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.

Key words

dyes - RNA probes - fluorescence - Gram-positive bacteria - fluorescence - mitochondria

Publication History

Received: 09 March 2023

Accepted after revision: 04 May 2023

Article published online:
12 June 2023

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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.