Synthesis of an Azo-Cyanine Dye as an RNA Probe for Distinguishing Gram-Positive Bacteria from Gram-Negative Bacteria

Abstract

We have developed a new cyanine fluorescent probe, Azo-ETO₃, that shows better RNA selectivity than commercial dyes in solution. Azo-ETO₃ can image the RNA of mitochondria in living cells and the RNA of the nucleolus and cytoplasm in fixed cells. As a practical application, Azo-ETO₃ emits strong fluorescence when interacting with Gram-positive bacteria, and it can be used to selectively label G⁺ bacteria in the presence of other bacteria. In addition, Azo-ETO₃ exhibits low toxicity and has essentially no major impact on bacterial colony growth. These properties could make it useful as a tool for distinguishing bacterial species.

Bacteria are the most numerous group of organisms and are ubiquitous in the environment and in the human body.[1] They can be divided into two groups, Gram-positive bacteria (G⁺ bacteria) and Gram-negative bacteria (G^– bacteria), according to the composition of their cell walls and cell membranes.[2] Pathogenic strains of bacteria pose a great threat to human health, causing large-scale infectious diseases and even death.[3] For instance, methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pneumoniae, and Bacillus cereus (all G⁺ bacteria) can cause a variety of diseases, including bloodstream infections, meningitis, pneumonia, and food poisoning.[4] G^– bacteria, such as Escherichia coli and Enterobacter cloacae can cause gastrointestinal diseases, skin infections, sepsis, etc.[5] Therefore, the rapid and selective identification of species of bacteria is an important issue in dealing with bacterial infection and developing antimicrobial strategies.

G⁺ bacteria and G^– bacteria are usually identified by conventional Gram staining. However, the operational complexity and biological incompatibility of Gram staining limit its range of applications. In addition, its accuracy is also affected by many factors, such as the size and integrity of the bacteria, sample complexity, and operator experience, which can lead to inaccurate staining.[6] Fluorescence analysis has become an indispensable identification tool in bacterial research because of its low cost, simplicity, rapidity, and high sensitivity. The reported methods for fluorescence identification of bacteria include lectin labeling, labeling with aggregation-induced-emission luminogens (AIEgens), and the use of luminescent nanomaterials,[7] which target wheat-germ agglutinin, lipoteichoic acid, and the cell membranes of G⁺ bacteria to distinguish G⁺ bacteria, respectively.

Nucleic acids (DNA and RNA) are indispensable components of lifeforms,[8] and they are certainly present in bacteria. Hexidium iodide, a commercial nucleic acid dye, can selectively stain G⁺ bacteria, but its selective staining of G⁺ bacteria over G^– bacteria is not significant.[9] Sunbul and co-workers have developed the NIR fluorophores SiRs,[10] which has been successfully applied to the visualization of RNA and super-resolution imaging of living bacteria by binding to the corresponding RNA aptamer SiRA. Another method of labeling RNA involves the use of fluorophore-quencher conjugates; with this method, Jäschke and co-workers designed an OFF–ON probe that images RNA in living bacteria by binding to SRB-2 aptamers.[11] However, these works were all based on cell-permeable fluorophores binding to RNA aptamers to image bacteria. To our knowledge, there are few small-molecule RNA probes that have been used for bacterial identification.

Thiazole orange-3 (TO-3) is a well-known and widely used nucleic acid fluorescent probe. When the dyes interact with nucleic acid, the torsion of their methine chain is inhibited, which causes an enhancement of fluorescence.[12] Unfortunately, TO-3 does not show a selective response between RNA and DNA. Azonia-skeleton heterocycles are often used as nucleic-acid intercalators,[13] especially for RNA.[14] Thus, it is feasible to integrate special scaffolds of TO-3 and an azonia-skeleton heterocycle into a single molecule.[12f] Given these considerations, we developed a novel asymmetric trimethine cyanine dye, Azo-ETO₃, based on TO-3 and pyrido[1,2-a]pyrimidinium ions (one of the azonia-skeleton heterocycles), which can selectively bind RNA and has a better RNA selectivity than the commercial RNA dye SYTO RNA Select Green. In vitro results showed that Azo-ETO₃ successfully stained mitochondria in live cells, as well as the cytoplasm and nucleolus of fixed cells. Furthermore, Azo-ETO₃ could selectively stain G⁺ bacteria without interacting with G^– bacteria. Thus, we reasoned that Azo-ETO₃ might be a good tool for distinguishing bacterial species.

The synthesis process of compound Azo-ETO₃ is shown in Scheme [1]. The molecular structures of Azo-ETO₃, TO-3, and all intermediate products were characterized by ESI-MS, ¹H NMR, and ¹³C NMR [Supplementary Information (SI), Figures S10–S14]. The absorption and emission spectra of Azo-ETO₃ in various solvents are shown in SI, Figure S1. Azo-ETO₃ showed almost no emission without nucleic acids in phosphate-buffered saline (PBS) (Φ_free = 0.003; SI, Table S1), probably because of the nonradiative energy loss by free rotation around the methine chain[15] between the benzothiazole and 1-azaquinolinium rings, like many trimethine cyanine dyes.

Figure [1] showed the absorption spectrum and fluorescence response of Azo-ETO₃ (5 μM) to 200 μg/mL yeast RNA or calf thymus DNA in 10 mM PBS (pH 7.4).

The fluorescence intensity of Azo-ETO₃ increased 33.7-fold on binding to RNA. However, when DNA was added to the Azo-ETO₃ solution under the same conditions, the enhancement factor of the fluorescence intensity decreased sharply (only 8.1-fold). Moreover, Azo-ETO₃ showed no fluorescence response to other analytes (Figure [2c]). Importantly, Azo-ETO₃ was much better than commercial SYTO RNA Select Green in terms of its nucleic acid selectivity. As shown in Figure [2b], SYTO RNA Select Green was not selective toward DNA or RNA at a lower nucleic acid concentration (<50 equiv), and the ratio of its maximum fluorescence intensity for RNA to its maximum fluorescence intensity for DNA was only 1.7:1, whereas the fluorescence intensity ratio for RNA/DNA of Azo-ETO₃ was 4.1:1 under the same conditions (Figure [2a]). This implies that commercial RNA-targeting fluorescent dyes cannot distinguish low concentrations of RNA from those of DNA in solution, whereas Azo-ETO₃ could be a prospective probe for detecting RNA in solution or complex biological environments. An experiment showed that Azo-ETO₃ is stable in the pH range 3.90–10.00 (SI, Figure S3). Therefore, the better selectivity and OFF–ON ability for RNA suggest that Azo-ETO₃ might be a potential probe for RNA detection.

In general, there are three binding modes of probes to nucleic acids: groove binding, embedding, and electrostatic attraction.[16] We used CD spectra to investigate the binding mode of Azo-ETO₃ to RNA. As shown in Figure [3a], circulating-tumor DNA (Ct-DNA) showed positive and negative peaks at 276 and 245 nm, respectively, in its CD spectra. When various concentrations of Azo-ETO₃ (0-0.5mM) were added to a solution of this DNA (0.5 mg/mL), the positive peak decreased in intensity. As shown in Figure [3b], Yeast RNA had a strong positive peak at 264 nm and a weak negative peak at 240 nm. There was an evident decrease in the intensities of both positive and negative peaks after Azo-ETO₃ (0–0.5 mM) was added to an RNA solution (0.5 mg/mL).

Then, the model structures of ssRNA (4A4T) and dsDNA (6DM7), obtained from the Protein Data Bank (PDB), were used to dock with Azo-ETO₃. We used the software AutoDock 4.2 to simulate the molecular docking, and the results showed that Azo-ETO₃ binds to the groove of RNA and DNA (Figure [3c]). The absorption spectra showed that there was no obvious hypochromic red shift after the interaction between Azo-ETO₃ and the nucleic acid (Figure [1a]). All these results indicate that the binding mode of Azo-ETO₃ to nucleic acids is the groove mode.

To explore the difference between Azo-ETO₃ and TO-3, we modeled the molecules by computational methods. As shown in SI, Figure S4, the E _gap of Azo-ETO₃ (2.65 eV) was larger than that of TO-3 (2.46 eV), causing a blue shift in its absorption and emission wavelengths (SI, Table S2), which is consistent with the experimental results. To further explain the selectivity of the probe to RNA, relatively tRNA (1ASY) and dsDNA (1BNA) were used for simulation and calculation. We used the local search parameters of AutoDock 4.2 to evaluate the difference in the Azo-ETO₃ and TO-3 binding energies between DNA and RNA. The energetics of molecular interactions with nucleic acids are exothermic, and the comparison results are shown in Table [1]. The binding energy of TO-3 with tRNA (5.36 kcal/mol) was lower than that of Azo-ETO₃ (6.29 kcal/mol), whereas the binding energy of TO-3 with dsDNA (5.58 kcal/mol) was a little higher than that of Azo-ETO₃ (5.14 kcal/mol). Therefore, the Δ_{free energy} of Azo-ETO₃ was much bigger than that of TO-3, which indicated that Azo-ETO₃ has a higher RNA selectivity. Moreover, the equilibrium constant calculated according to Δ_{free energy} is consistent with the trend of a titration experiment (SI, Table S2). Another docking result (SI, Figure S5) showed that the pyrido[1,2-a]pyrimidine part of Azo-ETO₃ can form a hydrogen bond with a nucleic acid, and the hydrogen bond formed with tRNA (2.3) is shorter than that formed with dsDNA (3.1).

Based on the specific recognition of RNA by Azo-ETO₃ in solution, its cellular uptake in cell staining was studied in vitro. First, the cytotoxicity of Azo-ETO₃ was examined by the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As shown in SI, Figure S6, the cell viability of A549 cells (human lung adenocarcinoma cells) treated with Azo-ETO₃ was almost identical to that of a control group, which suggests that the molecule has almost no cytotoxicity. RNA is generally distributed in the cytoplasm and nucleolus of cells. Next, to confirm that Azo-ETO₃ mainly stains RNA inside the cell, fixed MCF-7 cells (human breast cancer cells) were treated with DNase or RNase, which specifically digest DNA or RNA, respectively, and the fluorescence signal was observed by confocal laser-scanning microscopy (CLSM). As shown in Figure [4a], when the DNase was added into the fixed MCF-7 cells, the fluorescence signal of Azo-ETO₃ appeared in the nucleolus only, whereas when an RNase was added into the fixed MCF-7 cells, the fluorescence signal of Azo-ETO₃ in the nucleolus disappeared. Then, the permeability of Azo-ETO₃ in MCF-7 cells was studied. Azo-ETO₃ permeated the cells rapidly and dyed the cells within five minutes. Figure [4b] shows that the fluorescence signal appeared in the cytoplasm of live MCF-7 cells with a filamentous shape, the typical morphology of mitochondria. The cytoplasm and the nucleolus were the predominant areas stained by Azo-ETO₃ in fixed MCF-7 cells, which is likely due to binding of Azo-ETO₃ to RNAs.

To confirm the staining position of Azo-ETO₃ in live cells, we carried out co-stain experiments with two commercial dyes: Mito Track Green and Hoechst 33342. The fluorescence signal of Azo-ETO₃ (2 μM) displayed an excellent overlap with that of Mito Track Green (0.5 μM), demonstrating that the Azo-ETO₃ indeed stained the mitochondria of living A549 cells (Figure [5]).

These results showed that the fluorescence emission of the nucleolus was caused by binding of Azo-ETO₃ to RNA. Based on these results, from the point of view of molecular structure, Azo-ETO₃, like most mitochondrial probes, is merely a common cationic dye that preferentially collects in the mitochondria of living cells with a large ΔΨ_m.[17] When the cells were fixed, ΔΨ_m disappeared and Azo-ETO₃ stained RNA due to its properties as a nucleic acid dye.

A number of studies have demonstrated that positively charged aromatic rings can efficiently target bacteria.[18] To investigate the binding behavior of Azo-ETO₃ in various types of bacteria, we chose four kinds of bacteria, the G^– bacteria P. aeruginosa and E. coli and the G⁺ bacteria MRSA and B. subtilis, to study the fluorescence images produced with Azo-ETO₃. As shown in Figure [6], after incubating the bacteria with Azo-ETO₃ under identical conditions for 30 minutes, a fluorescence signal appeared in the G⁺ bacteria only.

Inspired by this result, and to further prove the validity of the above experimental results, we used mixed cultures containing MRSA, B. subtilis and E. cloacae to confirm the selectivity of the Azo-ETO₃ probe for G⁺ bacteria. As expected, like the staining for a single class of bacteria, the fluorescence signals were observed on MRSA and B. subtilis, which are all G⁺ bacteria (Figure [7a] and SI, Figure S9a). We then treated P. aeruginosa and E. cloacae with N-benzyl-N,N-dimethyldodecan-1-aminium bromide (a quaternary cationic surface-active broad-spectrum bactericide), which can alter the permeability of the cell membrane of bacteria. As shown in Figure [7b], due to the altered permeability of the cell membrane of the dead bacteria, the probe entered the interiors of the bacteria and caused the dead P. aeruginosa (G^– bacteria) and E. cloacae (G^– bacteria) to fluoresce brightly (SI, Figure S9b). The good biocompatibility of Azo-ETO₃ was also reflected in the bacterial growth. We chose two types of bacteria, S. aureus and E. coli, to test the bacterial toxicity, and the results showed that the viabilities of S. aureus and E. coli bacteria treated with Azo-ETO₃ were all over 85% (Figure [7c]). Then Azo-ETO₃ staining was performed for plate incubation, which is a commonly used method for observing the shape and type of bacterial colonies and for detecting the numbers of colonies. The results indicated that the bacteria remained alive and healthy in this procedure (Figure [7d]); photographs of the plate cultures are shown in SI, Figure S8.

Because bacteria have a negative surface charge, probes with positive charges tend to interact with bacteria. However, selective imaging of G⁺ bacteria should be related to differences in the envelopes of G⁺ bacteria and G^– bacteria. G⁺ bacteria have a thick peptidoglycan layer and a single cell membrane, whereas G^– bacteria have a thinner peptidoglycan layer and an external lipid membrane; this means that G^– bacteria have two layers of phospholipids. G⁺ bacteria do not display a barrier effect caused by an outer envelope structure, unlike G^– bacteria. In addition, there are pore channels in the outer envelope of G^– bacteria that can reduce the permeability of the membrane and prevent the inflow of small molecules. Thus, the cationic probe Azo-ETO₃ can enter the interior of G⁺ bacteria through electrostatic and hydrophobic interactions, and bind to RNA to produce fluorescence signals. From our results, we can confirm that Azo-ETO₃ is a viable probe for discriminating G⁺ bacteria from other bacteria, which might make the probe Azo-ETO₃ a good tool for distinguishing bacterial species in practice. All these results suggested that Azo-ETO₃ might be a potentially effective probe for the identification of G⁺ bacteria by targeting RNA.[18]

In this study, Azo-ETO₃ was successfully synthesized based on an asymmetric trimethine cyanine dye and a pyrido[1,2-a]pyrimidinium ion.[19] It shows good selectivity toward nucleic acids, a rapid response, red-light emission, and excellent biocompatibility. Azo-ETO₃, as a nucleic acid probe, can enter cells within a short time and stain the mitochondrial nucleic acids in living cells and the cytoplasm and nucleolus in fixed cells. Surprisingly, Azo-ETO₃ was able to achieve a specific distinction of G⁺ bacteria among G^– bacteria with the absence of any specific targeting groups. Such a highly selective nucleic acid probe has the potential to be a powerful tool in the study of G⁺ bacteria, and diseases and infections caused by G⁺ bacteria.

Conflict of Interest

The authors declare no conflict of interest.

• References and Notes

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• 19 Synthesis Details 2-Methyl-1,3-benzothiazole (2.98 g, 20.0 mmol) and EtI (3.74 g, 24.0 mmol) in MeCN were added to a 50 mL round-bottomed flask protected by N₂, and the mixture was heated to 80 °C with magnetic stirring for 6 h, then cooled to r.t. The precipitate was collected by filtration, and the filter cake was washed thoroughly with Et₂O and vacuum dried to give 1 as a whitish-gray solid powder; yield: 5.35 g (17.53 mmol, 87.78%). Product 1 (1 g, 3.28 mmol) and N,N'-Diphenylformamidine (643.08 mg, 3.28 mmol) were mixed in a 25 mL round-bottomed flask and the mixture was stirred and heated in an oil bath at 165 °C for 0.5 h. The resulting product was washed repeatedly with Et₂O to give product 2 as a violet solid powder; yield: 902.30 mg (67.44%). ¹H NMR (600 MHz, DMSO-d ₆): δ = 11.46 (d, J = 11.6 Hz, 1 H), 8.68 (t, J = 11.6 Hz, 1 H), 8.16 (d, J = 7.9 Hz, 1 H), 7.96 (d, J = 8.3 Hz, 1 H), 7.66 (t, J = 7.8 Hz, 1 H), 7.54 (t, J = 7.6 Hz, 1 H), 7.49–7.43 (m, 4 H), 7.22 (t, J = 6.9 Hz, 1 H), 6.36 (d, J = 12.1 Hz, 1 H), 4.48 (q, J = 7.2 Hz, 2 H), 1.39 (t, J = 7.2 Hz, 3 H). 70% aq HClO₄ (4.00 mL) was added to a solution of 2-aminopyridine (2.000 g, 21.25 mmol) and 4,4-dimethoxybutan-2-one (2.807 g, 21.25 mmol) in MeOH (5.0 mL), and the mixture was stirred at 25 °C for 24 h, during which time a precipitate of 3 formed. The precipitate was collected by filtration, washed with a small amount of ice-cold MeOH, and dried in a vacuum to give 3 as a light-pink powder; yield: 3.156 g (61%). ¹H NMR (400 MHz, DMSO-d ₆): δ = 9.43 (d, J = 4.5 Hz, 1 H), 9.35 (d, J = 7.0 Hz, 1 H), 8.73–8.66 (m, 1 H), 8.60 (d, J = 8.7 Hz, 1 H), 8.24 (t, J = 7.1 Hz, 1 H), 8.18 (d, J = 4.5 Hz, 1 H), 3.07 (s, 3 H). QTOF-HRMS: m/z [M⁺] calcd for C₉H₉N₂: 145.0760; found: 145.0760. 3-Ethyl-2-[(1E,3E)-3-(4H-pyrido[1,2-a]pyrimidin-4-ylidene)prop-1-en-1-yl]-1,3-benzothiazol-3-ium Iodide (Azo-ETO₃) Product 2 (0.385 g, 0.93 mmol) and product 3 (0.25 g, 1 mmol) were dissolved in pyridine (1.8 mL), Then N,N-diisopropylethylamine (DIPEA, 0.2 mL ) and acetic anhydride (0.2 mL) were added into the above solution. The mixture was stirred at room temperature for 4 h. After reaction, the mixture was poured into 100 mL ether. The suspension was filtrated and purified by silica gel column with CH _² Cl _² /CH _³ OH (10:1, v/v). The desired violet-black solid product Azo-ETO₃ was obtained (265.60 mg, 65.24%). Violet-black solid; yield: 265.60 mg (65.24%). ¹H NMR (400 MHz, DMSO-d ₆): δ = 9.05 (d, J = 7.4 Hz, 1 H), 8.57 (dd, J = 6.0, 1.8 Hz, 1 H), 8.17 (qd, J = 7.5, 7.0, 3.5 Hz, 3 H), 8.03–7.90 (m, 2 H), 7.74–7.67 (m, 2 H), 7.57–7.49 (m, 1 H), 7.37 (td, J = 8.0, 2.2 Hz, 1 H), 6.88 (d, J = 12.8 Hz, 1 H), 6.51 (dd, J = 12.5, 1.9 Hz, 1 H), 4.34 (q, J = 7.1 Hz, 2 H), 1.39–1.31 (m, 3 H). ¹³C NMR (101 MHz, DMSO-d ₆): δ = 162.65, 152.20, 151.04, 150.34, 145.93, 141.36, 138.65, 130.65, 128.35, 128.23, 125.50, 125.08, 123.33, 120.60, 113.20, 110.43, 101.70, 98.92, 41.54, 12.81. TOF-HRMS: m/z [M⁺] calcd for C₂₀H₁₈N₃S: 332.1216; found: 332.1216.

Corresponding Author

Publication History

Received: 09 March 2023

Accepted after revision: 04 May 2023

Article published online:
12 June 2023

© 2023. Thieme. All rights reserved

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• References and Notes

• 1 Furst AL, Francis MB. Chem. Rev. 2019; 119: 700
  CrossrefPubMedSearch in Google Scholar
• 2 Liu W, Li R, Deng F, Yan C, Zhou X, Miao L, Li X, Xu Z. ACS Appl. Bio Mater. 2021; 4: 2104
  CrossrefPubMedSearch in Google Scholar
• 3 Huang Y, Chen W, Chung J, Yin J, Yoon J. Chem. Soc. Rev. 2021; 50: 7725
  CrossrefPubMedSearch in Google Scholar
• 4a Sommer F, Bäckhed F. Nat. Rev. Microbiol. 2013; 11: 227
  CrossrefPubMedSearch in Google Scholar
• 4b Donaldson GP, Lee SM, Mazmanian SK. Nat. Rev. Microbiol. 2016; 14: 20
  CrossrefPubMedSearch in Google Scholar
• 4c Fan Y, Pedersen O. Nat. Rev. Microbiol. 2021; 19: 55
  CrossrefPubMedSearch in Google Scholar
• 5a Tao B, Lin C, Yuan Z, He Y, Chen M, Li K, Hu J, Yang Y, Xia Z, Cai K. Chem. Eng. J. (Amsterdam, Neth.) 2021; 403: 126182
  Search in Google Scholar
• 5b Pakbin B, Brück WM, Rossen JW. A. Int. J. Mol. Sci. 2021; 22: 9922
  CrossrefPubMedSearch in Google Scholar
• 6 Beveridge TJ. Biotech. Histochem. 2001; 76: 111
  CrossrefPubMedSearch in Google Scholar
• 7a Sizemore RK, Caldwell JJ, Kendrick AS. Appl. Environ. Microbiol. 1990; 56: 2245
  CrossrefPubMedSearch in Google Scholar
• 7b Holm C, Jespersen L. Appl. Environ. Microbiol. 2003; 69: 2857
  CrossrefPubMedSearch in Google Scholar
• 7c Kang M, Zhou C, Wu S, Yu B, Zhang Z, Song N, Lee MM. S, Xu W, Xu F.-J, Wang D, Wang L, Tang BZ. J. Am. Chem. Soc. 2019; 141: 16781
  CrossrefPubMedSearch in Google Scholar
• 7d Liu W, Gu H, Liu W, Lv C, Du J, Fan J, Peng X. Chem. Eng. J. (Amsterdam, Neth.) 2022; 450: 137384
  Search in Google Scholar
• 8 Stoddard BL, Khvorova A, Corey DR, Dynan WS, Fox KR. Nucleic Acids Res. 2018; 46: 1563
  CrossrefPubMedSearch in Google Scholar
• 9 Mason DJ, Shanmuganathan S, Mortimer FC, Gant VA. Appl. Environ. Microbiol. 1998; 64: 2681
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• 10 Wirth R, Gao P, Nienhaus GU, Sunbul M, Jäschke A. J. Am. Chem. Soc. 2019; 141: 7562
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• 11 Sunbul M, Jäschke A. Angew. Chem. Int. Ed. Engl. 2013; 52: 13401
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  CrossrefSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
• 16 Yao Q, Li H, Xian L, Xu F, Xia J, Fan J, Du J, Wang J, Peng X. Biomaterials 2018; 177: 78
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• 19 Synthesis Details 2-Methyl-1,3-benzothiazole (2.98 g, 20.0 mmol) and EtI (3.74 g, 24.0 mmol) in MeCN were added to a 50 mL round-bottomed flask protected by N₂, and the mixture was heated to 80 °C with magnetic stirring for 6 h, then cooled to r.t. The precipitate was collected by filtration, and the filter cake was washed thoroughly with Et₂O and vacuum dried to give 1 as a whitish-gray solid powder; yield: 5.35 g (17.53 mmol, 87.78%). Product 1 (1 g, 3.28 mmol) and N,N'-Diphenylformamidine (643.08 mg, 3.28 mmol) were mixed in a 25 mL round-bottomed flask and the mixture was stirred and heated in an oil bath at 165 °C for 0.5 h. The resulting product was washed repeatedly with Et₂O to give product 2 as a violet solid powder; yield: 902.30 mg (67.44%). ¹H NMR (600 MHz, DMSO-d ₆): δ = 11.46 (d, J = 11.6 Hz, 1 H), 8.68 (t, J = 11.6 Hz, 1 H), 8.16 (d, J = 7.9 Hz, 1 H), 7.96 (d, J = 8.3 Hz, 1 H), 7.66 (t, J = 7.8 Hz, 1 H), 7.54 (t, J = 7.6 Hz, 1 H), 7.49–7.43 (m, 4 H), 7.22 (t, J = 6.9 Hz, 1 H), 6.36 (d, J = 12.1 Hz, 1 H), 4.48 (q, J = 7.2 Hz, 2 H), 1.39 (t, J = 7.2 Hz, 3 H). 70% aq HClO₄ (4.00 mL) was added to a solution of 2-aminopyridine (2.000 g, 21.25 mmol) and 4,4-dimethoxybutan-2-one (2.807 g, 21.25 mmol) in MeOH (5.0 mL), and the mixture was stirred at 25 °C for 24 h, during which time a precipitate of 3 formed. The precipitate was collected by filtration, washed with a small amount of ice-cold MeOH, and dried in a vacuum to give 3 as a light-pink powder; yield: 3.156 g (61%). ¹H NMR (400 MHz, DMSO-d ₆): δ = 9.43 (d, J = 4.5 Hz, 1 H), 9.35 (d, J = 7.0 Hz, 1 H), 8.73–8.66 (m, 1 H), 8.60 (d, J = 8.7 Hz, 1 H), 8.24 (t, J = 7.1 Hz, 1 H), 8.18 (d, J = 4.5 Hz, 1 H), 3.07 (s, 3 H). QTOF-HRMS: m/z [M⁺] calcd for C₉H₉N₂: 145.0760; found: 145.0760. 3-Ethyl-2-[(1E,3E)-3-(4H-pyrido[1,2-a]pyrimidin-4-ylidene)prop-1-en-1-yl]-1,3-benzothiazol-3-ium Iodide (Azo-ETO₃) Product 2 (0.385 g, 0.93 mmol) and product 3 (0.25 g, 1 mmol) were dissolved in pyridine (1.8 mL), Then N,N-diisopropylethylamine (DIPEA, 0.2 mL ) and acetic anhydride (0.2 mL) were added into the above solution. The mixture was stirred at room temperature for 4 h. After reaction, the mixture was poured into 100 mL ether. The suspension was filtrated and purified by silica gel column with CH _² Cl _² /CH _³ OH (10:1, v/v). The desired violet-black solid product Azo-ETO₃ was obtained (265.60 mg, 65.24%). Violet-black solid; yield: 265.60 mg (65.24%). ¹H NMR (400 MHz, DMSO-d ₆): δ = 9.05 (d, J = 7.4 Hz, 1 H), 8.57 (dd, J = 6.0, 1.8 Hz, 1 H), 8.17 (qd, J = 7.5, 7.0, 3.5 Hz, 3 H), 8.03–7.90 (m, 2 H), 7.74–7.67 (m, 2 H), 7.57–7.49 (m, 1 H), 7.37 (td, J = 8.0, 2.2 Hz, 1 H), 6.88 (d, J = 12.8 Hz, 1 H), 6.51 (dd, J = 12.5, 1.9 Hz, 1 H), 4.34 (q, J = 7.1 Hz, 2 H), 1.39–1.31 (m, 3 H). ¹³C NMR (101 MHz, DMSO-d ₆): δ = 162.65, 152.20, 151.04, 150.34, 145.93, 141.36, 138.65, 130.65, 128.35, 128.23, 125.50, 125.08, 123.33, 120.60, 113.20, 110.43, 101.70, 98.92, 41.54, 12.81. TOF-HRMS: m/z [M⁺] calcd for C₂₀H₁₈N₃S: 332.1216; found: 332.1216.

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