Novel fluorescence detection reagent and technology for quantitatively detecting in-vivo and in-vitro CO

[0028] Example 1

[0029] Compound 3 synthesis

[0030] Compound 1 (1.56 g, 5 mmol) and compound 2 (1.30 g, 5 mmol) were dissolved in 5 ml of concentrated sulfuric acid, and at 90 o CHer heat reflow, TLC was monitored to the end of the reaction, remove the reaction, cooled to room temperature, poured into ice water, and added 1 ml of hydrochloric acid, see a large amount of precipitation, filtrate, collected, washing the filter, recrystallization with ethanol Deep green solid 1.50 g, the yield is 55.7%.

[0031] Compound 4 synthesis

[0032] At room temperature, Compound 3 (1.34 g, 2.5 mmol), hydrazine (1.64 mL, 25 mmol) and BOP (1.13 g, 2.6 mmol) were added to 50 ml of round bottom flask, and 10 ml of dichloromethane dissolved. The reaction system was stirred at room temperature, and TLC was monitored to the end of the reaction. The reaction was removed, the solvent was removed under reduced pressure. After purification of the column chromatography, the yellow solid was obtained from 1.12 g, and the yield was 78.3%. 1 H NMR (400 MHz, DMSO-D 6 ): δ = 7.93 (D, J = 7.6 Hz, 1H), 7.78 (T, J = 7.2, 1H), 7.58 (T, J = 7.2, 1H), 7.53 (S, 1H), 7.49 (D, J = 7.6 Hz, 1H), 7.41 (T, J = 7.8 Hz, 1H), 6.70-6.67 (m, 2H), 6.52 (S, 1H), 6.22 (D, J = 6.8 Hz, 1H), 6.15 (S, 1H), 5.47 (S, 1H), 3.38 (Q, J = 6.8 hz, 4h), 3.31 (q, J = 6.8 hz, 4h), 1.17-1.13 (m, 12h).

[0033] Synthesis of target molecular probe

[0034] Compound 4 (550.65 mg, 1 mmol) and 2-pyridine formaldehyde (240.05 mg, 1 mmol) were dissolved in 10 ml of anhydrous methanol, and the mixture was completed at room temperature to end. After the end of the reaction, the solvent was removed under reduced pressure. After purification of the column chromatography, the red solid was 638.63 mg, and the yield was 81.7%. 1 H NMR (400 MHz, CDCL 3 : δ = 9.13 (D, J = 7.2, 1H), 8.48 (T, J = 8.2, 1H), 8.01 (T, J = 8.2, 1H), 7.91 (D, J = 7.6 Hz, 1H), 7.82 (T, J = 7.2, 1H), 7.60-7.56 (M, 2H), 7.50 (S, 1H), 7.43-7.40 (m, 2H), 7.18 (D, J = 7.6Hz, 1H), 6.88 (D, J = 8.2 Hz, 1H), 6.69 (D, J = 8.0 Hz, 1H), 6.54 (S, 1H), 6.20 (D, J = 6.8 hz, 1H), 6.13 (S, 1H), 5.47 (S, 1H), 4.39 (S, 3H), 3.41 (Q, J = 7.2Hz, 4H), 3.31 (q, J= 7.1 Hz, 4H), 1.16 (T, J = 7.0 hz, 6h), 1.21 (t, J = 7.1 Hz, 6H). HRMS-ESI M / Z: Calc. For [m-i] + : 654.3074; Found, 654.3719.

[0035] Example 2

[0036] Response of fluorescence detection for different concentrations CO

[0037] The fluorescent detection test agents prepared by the aqueous solution of 5 ml of a concentration of 1 mM CO and a concentration of 1 mm were prepared were used as a spare. Dilution was diluted with PBS buffer solution (10 mM, pH = 7.4), the concentration of 10 μm contained in the solution was adjusted to the solution, and the concentration of CO was 0 μm, 1 μm, 2 μm, 4 μm, 7 μm, 10 μm, and. After 20 min at room temperature, the fluorescence spectrum of different systems was tested in 10 mm cuvette. figure 1. Calculate the fluorescence intensity in each system and establish a standard curve of fluorescence intensity and CO concentration. Such as figure 2 As shown, when the concentration of CO is in the range of 0-4 μm, the fluorescence intensity is a good linear relationship with the concentration of CO, and the regression linear equation is Y = 18.56897X + 6.60021, the linear correlation coefficient is: 0.99153, and calculate the test The limit (LOD) is 1.02 μm (S / N = 3), indicating that the fluorescent probe has good sensitivity.

[0038] Example 3

[0039] Psychic detection reagent to CO selectivity

[0040] The PBS aqueous solution of various conventional ions and amino acids prepared for 5 ml of concentration of 50 mm and a concentration of a fluorescent detection tester prepared as 1 mM Example 1 were used as spare. Dilution was diluted with PBS buffer solution (10 mM, pH = 7.4), adjusted to a solution of a probe compound having a concentration of 10 μm, CO concentration of 30 μm, and O 2 - , OH, H 2 O 2 , HOCL, OONOO - , CN - H 2 S, GSH concentration is 100 μm. After 20 min at room temperature, the fluorescence spectrum of different systems was tested in 10 mm cuvette. image 3. The results showed that other objects to be tested had little effect on the fluorescence of the fluorescent molecular probe, while CO was added to increase the fluorescence of the fluorescent molecule probe.