Novel Fluorescent Azacyanine Compounds: Improved Synthesis and Optical Properties

Abstract

Benzothiazoles are known to possess a number of biological activities and therefore are considered to be an important scaffold in the design and synthesis of pharmacophores. In this study, an improved synthesis method for novel fluorescent benzothiazole-based cyclic azacyanine (CAC) dyes bearing different electron-donating/withdrawing groups on their scaffold is presented. The improved method enabled us to increase the synthesis yield for the previously reported CACs. More importantly, it allowed us to synthesize new CAC dyes that were not synthesizable with the previously reported method. The synthesized dyes were characterized by ¹H and ¹³C NMR spectroscopy, elemental analysis, and mass spectrometry and their optical (absorption and fluorescence) properties were investigated. All of the synthesized CACs were found to be displaying strong absorption within the range of 387–407 nm. The spectral shifts observed in the absorption and fluorescence measurements suggested that the spectroscopic and optical properties of CACs can be directly modulated by the nature of the electron-donating/withdrawing substituents. The fluorescence quantum yields (QYs) of the unsubstituted (parent CAC) and substituted CACs were also measured and compared. The fluorescence QY of CACs with electron-donating substituents (methoxy or ethoxy) was found to be at least four times higher than that of the parent CAC with no substitutions.

Introduction

The synthesis of novel heterocyclic cyanine dyes bearing various structural elements is of immense interest because of their potential mainly as fluorescent visualization and labeling agents. Cyanine dyes with their remarkable physicochemical characteristics including high photochemical stabilities, excellent molar absorption coefficients, narrow absorption bands, and high fluorescence intensities have found a wide range of applications in numerous fields such as single-molecule fluorescence studies,^1,2 DNA labeling/detection,^3,4 sensing of various biomolecules,⁵ cancer targeting,⁶ laser materials,⁷ and solar cells.^8,9 Cyclic azacyanines (CACs) are nitrogen-containing heterocyclic benzothiazole/benzimidazole analogues which were first synthesized by Haddadin and co-workers as chloride-selective ion channel modulators.^10,11 CACs are positively charged heterocyclic compounds with one or more nitrogen atoms and have recently emerged in a wide variety of fields (with respect to their spectral characteristics) including surface treatment of optical recording media,¹² textile industry (as coloring agents),¹³ molecular recognition,^14,15 biological applications, and biomedical imaging.^16,17 Additionally, much effort has recently been put on the green/modified synthesis, spectral characterization, and other applications of numerous novel azacyanine dyes.¹⁸ Patra et al.¹⁹ investigated the spectroscopic and photophysical properties of three novel azacyanine dyes in various solvents to understand the solvent effect on the optical properties and energy states of CACs. They demonstrated that introduction of electron-donating/withdrawing groups at specific positions on the azacyanine scaffold can lead to the modulation of its spectroscopic properties. An electron-donating (methoxy; −OCH₃) group had been shown to increase the fluorescence lifetime and influence the radiative process, whereas an electron-withdrawing (fluorine; −F) group had been shown to slightly increase the lifetime of the excited state and significantly enhance the radiative process with respect to the parent sulfur-containing CAC. Patra and co-workers also investigated the absorption and fluorescence properties of CACs both experimentally and theoretically. In line with their previous work, they also established the stabilization of the positive charge on the CAC ring by the electron-donating groups and, vice versa, the destabilization of the positive charge by the electron-withdrawing groups.²⁰

In a later study, Tasior et al.¹⁷ reported on the significant influence of electron-donating groups on the internal charge transfer and fluorescence quantum yield (QY) of V-shaped azacyanines. All these findings revealed that the photophysical properties of azacyanines can explicitly be controlled via the substitution of electron-donating/withdrawing groups. The same group also reported the synthesis and study of the optical properties of two bis(styryl)-azacyanines.²¹ In this case, the combination of high two-photon absorption cross sections and strong fluorescence QY resulted in good two-photon brightness which opened up the possibility of the use of these dyes as bioimaging agents.

The use of benzothiazole/benzimidazole derivatives as effective biological markers in DNA detection has also drawn considerable attention in recent years because of their positively charged planar structure.⁴ So far, several derivatives of these compounds had already been reported with improved biological activities and optical properties.^22,23

Accordingly, during the last years, the major focus of our research group has been the investigation of the interactions of azacyanines with different nucleic acid structures.^14,15,24,25 Among the azacyanines investigated, only 2 of them named azacyanine4 (Aza4) and azacyanine5 (Aza5) were benzothiazole derivatives, and both of them were found to be binding to the human telomeric sequence tel24 and poly(A) with high affinity.^14,15 They were also recently evaluated for their potential as topoisomerase II inhibitors.²⁶ However, one downside of the azacyanine synthesis via the previously reported method was the low yields. In addition, the reported procedure was not suitable for the synthesis of the derivatives. Therefore, in an attempt first to increase their yield of synthesis and second to broaden the repertoire of azacyanine dyes with different electron-donating and electron-withdrawing groups, we directed our efforts toward progressing the current synthesis method to obtain a series of azacyanine dyes as novel analogues of “Aza4”.

Here, the synthesis of Aza4 derivatives with different electron-donating/withdrawing substituents in the benzothiazole moiety with moderate yields was reported and their optical properties were investigated. It is demonstrated that the presence of substituents on the CAC impacts their spectroscopic properties significantly. The fluorescence QY of CACs with electron-donating substituents (methoxy or ethoxy) is shown to be at least four times as high as that of the parent CAC (without any electron-donating/withdrawing substituent). Likewise, the fluorescence QY of CACs with the electron-withdrawing substituents was lower.

Results and Discussion

CACs azacyanine4 (Aza4, 2a) and azacyanine5 (Aza5, 2b) which were initially synthesized by Haddadin and co-workers^10,11 were also synthesized by our research group several times to investigate their interactions with nucleic acids. During our synthesis, our yields were about 10 and 20% for Aza4 and Aza5, respectively.^14,15 In order to enrich our repertoire and understanding of the effect of electron-donating and electron-withdrawing groups on the optical properties of CACs, we initially tried to synthesize CACs with different functional groups on the benzothiazole ring using the previously established synthesis method. However, our initial trials failed, especially with the CACs bearing electron-withdrawing groups or electron-donating groups at position 4. In some of our synthesis, even though we observed the formation of the product during the reaction through the color change monitoring and UV–vis measurements, we were not able to obtain the product afterward. Therefore, first, we began contemplating several synthesis possibilities, starting with the optimization of parameters such as the reaction temperature, reactant ratio, and solvent amount. Again, our efforts were in vain. Next, we started to explore different reaction conditions using different solvents and reagents. The solvent effect on the one-pot synthesis of the target CACs was tested using different pure solvents including dimethylformamide, tetrahydrofuran, acetonitrile, 1-butanol, 2-butanone, chloroform, isoamyl alcohol, toluene, pyridine, dimethylacetamide, and their mixtures with 2-(2-ethoxy)ethanol (diethylene glycol monoethyl ether) at different ratios. Besides, bromoform was individually tested instead of diiodomethane (as the reagent) during the optimization of the synthesis. Compared to the use of pure organic solvents, the use of binary organic solvent mixtures slightly promoted the yield of Aza4 (parent CAC) and Aza5 but showed negligible influence on the success of the synthesis of other CACs with an electron-donating methyl group at position 6 or with the methoxy group at position 4. In addition, despite a relatively satisfactory yield based on the weigh-ins, more than one product was observed in the ¹³C and ¹H NMR spectra. There was also a deviation in the results of the elemental analysis from the expected values. One of the products observed in NMR was confirmed to be the desired product via MS. However, recharacterization of the sample via NMR after several weeks revealed the decomposition of the desired product over time.

These results indicated that one should be careful and should take such a possibility into consideration, especially with the synthesis of those with functional groups at position 4. As a result, among all the tested binary solvent mixtures and/or reagents, the best result was achieved in 2-(2-ethoxy)ethanol/pyridine (4:1 v/v) using CH₂I₂ as a reagent, which enhanced the yields of Aza4 and Aza5 CACs (more than 10%) with the desired characteristics and physiochemical properties.

However, our attempts to synthesize CACs with the electron-withdrawing substituents also led to time-dependent decomposition of the products. Subsequently, repeating the synthesis under an acidic medium in our synthetic route under the optimized conditions using glacial acetic acid was found to greatly promote the yield of all CACs. In other words, all of the desired CAC derivatives were synthesized successfully using a binary solvent mixture [2-(2-ethoxy)ethanol/pyridine: 4:1 v/v] and under mildly acidic conditions (Table 1). Spectral data for the synthesized CACs were monitored weekly/monthly and no decomposition was observed.

Table 1. Spectral Data and the Yield for the Synthesized CAC Dyes (2a–2g).

synthesized CAC	R1	R2	yield (%)	λmax (nm)	ε (M–1 cm–1)a± SDb
2a	H	H	41	387	26,000 ± 2600
2b	H	OCH3	44	407	27,600 ± 2000
2c	OCH3	H	27	400	21,800 ± 1900
2d	H	CH3	45	392	31,000 ± 1100
2e	H	OCH2CH3	31	408	26,600 ± 2800
2f	H	F	34	387	29,500 ± 1700
2g	H	Cl	60	390	31,600 ± 2500

^aMeasured in DMSO.

^bSD: standard deviation.

Azacyanine4 (2a), azacyanine5 (2b), and the other new CAC dyes (2c–2g) were synthesized through a simple one-step path, as depicted in Scheme 1. A proposed plausible mechanism for the CAC synthesis using the parent compound (2a) as the model is depicted in Scheme 2.²⁷ All of the synthesized compounds were characterized by NMR spectroscopy (¹H and ¹³C), elemental analysis, and mass spectrometry (details are shown in the Materials and Instrumentation section). The yields of the synthesized CACs are listed in Table 1. Clearly, all of the synthesized compounds exhibited relatively satisfactory results in terms of the product yields. The highest product yield was observed for the compound 2g (60%).

Scheme 1. Path to Synthesize CAC Dyes.

Reagents and conditions for all of the synthesized azacyanines: 2-(2-ethoxy)ethanol/pyridine (4:1 v/v) under slightly acidic conditions, diiodomethane/200 °C.

Scheme 2. Proposed Reaction Mechanism for the Formation of Parent CAC (Aza4, 2a).

The presence of electron-donating groups is possibly leading to a more stable state with lower energy by donating electron density to the π system of the CACs and stabilizing the positive charge on the nitrogen atom.^19,28 Accordingly, among the electron-donating groups, only the alkyl substituent bound to position 6 of 1d (starting material of 2d) cannot donate the lone-pair electrons to the π system. However, it could still increase the electron density of the ring system, as evident from 45% product yield of 2d. In the case of the compound 2c, the methoxy group attached to position 4 is believed not able to stabilize the ring system as effectively as the other electron-donating groups, and therefore, it exhibits the lowest product yield (27%). Besides, the position 4 is believed to be creating steric hindrance for the dimerization reaction (can also be observed from the proposed reaction mechanism) and less stability²⁹ resulting in a lower yield. This can be considered as the main reason for the fact that our efforts for the synthesis of the CAC derivative with the methyl substituent at position 4 failed to yield the final product.

On the other hand, the presence of electron-withdrawing groups is leading to a less-stable structure by decreasing the electron density on the aromatic ring.³⁰ They are neither able to stabilize the positive charge on the nitrogen nor contribute to the conjugation of the π system. Therefore, the presence of electron-withdrawing functional groups could decrease the reactivity of the molecule. However, in this case, −Cl and −F as electron-withdrawing substituents (which are exceptions of electron-withdrawing groups with an ability to donate their lone pairs to the π system of aromatic rings³¹) can still induce electron density and enhance the reactivity. Hereby, the product yield of the 2f and 2g compounds (34 and 60%, respectively) was in an acceptable range and close to the yield of the parent CAC with no functional group (2a). Because the electronegativity of fluorine is greater than that of chlorine, the yield of compound 2f was lower than that of the 2g.

Once the synthesis of CACs was determined to be successful via ¹H and ¹³C NMR, elemental analysis, and mass spectroscopy, their optical properties were investigated using UV–vis absorption and fluorescence spectroscopy techniques. All the synthesized CAC dyes (2a–2g) were found to be displaying intense and broad absorptions (ε > 20,000 M^–1 cm^–1) in the visible region (λ_max: 387–408 nm, Table 1). As seen, the maximum absorption wavelength for the synthesized CACs rises in the order 2a = 2f < 2g < 2d < 2c < 2b < 2e, probably as a consequence of the presence of the electron-withdrawing (−F and −Cl) and electron-donating (−CH₃, −OCH₃, and −OCH₂CH₃) substituents at the specific positions on the synthesized CAC scaffold in relation to 2a (CAC with no functional group).

Figure 1 depicts the normalized UV–vis absorption, fluorescence emission, and excitation spectra of the synthesized CAC dyes at the same concentration [30.0 μM, in dimethyl sulfoxide (DMSO)] at room temperature. Based on the results, Aza4 (2a, Figure 1A) as the parent compound (without any electron-donating/withdrawing substituent) showed a broad absorption spectrum between 330 and 420 nm (S₀–S₁ transition) with two observable vibronic bands at around 387 and 396 nm. Also, a maximum emission intensity at 446 nm was observed (Table 2) in its fluorescence emission spectrum. The presence of the electron-donating (−OCH₃ group as in 2b and 2c, −CH₃ and −OCH₂CH₃ as in 2d and 2e, respectively) and an electron-withdrawing group (−Cl as in 2g) resulted in a red shift in the absorption spectra of these synthesized CACs. The maximum absorbance (max λ_abs/nm) and maximum emission (max λ_em/nm) wavelengths as well as the Stokes’ shifts (in cm^–1) of the synthesized CAC dyes are tabulated in Table 2.

Figure 1.

UV–vis absorption, excitation, and fluorescence emission spectra of the synthesized CACs in DMSO; (A) parent CAC-2a; (B) 2b; (C) 2c; (D) 2d; (E) 2e; (F) 2f; and (G) 2g. For all the synthesized CACs: black, blue, and red lines represent the normalized absorption, excitation, and emission spectra, respectively.

Table 2. Optical and Photophysical Properties of the Synthesized CAC Compounds (2a–2g).

synthesized CAC	λabs max (nm)	λem max (nm)	Stokes’ shift (cm–1)	fwhm (nm)a	QY (ΦF %)b
2a	387	446	3418	63	17.0
2b	407	478	3650	70	70.0
2c	400	474	3903	72	26.4
2d	392	458	3676	63	47.5
2e	408	486	3934	70	71.0
2f	387	447	3468	64	13.4
2g	390	456	3711	64	17.7

^afwhm: full width at half-maximum for the fluorescence band.

^bMeasured in DMSO.

As can be seen, 2b and 2e displayed a similar spectral behavior (Figure 1B and E). In both, the maximum absorption was around 407 and 408 nm, respectively, and a transition from S₀ to the two different vibrational energy levels of S₁ was observed. Moreover, the highest red shift compared to the maximum λ_abs of Aza4 (2a) was observed in 2e. This observation is probably due to the effect of the “ethoxy” substituent as the strong activating electron-donating group capable of donating the lone-pair electrons to the π system of adjacent carbons and increasing the electron density on the fused ring system by resonance effect accompanied by the delocalization of the electrons.³² In the absorption spectrum of 2c, the peak intensity of the transition from S₀ to the higher vibrational level of S₁ has sharply decreased and two separate peaks were present at around 330 nm (the absorption maximum for the S₀–S₂ transition in the 300–350 region) and 400 nm, respectively. Evidently, in this case, the spectral region for the S₀–S₁ transition was different from those obtained for the other synthesized CACs which might be attributed to the presence of an electron-donating functional group (methoxy) at position 4. This also shows that the same substituent exerted very different effects in different positions. As mentioned previously, the synthesis of compounds with functional groups at position 4 was unsuccessful compared to the synthesis of compounds with functional groups at position 6. Figure 2, displaying the UV–vis absorption spectra of the synthesized CACs in DMSO, reveals the difference more evidently. Besides, 2a, 2d, 2f, and 2g exhibit a similar spectral region and vibrational structured transitions for the S₀–S₁ transition (two vibronic bands were observed). However, a slight red shift was observed in the absorption spectra of 2d and 2g relative to 2a. The red shift observed in the absorption spectra of all the substituted CACs (except 2f) is probably due to π-donation into the cationic CAC π-system.²⁰ Obviously, 2b and 2e exhibited a greater red shift relative to Aza4 and the highest red shift (compared to the other synthesized CACs) was observed for 2e (21 nm). Because the methoxy group has a similar electron-donating behavior to ethoxy, the compound 2b appeared to have a similar red shift in the absorption spectrum and the absorption maximum was shifted about 20 nm compared to the parent CAC (2a). Although a similar trend was expected in the absorbance spectrum of 2c (in the case of the electron-donating methoxy group at position 4), the red shift was only 13 nm with respect to 2a. The main reason behind this observation is probably the fact that the charge delocalization, and thus the stabilization of the resonance structures, is greater when the substituent is at position 6. As a result, 2b has more stabilized resonance forms of the negative charge delocalization and less energy is required to excite it with respect to 2c. When the methyl group was introduced as a substituent to the parent compound (2a), a relatively similar absorption spectrum was obtained for 2d, with a red shift in the absorption maxima of about 5 nm.

Figure 2.

UV–vis absorption spectra of the synthesized CAC dyes at 30.0 μM concentration in DMSO at room temperature.

Methyl is also an electron-donating group, but it has no lone-pair electrons to contribute to the resonance structure. Therefore, it can only activate the ring by inductive effect and increase the electron density. Hence, the contribution of the methyl group to the stabilization of CAC dyes is the least among the electron-donating functional groups. Based on the fact that halogens can act as π-donors in addition to being σ-withdrawing groups,³³ the synthesized CACs with electron-donating groups were expected to show obvious and longer red shifts than CACs with electron-withdrawing moieties. When the electron-donating groups were replaced by electron-withdrawing functional groups such as −F and −Cl, the spectrum resembled more to the parent compound without any obvious red shift (2f, probably because of a greater electronegativity) or with a minor red shift in the absorption spectrum (2g, 3 nm red shift). −F and −Cl can deactivate the aromatic ring by decreasing the electron density because of the electronegativity and the simultaneous activation of the ring by lone-pair donation through position 6.

The fluorescence emission spectra of the synthesized CACs are given in Figure 3. Like the CAC with no functional group (2a), the intense and broad emissions (in the wavelength region of 400–650 nm) were observed in the fluorescence spectra of the substituted compounds and demonstrated significant substitution-dependent shifts in their emission maxima. It was found that the emission spectra of the compounds 2b–2g have two trends. The spectra of 2b and 2e exhibited similar emission bands in the 410–650 nm range by around 32 and 40 nm shift, respectively, and up to 3934 cm^–1 Stokes’ shifts (max λ_em = 478 and 486 nm for 2b and 2e, respectively). On the contrary, the emission spectra of 2d, 2f, and 2g in the same wavelength range demonstrated a slightly different character having maxima in the range of 446–456 nm and Stokes’ shifts up to 3711 cm^–1 (Figure 3, Table 2). The Stokes’ shift for the electron-withdrawing substituted CACs demonstrated an increase from 3418 cm^–1 (for 2a) to 3711 cm^–1 (for 2g), whereas it increased for the methoxy- and ethoxy-substituted CACs (2b and 2e) to 3650 and 3934 cm^–1, respectively (see Table 2). It is important to note that the fluorescence maximum of 2c broadly shifted to a shorter wavelength (474 nm, when compared to 2b and 2e) probably arising from the presence of the electron-donating group at position 4.

Figure 3.

Fluorescence emission spectra of the synthesized CAC dyes at 10.0 μM concentration in DMSO at room temperature, upon excitation at their maximum absorbance wavelengths.

It can be concluded that when an electron-withdrawing group such as −F or −Cl is introduced as a substituent in the CAC structure, a similar fluorescence spectrum is obtained without a significant red shift in the emission maxima (in relation to 2a). The spectral shifts in the absorption and emission are probably because of the different stabilization of the positive charge on the nitrogen atom by the electron-withdrawing/donating groups.¹⁹ The electron-donating groups are thought to be stabilizing the positive charge and favoring the conjugation, resulting in a decrease in the π–π* or n−π* energy gap, meaning the shift of absorption to higher wavelengths. The electron-withdrawing groups do not have such a stabilization effect on the positive charge of the nitrogen atom and therefore do not change the energy gap. It is usual to see such shifts in the positions of absorption maxima, depending on the identity of the substitution.^34,35 Very similar effects were observed by Enchev et al.¹⁸ They reported that although replacing the hydrogen with a chlorine was shifting the absorption maxima of their parent azacyanine by only 9 nm, replacing it with a methoxy group was shifting it about 30 nm. On the other hand, broadness of the absorption peaks is due to the presence of several vibrational and rotational states corresponding to the ground state and the excited states.^36,37 The presence and the identity of the substitution of the ring system might be one of the possible reasons behind the observed differences in broadness. However, how these groups are affecting the interactions between the solvent and the molecule, and the orientation of the dipoles in the solvent might also be contributing to the observed changes in broadness.³⁸⁻⁴⁰

The measured fluorescence QYs of the synthesized CACs are summarized in Table 2, and the digital photograph of the synthesized CACs taken under UV light under the same conditions is depicted in Figure 4. Hence, the trend in the fluorescence QY of the synthesized CACs can be ordered as 2e > 2b > 2d > 2c > 2a > 2g > 2f. Surprisingly, small fluorescence QYs were observed for the compounds 2f and 2g (Table 2) and the highest QY belonged to the compound 2e which was relatively high compared to the QYs of other CACs and cyanine dyes reported in the literature.^18,19,33 In other words, this shows that while the electron-withdrawing groups were decreasing the QYs, the electron-donating groups were enhancing it. Among the synthesized CACs, 2e and 2b are anticipated to be the best potential candidates for the optical detection and imaging platforms based on their high QYs. An analogous platform was recently developed utilizing 2b by our group.⁴¹

Figure 4.

Digital photograph of the synthesized CAC dyes taken under a 365 nm UV lamp.

Conclusions

In conclusion, an improved synthesis method based on the use of the binary solvent mixture, 2-(2-ethoxy)ethanol/pyridine (4:1 v/v) in a slightly acidic medium, for the synthesis of CAC dyes was developed in this study. The improved method enabled the synthesis of novel CACs with different electron-donating/withdrawing groups in their benzothiazole ring. It was found that introducing electron-donating/withdrawing substituents onto the benzothiazole scaffold was affecting their synthesis and spectroscopic/optical properties. Compared to the other synthesized CACs, compound 2c exhibited the lowest product yield probably because of the methoxy group being located at position 4. Except for the compound 2f, a red shift was observed in the absorption spectra for all the CACs whether they are bearing an electron-donating or electron-withdrawing group. However, the synthesized CACs with electron-donating groups displayed greater red shifts than the CACs with electron-withdrawing groups. The highest red shift was observed in 2e because of the effect of the ethoxy substituent. The compounds 2f and 2g exhibited lesser red shifts than 2e and 2b. Despite the fact that 2f possesses two evident electron-withdrawing groups (2,8-F), its absorption spectrum was quite similar to the spectrum of 2a and did not exhibit any obvious red shift. The compound 2c also displayed a similar red shift to 2g. However, its spectrum was completely different than the spectrum of all other CACs more likely because of the functional group being at position 4.

In terms of the fluorescence spectra, all the synthesized CAC dyes, except 2f, showed intense and broad emission and substitution-dependent shifts in their emission maxima. The type of the substituent also had a notable impact on the QYs. The compounds with electron-donating groups had higher QYs in general where the highest fluorescence QY was observed for the compound 2e. More importantly, its QY was relatively high compared to the QYs of other CACs and cyanine dyes reported. All the observations confirm that the compounds 2e and 2b can be plausible candidates for optical imaging applications. More importantly, the improved synthesis method reported here opens up new possibilities in the synthesis of new CAC dyes.

Materials and Instrumentation

2-Aminobenzothiazole (1a, 97%), 2-(2-ethoxy)ethanol (99.0%), perylene (≥99%), bromoform (CHBr₃, 99%), and dichloromethane (≥99%) were obtained from Sigma-Aldrich (St. Louis, USA). 2-Amino-6-methoxybenzothiazole (1b, 98%) and 2-amino-4-methoxybenzothiazole (1c, 97%) were purchased from Acros Organics (New Jersey, USA). 2-Amino-6-methylbenzothiazole (1d, 99%), 2-amino-6-ethoxybenzothiazole (1e, 99%), and 2-amino-6-fluorobenzothiazole (1f, 98%) were supplied by Alfa Aesar (Kandel, Germany). 2-Amino-6-chlorobenzothiazole (1g, 98%) was purchased from TCI (Tokyo Chemical Industry, Tokyo, Japan). DMSO (99.8%) and cyclohexane (99.5%) were obtained from AppliChem GmbH (Darmstadt, Germany). Deuterated dimethyl sulfoxide (DMSO-d₆) for NMR spectroscopy, pyridine (spectroscopic grade; 99.0%), acetone, methanol (≥99.8%), dichloromethane, glacial acetic acid (≥99%), and all other organic solvents were acquired from Merck (Darmstadt, Germany). Diiodomethane (CH₂I₂; 99.0%) was ordered from abcr GmbH (Karlsruhe, Germany). All the starting materials and solvents were of analytical/spectroscopic grade and used without additional purification.

All the reported NMR (¹H and ¹³C) spectra of the synthesized compounds were recorded on a 400 MHz Bruker AVANCE III NMR spectrometer (Bruker, Karlsruhe, Germany) and the chemical shifts were expressed in ppm (δ) downfield from tetramethylsilane (internal standard). Coupling constants (J) are quoted in Hertz (Hz). High-resolution mass spectra (HRMS) were obtained via electrospray ionization with time-of-flight (ToF) detection using a Waters SYNAPT G1 MS spectrometer (Waters, Milford, MA. USA). Elemental analysis (C, H, N, and S) was performed on a Leco analyzer (CHNS-932, USA). A Cary 8454 UV–vis photodiode array spectrophotometer (Santa Clara, CA, USA) was used to collect the absorption spectra of the synthesized CACs at room temperature. Fluorescence intensity measurements were performed at room temperature using an Agilent Cary Eclipse spectrofluorometer (Agilent Technologies, USA). The excitation and emission slit widths were set at 2.5 and 5.0 nm, respectively. The spectral data were collected using Chemstation software and data processing/analysis was performed using IGOR Pro software.

General Information

Molar Extinction Coefficients (ε) and Fluorescence QY Measurements

The molar extinction coefficients (ε) and fluorescence QYs (ΦF) of all the synthesized derivatives were calculated with the well-established approaches using the absorbance wavelengths (λ_max) of the as-synthesized CAC standard solutions.

For ε measurements, first, each of the synthesized CAC derivatives was weighed at five different amounts (5.0, 10.0, 15.0, 20.0, and 25.0 mg) and diluted to 50.0 mL with DMSO. Second, five different stock solutions were prepared for each CAC and each stock solution was diluted to six different solutions using different dilution ratios. Their UV–vis absorption values were measured at their maximum absorption wavelength. Afterward, the graphs of the absorbance versus the concentration of the samples were plotted and the slopes of five plots of the synthesized compounds were averaged to obtain their molar extinction coefficients (ε) as assessed by Beer–Lambert law (Beer’s law).

The fluorescence QYs of the synthesized CACs (in DMSO) were measured under dilute conditions with a relative optical method⁴² using perylene (QY = 0.94; in cyclohexane⁴³) as a reference standard. To avoid concentration effects including internal reflections, self-quenching, and secondary inner filter effect (reabsorption of the emission), the sample/standard concentration ranges were carefully adjusted such that the absorbance values would all be kept between 0.02 and 0.1. Also, both solvents were checked for background fluorescence prior to the experiments. The excitation wavelength used for the synthesized CACs and the standard sample was 410 nm, and the fluorescence QYs were determined using the following equation⁴⁴

1

where Φ is the fluorescence QY (the subscripts ST and x denote the standard and the synthesized CAC, respectively). Grad_x and Grad_ST are the measured integrated emission intensity of the CAC and the standard sample, respectively, and η is the refractive index of the solvents.

General Procedure for the Synthesis of CACs

The general one-step procedure for the synthesis of “Aza4” and other CAC dyes as their analogues were as follows:

A total of 1.0 g of the starting material was taken in a dry round-bottom flask equipped with a magnetic stirrer. A mixture of 5.0 mL of pyridine and 20.0 mL of 2-(2-ethoxyethoxy) ethanol was added to it and stirred carefully at room temperature to get a clear solution. Next, 600.0 μL of glacial acetic acid was added to the reaction mixture and the solution was heated to 200 °C. Afterward, diiodomethane (2.0 g, 600.0 μL, 7.5 mmol) was immediately injected into the above-mentioned mixture and refluxed at 200 °C for 15 min under a gentle flow of nitrogen. Upon completion of the reaction, the mixture was allowed to cool down to room temperature. Finally, the precipitated solid was filtered off and washed thoroughly (several times) with pure acetone and dichloromethane and dried at 65 °C overnight. The final obtained products were recrystallized from methanol (20% v/v; MeOH–water) and characterized. Because of its lower polarity (in relation to alcohols) and the good solubility of the CACs, DMSO was preferred as a solvent to measure the molar extinction coefficients. The stock solutions (1 mM) of the synthesized CACs were prepared in DMSO and the working solutions were prepared after dilution with DMSO to 10.0 and 30.0 μM during recording the fluorescence and absorption spectra, respectively.

Azacyanine4: (Dibenzo[4,5]thiazolo[3,2-a]benzo[4,5]thiazolo[2,3-d][1,3,5]triazin-12-ium) (2a)

The product was obtained as a reddish-brown solid compound; ¹H NMR (400 MHz, DMSO-d₆): δ 6.6623 (s, 2H), δ 7.6886, 7.6679, 7.6497 (t, 2H), δ 7.8637, 7.843, 7.8223 (t, 2H), δ 7.9609, 7.9402 (d, 2H), δ 8.2708, 8.2514 (d, 2H); ¹³C NMR (125 MHz, DMSO-d₆): δ 61.40, 114.11, 124.83, 125.29, 127.65, 129.58, 139.97, 167.64; HRMS (m/z): calcd for [M + H]⁺, 296.0318; found, 296.0316; elemental analysis experimental: calcd for C₁₅H₁₀N₃S₂I: C, 42.56%; H, 2.38%; N, 9.93%; S, 15.15%. Found: C, 41.76%; H, 2.49%; N, 9.74%; S, 14.86%. Yield: 41%; QY: ΦF = 0.170, λ_em = 446 nm; UV–vis (DMSO): ε = 26,000 M^–1 cm^–1, λ_max = 387 nm.

Azacyanine5: (3,9-Dimethoxy-13H-benzo[4,5]thiazolo[3,2-a]benzo[4,5]thiazolo[2,3-d][1,3,5]triazin-12-ium) (2b)

The product was obtained as a shinny yellow solid compound; ¹H NMR (400 MHz, DMSO-d₆): δ 4.058 (s, 6H), δ 7.165 (s, 2H), δ 7.393, 7.372 (d, 2H), δ 7.561, 7.540, 7.512 (t, 2H), δ 7.745, 7.726 (d, 2H); ¹³C NMR (125 MHz, DMSO-d₆): δ 56.08, 60.46, 108.32, 113.96, 125.51, 129.88, 158.04, 165.08; HRMS (m/z): calculated for [M + H]⁺, 356.527; found, 356.515; elemental analysis experimental: calcd for C₁₇H₁₄N₃O₂S₂I: C, 42.24%, H, 2.92%, N, 8.69%, S, 13.26%. Found: C, 42.03%; H, 3.06%; N, 8.84%; S, 13.24%. Yield: 44%; QY: ΦF = 0.700, λ_em = 478 nm; UV–vis (DMSO): ε = 27,600 M^–1 cm^–1, λ_max = 407 nm.

Azacyanine-4-methoxy: (5,7-Dimethoxy-13H-benzo[4,5]thiazolo[3,2-a]benzo[4,5]thiazolo[2,3-d][1,3,5]triazin-12-ium) (2c)

The product was obtained as a brown solid compound; ¹H NMR (400 MHz, DMSO-d₆): δ 6.662 (s, 2H), δ 7.689, 7.668, 7.650 (t, 2H), δ 7.864, 7.843, 7.822 (t, 2H), δ 7.961, 7.940 (d, 2H), δ 8.271, 8.251 (d, 2H); ¹³C NMR (125 MHz, DMSO-d₆): δ 57.194, 64.32, 111.189, 116.19, 125.2, 127.6, 147.8, 165.81; HRMS (m/z): calcd for [M + H]⁺, 356.0527; found, 356.0503; elemental analysis experimental: calcd for C₁₇H₁₄N₃O₂S₂I: C, 42.24%; H, 2.92%; N, 8.69%; S, 13.26%. Found: C, 41.81%; H, 3.08%; N, 8.82%; S, 13.17%. Yield: 27%; QY: ΦF = 0.264, λ_em = 474 nm; UV–vis (DMSO): ε = 21,800 M^–1 cm^–1, λ_max = 400 nm.

Azacyanine-6-methyl: (3,9-Dimethyl-13H-benzo[4,5]thiazolo[3,2-a]benzo[4,5]thiazolo[2,3-d][1,3,5]triazin-12-ium) (2d)

The product was obtained as a pale-yellow solid compound; ¹H NMR (400 MHz, DMSO-d₆): δ 2.50 (s, 6H), δ 6.5945 (s, 2H), δ 7.667, 7.664, 7.6453, 7.643 (dd, 2H), 7.8267, 7.8059 (d, 2H), 8.04 (s, 2H); ¹³C NMR (125 MHz, DMSO-d₆): δ 20.858, 38.847, 39.054, 39.263, 39.472, 39.680, 39.890, 40.097, 112.729, 112.921, 123.885, 124.009, 129.607, 134.021, 136.727; HRMS (m/z): calcd for [M + H]⁺, 324.629; found, 324.618; elemental analysis experimental: calcd for C₁₇H₁₄N₃S₂I: C, 45.24%, H, 3.13%, N, 9.31%, S, 14.21%. Found: C, 44.52%; H, 3.32%; N, 9.52%; S, 14.18%. Yield: 45%; QY: ΦF = 0.475, λ_em = 458 nm; UV–vis (DMSO): ε = 31,000 M^–1 cm^–1, λ_max = 392 nm.

Azacyanine-6-ethoxy: (3,9-Diethoxy-13H-benzo[4,5]thiazolo[3,2-a]benzo[4,5]thiazolo[2,3-d][1,3,5]triazin-12-ium) (2e)

The product was obtained as a yellow solid compound; ¹H NMR (400 MHz, DMSO-d₆): δ 1.3723, 1.3898, 1.3723 (t, 6H), δ 4.1503, 1503, 4.1327, 4.1152 (q, 4H), δ 6.5531 (s, 2H), δ 7.4235, 7.4289, 7.4126, 7.4063 (dd, 2H), δ 7804, 7.7918 (d, 2H), δ 7.848, 7.8417 (d, 2H); ¹³C NMR (125 MHz, DMSO-d₆): δ 14.467, 38.881, 39.080, 39.298, 39.507, 39.715, 40.113, 60.468, 64.167, 108.773, 113.998, 116.638, 124.383, 129.807, 157.114, 164.540; HRMS (m/z): calcd for [M + H]⁺, 384.0840; found, 384.0829; elemental analysis experimental: calcd for C₁₉H₁₈N₃O₂S₂I: C, 47.60%; H, 3.78%; N, 8.77%; S, 13.38%. Found: C, 43.98%; H, 3.74%; N, 8.37%; S, 13.36%. Yield: 31%; QY: ΦF = 0.710, λ_em = 486 nm; UV–vis (DMSO): ε = 26,600 M^–1 cm^–1, λ_max = 408 nm.

Azacyanine-6-fluoro: (3,9-Difluoro-13H-benzo[4,5]thiazolo[3,2-a]benzo[4,5]thiazolo[2,3-d][1,3,5]triazin-12-ium) (2f)

The product was obtained as a creamy yellow solid compound; ¹H NMR (400 MHz, DMSO-d₆): δ 6.645 (s, 2H), δ 7.826, 7.820, 7.803, 7.797, 7.781, 7.746 (dt, 2H), δ 7.963, 7.953, 7.941, 7.930 (m, 2H), δ 8.204, 8.198, 8.183, 8.177 (dd, 2H); ¹³C NMR (125 MHz, DMSO-d₆): δ: 60.944, 111.424, 111.726, 114.712, 114.813, 116.642, 116.910, 125.585, 125.719, 132.992, 158.662, 161.090, 166.704; HRMS (m/z): calcd for [M + H]⁺, 332.0115; found, 332.0128; elemental analysis experimental: calcd for C₁₅H₈N₃F₂S₂I: C, 39.23%; H, 1.76%; N, 9.15%; S, 13.96%. Found: C, 39.11%; H, 1.97%; N, 9.05%; S, 14.08%. Yield: 34%; QY: ΦF = 0.134, λ_em = 447 nm; UV–vis (DMSO): ε = 29,450 M^–1 cm^–1, λ_max = 387 nm.

Azacyanine-6-chloro: (3,9-Dichloro-13H-benzo[4,5]thiazolo[3,2-a]benzo[4,5]thiazolo[2,3-d][1,3,5]triazin-12-ium) (2g)

The product was obtained as a yellowish-brown solid compound; ¹H NMR (400 MHz, DMSO-d₆): δ 6.638 (s, 2H), δ 7.9195, 7.8975 (d, 2H), δ 7.987, 7.981, 7.645, 7.643 (dd, 2H), δ 7.391, 8.386 (d, 2H); ¹³C NMR (125 MHz, DMSO-d₆): δ 60.502, 114.580, 124.068, 125.743, 129.069, 130.935, 134.994, 166.946; HRMS (m/z): calcd for [M + H]⁺, 363.953; found, 363.954; elemental analysis experimental: calcd for C₁₅H₈N₃Cl₂S₂I: C, 36.60%; H, 1.64%; N, 8.54%; S, 13.03%. Found: C, 35.74%; H, 1.82%; N, 9.49%; S, 12.71%. Yield: 60%; QY: ΦF = 0.177, λ_em = 456 nm; UV–vis (DMSO): ε = 31,600 M^–1 cm^–1, λ_max = 390 nm.

Author Contributions

K.D. and A.G. contributed equally. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.