive in neutral solutions, treatment with acids or bases like trifluoroacetic acid (TFA) or 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) reveals distinct luminescence at wavelengths of 540 nm or 630 nm in propan-2-ol, respectively. Turn-ON emission can be detected at pH values as mild as pH = 5.31 or 8.70. Quantum yields in propan-2-ol are substantial for protonated (Φf = 0.058) and deprotonated (Φf = 0.059) species. Photometrically, pKa1 of 3.5 and pKa2 of 10.5 were determined in propan-2-ol. With lipophilic polyether sidechains and hydrophilic protonation and deprotonation sites the molecule can be regarded as amphipolar, which results in good solubility properties for different organic solvents. In aqueous media, an organic co-solvent like propan-2-ol (35%) or tetrahydrofuran (25%) is needed, and the solution can be diluted with pure water without precipitation of the compound. At higher concentrations of water, a turbid solution is formed, which indicates the formation of micellar structures or clusters. With dynamic light scattering we could show that these clusters increase in size with increasing water content.

Herein, we disclose a novel type of versatile amphiprotic 2,4-diarylpyrano [2,3b]indole system suitable for the detection of both acids as well as bases through turn-ON luminescence in organic and aqueous solutions. Besides the synthesis, photophysical characterization and particle size analysis of an amphiprotic 2,4-diarylpyrano [2,3-b]indole are presented and discussed.
Herein, we disclose a novel type of versatile amphiprotic 2,4-diarylpyrano [2,3b]indole system suitable for the detection of both acids as well as bases through turn-ON luminescence in organic and aqueous solutions. Besides the synthesis, photophysical characterization and particle size analysis of an amphiprotic 2,4-diarylpyrano [2,3-b]indole are presented and discussed.

Synthesis of the 2,4-diarylpyrano[2,3-b]indole Chromophores
Based on our previous synthetic protocols [61,62], a novel generation of halochromic 2,4-diarylpyrano [2,3-b]indoles containing a hydroxyl group has been created using our potassium fluoride mediated deprotection-alkylation one-pot reaction. Starting from secondary amide 1 [62] and terminal alkyne 2a [63], the O-acyl protected 2,4diarylpyrano [2,3-b]indole 3a is formed by a Pd-and Cu-catalyzed domino insertioncoupling-cycloisomerization reaction in moderate yields of up to 54% (Scheme 2). Alternatively, 2,4-diarylpyrano [2,3-b]indole 3b containing a 2-tetrahydropyranyl (THP) group can be synthesized by the coupling of alkyne 2b in 73% yield.  The solubilizing swallowtail substituent can be introduced by fluoride-induced desilylative cleavage of the (trimethylsilyl)ethyl group of 2,4-diarylpyrano [2,3-b]indoles 3 [62]. Interestingly, KF simultaneously acts as a desilylation and deacylation agent and for substrate 3a (PG = Ac) both Oand S-protecting groups are concomitantly cleaved in the presence of an excess of potassium fluoride and the subsequent addition of the swallowtail tosylate 4 selectively leads S-alkylation furnishing hydroxy 2,4-diarylpyrano [2,3-b]indole 5 in 30% yield in a one-pot fashion (Scheme 3). Activated molecular sieves, added at the beginning of the reaction, prove to be beneficial to maintain the thiolate intermediate, which is sensitive against moisture. In contrast, substrate 3b (PG = THP) is transformed to the targeted hydroxy 2,4-diarylpyrano [2,3-b]indole 5 in two separate steps: first the desilylative S-alkylation with the swallowtail tosylate 4 in 79% yield and then a PTSA-mediated deprotective transacetalization [64] of the THP-protected S-alkyl derivative 6 furnishes the target compound 5 in 18% yield. Due to the lower overall yield of the last step, the first synthetic route starting from acyl-protected 2,4-diarylpyrano [2,3-b]indole 3a is preferred. Also, this route is more elegant, since deprotection of the hydroxyl group takes place in the same reaction vessel. For further investigations, no distinctions were made between the products of the two different synthetic routes.
In addition, 2,4-diarylpyrano[2,3-b]indole 5 shows a peculiar response to deprotonation of the phenolic hydroxy group in solution with stronger bases, such as 1,8diazabicyclo [5.4.0]undec-7-ene (DBU), which to this date has not been reported for this type of class. Upon deprotonation in solution, the resulting phenolate oxygen exerts a very strongly electron donating character, leading to an extended π-conjugated push-pull system displaying a bright red emission (Scheme 4, Figure 1 right). It is worthy of men-  In addition, 2,4-diarylpyrano[2,3-b]indole 5 shows a peculiar response to deprotonation of the phenolic hydroxy group in solution with stronger bases, such as 1,8diazabicyclo [5.4.0]undec-7-ene (DBU), which to this date has not been reported for this type of class. Upon deprotonation in solution, the resulting phenolate oxygen exerts a very strongly electron donating character, leading to an extended π-conjugated push-pull system displaying a bright red emission (Scheme 4, Figure 1 right). It is worthy of mention, that both protonation and deprotonation are reversible upon the addition of TFA or DBU, respectively.
Starting at a given concentration (c = 1.3 × 10 −3 mol•L −1 ), the mixture of 2,4diarylpyrano[2,3-b]indole 5 and corresponding solvent were stirred for 30 min and sonicated for 10 min. Solubility upon eyesight was indicated with "+", whereas the presence of undissolved compound was indicated with "-". At first, organic solvents from lesser to higher polarity according to Reichardt's polarity scale [65] were employed (Table  1). Specifically, non-toxic solvents were of interest, opening the possibility for an application as a halochromic chemosensor for protons and the absence of such in biological systems.  In addition, 2,4-diarylpyrano[2,3-b]indole 5 shows a peculiar response to deprotonation of the phenolic hydroxy group in solution with stronger bases, such as 1,8diazabicyclo [5.4.0]undec-7-ene (DBU), which to this date has not been reported for this type of class. Upon deprotonation in solution, the resulting phenolate oxygen exerts a very strongly electron donating character, leading to an extended π-conjugated push-pull system displaying a bright red emission (Scheme 4, Figure 1 right). It is worthy of mention, that both protonation and deprotonation are reversible upon the addition of TFA or DBU, respectively.
Starting at a given concentration (c = 1.3 × 10 −3 mol•L −1 ), the mixture of 2,4diarylpyrano[2,3-b]indole 5 and corresponding solvent were stirred for 30 min and sonicated for 10 min. Solubility upon eyesight was indicated with "+", whereas the presence of undissolved compound was indicated with "-". At first, organic solvents from lesser to higher polarity according to Reichardt's polarity scale [65] were employed (Table  1). Specifically, non-toxic solvents were of interest, opening the possibility for an application as a halochromic chemosensor for protons and the absence of such in biological systems.
Starting at a given concentration (c = 1.3 × 10 −3 mol·L −1 ), the mixture of 2,4diarylpyrano[2,3-b]indole 5 and corresponding solvent were stirred for 30 min and sonicated for 10 min. Solubility upon eyesight was indicated with "+", whereas the presence of undissolved compound was indicated with "-". At first, organic solvents from lesser to higher polarity according to Reichardt's polarity scale [65] were employed (Table 1). Specifically, non-toxic solvents were of interest, opening the possibility for an application as a halochromic chemosensor for protons and the absence of such in biological systems.
As seen in Table 1, (un)branched alkanes like n-pentane and unbranched ethers like 2-methoxy-2-methylpropane (MTBE) are not suitable for solubilizing 2,4-diarylpyrano[2,3b]indole 5, whereas other non-polar solvents like benzene and toluene are able to dissolve 2,4-diarylpyrano[2,3-b]indole 5 completely. This can be plausibly rationalized by π-π interactions between molecules 5 and the aromatic solvent, preventing them from aggregation. More polar solvents, such as 1,4-dioxane, acetone, and propan-2-ol, are all suitable for further investigations, as well as chlorinated solvents, such as chloroform. Most of the solvents able to dissolve chromophore 5 lead to intense luminescence after addition of an excess of TFA observed by the naked eye, but only a few solvents enable luminescence in the deprotonated state. Mainly protic solvents of higher polarity, such as propan-2-ol, ethanol or methanol, show noticeable luminescence, thus narrowing the applicable solvent spectrum for switching in the polarity range. For further photophysical investigations, propan-2-ol is employed as a protic solvent with the most intense luminescence in both protonated and deprotonated states for the naked eye.
Unfortunately, water alone, as the most polar protic solvent, is not able to solubilize chromophore 5. Therefore, water mixtures with different polar co-solvents such as THF and propan-2-ol were prepared as well as selected aqueous systems and mixtures of compound 5 at different pH values (Table 2). Again, solubility at a specified concentration (c = 1.3 × 10 −3 mol·L −1 ) was indicated with "+", whereas the presence of undissolved compound was indicated with "-".
While biologically important buffers [66,67] like HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid) or MOPS (3-morpholinopropane-1-sulfonic acid) did not facilitate the water solubility of dye 5, a clear trend can be identified for aqueous systems at different pH values ( Table 2). The more basic or acidic the solution, the better dye 5 is soluble. Below pH 3 and above pH 11, complete solubility was achieved. This can be rationalized by the structure and functional groups of 2,4-diarylpyrano[2,3-b]indole 5. Below pH 3, complete protonation at the nitrogen atom of the indole moiety occurs and the positive charge causes repulsion and, hence, less π-π-stacking between the molecules and better solubility in aqueous media. The same principle holds true for aqueous solutions with pH >11. Through deprotonation with a strong organic base such as DBU, a phenolate is created, resulting in complete dissolving of dye 5 under these conditions. However, at higher pH values above 13, degradation of the 2,4-diarylpyrano [2,3-b]indoles can be observed [62]. Moreover, the pK a1 value for indole nitrogen atom protonation and the pK a2 value for phenol deprotonation can be estimated by the solubility. Since solubility starts below pH 3 and above pH 11, a pK a1 value of around 3 and pK a2 of around 11 can be roughly assumed. This qualitative estimation can be quantified by UV/Vis photometric determination of both pK a values (see Section 2.4). For the mixtures of organic co-solvent and water, 2,4-diarylpyrano[2,3-b]indole 5 is dissolved in each organic solvent and this solution is rapidly injected in the corresponding amount of water. In Table 2, the lowest amount of co-solvent, which is needed for solubilizing 2,4-diarylpyrano[2,3-b]indole 5 in aqueous media is presented. Hereby, the fraction of water is incrementally increased in steps of 5 vol-% until formation of clusters or turbid solutions can be observed indicating too high and non-suitable water fractions ( Figure 2). At excessive fractions, the formation of clusters occurs rapidly just seconds after the mixing of the two solvents. Since we expect that size and appearance of the clusters depend strongly on the solvent mixture, we used dynamic light scattering (DLS) to analyze the different samples.

Particle size of 2,4-diarylpyrano[2,3-b]indole 5 in Aqueous Solutions
DLS was used to study the hydrodynamic size and size distribution with regard to the dependence of the water content in the solvent mixture. For this 2,4-diarylpyrano[2,3b]indole 5 was dissolved in different propan-2-ol/water mixtures. The water content of the mixtures was varied from 75 up to 95 vol-% in 5 vol-% steps. All samples were sonicated for 30 min and filtered using a filter with 5 μm pore size before the DLS measurement. This was mostly done to remove dust and potentially very large impurities or aggregates. We want to highlight that the filtered samples did not show any precipitation in the course of several hours, i.e., much longer than the measurement times in DLS. The size distributions of all measured samples were obtained at a 110° scattering angle by CONTIN analysis [68] are shown in Figure 3.

Particle size of 2,4-diarylpyrano[2,3-b]indole 5 in Aqueous Solutions
DLS was used to study the hydrodynamic size and size distribution with regard to the dependence of the water content in the solvent mixture. For this 2,4-diarylpyrano[2,3b]indole 5 was dissolved in different propan-2-ol/water mixtures. The water content of the mixtures was varied from 75 up to 95 vol-% in 5 vol-% steps. All samples were sonicated for 30 min and filtered using a filter with 5 µm pore size before the DLS measurement. This was mostly done to remove dust and potentially very large impurities or aggregates. We want to highlight that the filtered samples did not show any precipitation in the course of several hours, i.e., much longer than the measurement times in DLS. The size distributions of all measured samples were obtained at a 110 • scattering angle by CONTIN analysis [68] are shown in Figure 3.
for 30 min and filtered using a filter with 5 μm pore size before the DLS measurement. This was mostly done to remove dust and potentially very large impurities or aggregates. We want to highlight that the filtered samples did not show any precipitation in the course of several hours, i.e., much longer than the measurement times in DLS. The size distributions of all measured samples were obtained at a 110° scattering angle by CONTIN analysis [68] are shown in Figure 3. At the lowest water concentration of 75 vol-% (purple) we find a monomodal size distribution with an average cluster size of approximately 140 nm in diameter. The narrow width of the distribution indicates rather low polydispersity. In contrast, when increasing the water content, the size distribution of the clusters shifts to larger sizes with increased polydispersity. For water contents of 85 vol-% and above, additional contributions become visible and the distribution functions become at least bimodal with a contribution At the lowest water concentration of 75 vol-% (purple) we find a monomodal size distribution with an average cluster size of approximately 140 nm in diameter. The narrow width of the distribution indicates rather low polydispersity. In contrast, when increasing the water content, the size distribution of the clusters shifts to larger sizes with increased polydispersity. For water contents of 85 vol-% and above, additional contributions become visible and the distribution functions become at least bimodal with a contribution from larger aggregates in the micrometer regime. Nevertheless, even these samples were found to be colloidally stable, at least in the course of hours. The size of the aggregates varies strongly between samples and cannot be precisely determined. In particular, the sample at a water concentration of 95 vol-% contains large and polydisperse aggregates that dominate the light scattering. This finding is in good agreement with our turbidity observations previously shown in this work. Nevertheless, we attempted analysis from angle-dependent DLS (see Figure S19, Supporting Information) to study the diffusive behavior of the smaller cluster phase. For these we find translational diffusion that allowed for a more quantitative size analysis. The results are reported in Table S3 (Supporting Information).
In addition, the stability of the clusters and aggregates over time was investigated. For this the measurements were repeated after one month. We found a significant increase in size and polydispersity after this aging time (see Figure S20, Supporting Information). This is a strong indication that the clusters are not stable in these solvent mixtures over a long period of time.
For this the measurements were repeated after one month. We found a significant increase in size and polydispersity after this aging time (see Figure S20, Supporting Information). This is a strong indication that the clusters are not stable in these solvent mixtures over a long period of time.

Photophysical Properties of 2,4-diarylpyrano[2,3-b]indole 5
Although non-luminescent in the solid state and in solution, 2,4-diarylpyrano[2,3b]indole 5 displays peculiar luminescence under certain conditions, rendering it a turn-ON luminophore sensitive to acidic and basic solutions as well as for metal cations. Herein, the photophysical properties of 2,4-diarylpyrano[2,3-b]indole 5 in propan-2-ol under neutral, acidic, and basic conditions are discussed (Table 3, Figures 4 and 5).    In the ground state, the neutral dye 5 shows an expected absorption behavior comparable to 2,4-diarylpyrano[2,3-b]indoles with an 4-anisyl group at position 2 (λmax,abs = 369 nm) [62]. Upon protonation, the absorption maximum shifts from 372 to 455 nm. Moreover, the absorption maximum can be shifted even further bathochromically upon deprotonation, i.e., the absorption maximum is shifted from 372 to 533 nm. Instead of a cyanine chromophore in the acidic medium, an oxonol anion chromophore is formed in As seen in Table 2 affected by water as a solvent. After replacing about 2/3 of the organic solvent by water, molar decadic absorption coefficients ε for all 2,4-diarylpyrano[2,3-b]indole species 5, 5 + H + , and 5 − H + increase up to 70% (Table 4). Table 4. Photophysical data of 2,4-diarylpyrano[2,3-b]indole 5, 5 + H + , and 5 − H + recorded in propan-2-ol/water (c 0 (5) = 10 −5 -10 −7 mol·L −1 ) at T = 293 K. Protonated and deprotonated spectra were recorded with an excess amount of TFA or DBU (c(TFA/DBU) = 10 −2 mol·L −1 ). For this the measurements were repeated after one month. We found a significant increase in size and polydispersity after this aging time (see Figure S20, Supporting Information). This is a strong indication that the clusters are not stable in these solvent mixtures over a long period of time.
As seen in Table 2, 2,4-diarylpyrano[2,3-b]indole 5 is soluble in mixtures of water and propan-2-ol up to 65:35 vol-%, raising the question, how the photophysical properties are affected by water as a solvent. After replacing about 2/3 of the organic solvent by water, molar decadic absorption coefficients ε for all 2,4-diarylpyrano[2,3-b]indole species 5, 5 + H + , and 5 − H + increase up to 70% (Table 4).     For chemosensors, the analyte as well as the spectrum of action are essential and define their possible application. In the case of 2,4-diarylpyrano[2,3-b]indole 5, besides metal cations, the main analytes are acidic and basic solutions due to its amphiprotic character. For amphiprotic molecules, the spectrum of action is defined by its two pKa   For chemosensors, the analyte as well as the spectrum of action are essential and define their possible application. In the case of 2,4-diarylpyrano[2,3-b]indole 5, besides metal cations, the main analytes are acidic and basic solutions due to its amphiprotic character. For amphiprotic molecules, the spectrum of action is defined by its two pKa For chemosensors, the analyte as well as the spectrum of action are essential and define their possible application. In the case of 2,4-diarylpyrano[2,3-b]indole 5, besides metal cations, the main analytes are acidic and basic solutions due to its amphiprotic character. For amphiprotic molecules, the spectrum of action is defined by its two pK a values, which can be determined by UV/Vis absorption titration and their change can be monitored in the presence of an analyte.
For the determination of pK a1 of 2,4-diarylpyrano[2,3-b]indole 5, absorption spectra at various pH values in a range from 7 to 1.3 can be recorded upon addition of TFA in propan-2-ol. Propan-2-ol as a solvent is completely miscible with both TFA and DBU and the spectra are less noisy in comparison to other solvents, such as THF. In addition, propan-2-ol/water is also an aqueous system, where 2,4-diarylpyrano[2,3-b]indole 5 is soluble and, thus, can be employed as chemosensor.
The spectra at different pH values display isosbestic points, indicating that deprotonation proceeds directly without an intermediate (Figure 8) [70]. The involved species are 5 and 5 + H + furnishing a pK a1 value of 3.5 for the deprotonation of 5 + H + (for details, see Supporting Information).  Likewise, the pKa2 value of the phenolic hydroxy group can be determined by titration with DBU as a base. Again, the occurrence of isosbestic points indicates the direct transition from species 5 to 5 − H + without an intermediate (Figure 9) furnishing the corresponding pKa value of 10.5 for the deprotonation of 5 (for details, see Supporting Information). The average of both pKa values accounts for an isoelectric point of pH = 7 for compound 5. The calculated isoelectric point is in agreement with previous observations that neutral 2,4-diarylpyrano[2,3-b]indole 5 is insoluble in water at this pH region and facilitates aggregation.  Likewise, the pK a2 value of the phenolic hydroxy group can be determined by titration with DBU as a base. Again, the occurrence of isosbestic points indicates the direct transition from species 5 to 5 − H + without an intermediate (Figure 9) furnishing the corresponding pK a value of 10.5 for the deprotonation of 5 (for details, see Supporting Information). The average of both pK a values accounts for an isoelectric point of pH = 7 for compound 5. The calculated isoelectric point is in agreement with previous observations that neutral 2,4-diarylpyrano[2,3-b]indole 5 is insoluble in water at this pH region and facilitates aggregation.
Furthermore, the emission properties at different pH values can be investigated. Upon titration, the pH detection limit under acidic and basic conditions is first determined (Figures 10 and 11). For the addition of TFA, luminescence is turned on at pH values smaller than 5.31, as for the addition of DBU, luminescence turns on at pH values higher than 8.70. This leads to a blind spot of the chemosensor 5 between pH 5.31-8.70, where no significant emission takes place.
Finally, the proper reversibility after binding an analyte and, thus, the accuracy of the results have to be ascertained. For 2,4-diarylpyrano[2,3-b]indole 5, the reversibility is tested by an alternating addition of an excess amount of TFA and DBU to a neutral solution of 2,4-diarylpyrano [2,3-b]indole 5 in propan-2-ol. After the first cycle of addition of TFA, DBU, and again TFA to 2,4-diarylpyrano[2,3-b]indole 5, a decrease in absorbance can be seen (Figure 12), probably due to ion strength of [DBU-H + ][TFA − ] and its interaction with the chromophore. Furthermore, a comparably smaller decrease in absorbance after each cycle can be attributed to dilution by increasing the amount of acid and base, respectively. The color change during these cycles occurs considerably fast, allowing a rapid readout just in seconds after addition of the analyte. After five cycles, no further effects besides small decreases in absorbance can be seen and, therefore, a good reversibility of this chemosensor can be concluded. Likewise, the pKa2 value of the phenolic hydroxy group can be determined by titration with DBU as a base. Again, the occurrence of isosbestic points indicates the direct transition from species 5 to 5 − H + without an intermediate (Figure 9) furnishing the corresponding pKa value of 10.5 for the deprotonation of 5 (for details, see Supporting Information). The average of both pKa values accounts for an isoelectric point of pH = 7 for compound 5. The calculated isoelectric point is in agreement with previous observations that neutral 2,4-diarylpyrano [2,3-b]indole 5 is insoluble in water at this pH region and facilitates aggregation. Furthermore, the emission properties at different pH values can be investigated. Upon titration, the pH detection limit under acidic and basic conditions is first determined (Figures 10 and 11). For the addition of TFA, luminescence is turned on at pH values smaller than 5.31, as for the addition of DBU, luminescence turns on at pH values higher than 8.70. This leads to a blind spot of the chemosensor 5 between pH 5.31-8.70, where no significant emission takes place.

General Considerations
Commercially available chemicals were purchased reagent grade and used without further purification. [63,71,72], swallowtail-tosylate (Sw-OTs, 4) [71][72][73], and 2-(4-ethynylphenoxy)tetrahydro-2H-pyran (6) [74,75], were prepared according to literature protocols. Reactions were performed in oven-dried Schlenk tubes under a nitrogen atmosphere unless stated otherwise. Dry solvents were dried through a solvent purification system (MB-SPS-800, M. Braun Inertgas-Systeme, Garching, Germany). Reaction progress was qualitatively monitored using silica gel layered aluminium foil (60 F254, Merck, Kenilworth, NJ, USA). For detection, UV light of wavelengths 254 nm and 365 nm as well as an aqueous potassium permanganate solution was used. Column chromatography was performed using flash technique under pressure of 2 bar with silica gel 60, mesh 230-400 (Macherey-Nagel, Düren, Germany). 1 H and 13 C NMR spectra were measured on an Avance III-300 (Bruker, Billerica, MA, USA) and Avance III-600 (Bruker, Billerica, MA, USA) spectrometer. Chemical shifts (δ) were referenced to the internal solvent signal: CDCl3 ( 1 H δ 7.26, 13 C δ 77.2). Multiplicities are stated as: s (singlet), d (doublet), dd (doublet of doublet), t (triplet), and m (multiplet). Coupling constants (J) are given in Hz. The assignment of primary (CH3), secondary (CH2), tertiary (CH) and quaternary carbon nuclei (Cquat) was made using DEPT-135 spectra. Mass spectrometric investigations were carried out at the Department of Mass Spectrometry of the Institute of Inorganic and Structural Chemistry, Heinrich-Heine-Universität Düsseldorf. IR spectra were recorded using an IR Affinity-1 spectrometer (Shimadzu, Kyoto, Japan) via the attenuated total reflection (ATR) method. The intensities of the IR bands are abbreviated as w (weak), m (medium), s (strong). Melting points (uncorrected) were measured using a Melting Point B-540 (Büchi, Oldham, UK). Combustion analyses were measured on a Series II Analyser 2400 (Perkin Elmer, Waltham, MA, USA) at the Institute of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-Universität Düsseldorf. Absorption spectra were recorded on a UV/Vis/NIR Lambda 19 spectrometer (Perkin Elmer, Waltham, MA, USA). Molar extinction coefficients ε were determined by absorption measurements at five different concentrations. Emission spectra were recorded using a F-7000 spectrometer (Hitachi, Tokyo, Japan) and corrected with spectra of the solvent mixtures. Excitation slit was set to 5.0 nm and emission slit was set to 10 nm. PMT Voltage was set to 350 V and scan speed was set to 240 nm/min. Absolute quantum yields were determined using a

General Considerations
Commercially available chemicals were purchased reagent grade and used without further purification. N-(2-iodophenyl)-3-(4-((2-(trimethylsilyl)ethyl)thio)phenyl)propiolamide (1) [62], 4-ethynylphenyl acetate (2) [63,71,72], swallowtail-tosylate (Sw-OTs, 4) [71][72][73], and 2-(4-ethynylphenoxy)tetrahydro-2H-pyran (6) [74,75], were prepared according to literature protocols. Reactions were performed in oven-dried Schlenk tubes under a nitrogen atmosphere unless stated otherwise. Dry solvents were dried through a solvent purification system (MB-SPS-800, M. Braun Inertgas-Systeme, Garching, Germany). Reaction progress was qualitatively monitored using silica gel layered aluminium foil (60 F 254 , Merck, Kenilworth, NJ, USA). For detection, UV light of wavelengths 254 nm and 365 nm as well as an aqueous potassium permanganate solution was used. Column chromatography was performed using flash technique under pressure of 2 bar with silica gel 60, mesh 230-400 (Macherey-Nagel, Düren, Germany). 1 H and 13 C NMR spectra were measured on an Avance III-300 (Bruker, Billerica, MA, USA) and Avance III-600 (Bruker, Billerica, MA, USA) spectrometer. Chemical shifts (δ) were referenced to the internal solvent signal: CDCl 3 ( 1 H δ 7.26, 13 C δ 77.2). Multiplicities are stated as: s (singlet), d (doublet), dd (doublet of doublet), t (triplet), and m (multiplet). Coupling constants (J) are given in Hz. The assignment of primary (CH 3 ), secondary (CH 2 ), tertiary (CH) and quaternary carbon nuclei (C quat ) was made using DEPT-135 spectra. Mass spectrometric investigations were carried out at the Department of Mass Spectrometry of the Institute of Inorganic and Structural Chemistry, Heinrich-Heine-Universität Düsseldorf. IR spectra were recorded using an IR Affinity-1 spectrometer (Shimadzu, Kyoto, Japan) via the attenuated total reflection (ATR) method. The intensities of the IR bands are abbreviated as w (weak), m (medium), s (strong). Melting points (uncorrected) were measured using a Melting Point B-540 (Büchi, Oldham, UK). Combustion analyses were measured on a Series II Analyser 2400 (Perkin Elmer, Waltham, MA, USA) at the Institute of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-Universität Düsseldorf. Absorption spectra were recorded on a UV/Vis/NIR Lambda 19 spectrometer (Perkin Elmer, Waltham, MA, USA). Molar extinction coefficients ε were determined by absorption measurements at five different concentrations. Emission spectra were recorded using a F-7000 spectrometer (Hitachi, Tokyo, Japan) and corrected with spectra of the solvent mixtures. Excitation slit was set to 5.0 nm and emission slit was set to 10 nm. PMT Voltage was set to 350 V and scan speed was set to 240 nm/min. Absolute quantum yields were determined using a Spectrofluorometer FS5 (Edinburgh Instruments, Livingston, UK). Light scattering experiments were performed on a 3D LS Spectrometer (LS Instruments, Fribourg, Switzerland) in 2D mode. A HeNe-Laser was used as a light source and two Avalanche photodiodes in cross-correlation mode were used for the detection of scattered light. The samples were placed in a refractive index matching bath filled with decalin. The temperature control was realized with a JULABO CF31 and a PT100 probe near the sample position. The samples were prepared in Fisherbrand culture tubes (Fisher Scientific GmbH, Schwerte, Germany) with an outer diameter of 1 cm. Measurements were performed over a range of scattering angles of 30 • to 140 • in 10 • steps. Three measurements with acquisition times between 10 and 60 s, depending on the scattering intensity of the sample, were performed at each angle. The temperature for all measurements was set to 25 • C. The data were analyzed using the CONTIN algorithm [68] as implemented in AfterALV v.1.0e (Dullware), The Netherlands.
The NMR data were identical with the sample prepared under Section 3.4.

Conclusions
The Pd/Cu-catalyzed domino insertion-coupling-cycloisomerization of alkynoyl oiodo anilides and terminal arylacetylenes furnishes a novel 2,4-diarylpyrano [2,3-b]indole, which is employed as a substrate for the preparation of a versatile amphiprotic turn-ON luminophore using a fluoride mediated double deprotection-alkylation process in a one-pot fashion. Solubility screenings reveal that the chemosensor can be used in most of organic solvents including DMSO, propan-2-ol and ethanol. Furthermore, aqueous mixtures of up to a 75:25 vol-% water-co-solvent ratio are accessible. The photophysical properties of this hydroxy-2,4-diarylpyrano [2,3-b]indole are dependent on the pH of the solvent system. Here, yellow or red luminescence is turned on under acid or basic conditions, respectively, allowing a sensitive read-out against a dark background and a reversible fine tuning of the absorption and emission characteristics. As an improvement over the previous second generation of 2,4-diarylpyrano[2,3-b]indoles, higher quantum yields of up to Φ f = 0.058 and Φ f = 0.059 in propan-2-ol for the protonated and deprotonated species respectively have been detected. In an aqueous mixture of 75:25 vol-% water/co-solvent ratio, clusters of approximately 140 nm in diameter are formed. These clusters start to rapidly form aggregates at higher concentrations of water. Further studies on the formation of clusters in solution as well as their application in biological systems are currently underway.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules27072354/s1, Supporting Information: Synthetic and analytic details, and 1 H and 13 C NMR spectra of compounds 3, 5, and 6, UV/Vis and fluorescence analyses, and dynamic light scattering analysis.