A rhodamine derivative fluorescent probe, preparation method and application thereof

Abstract

The present invention provides a rhodamine derivative fluorescent probe, a preparation method thereof and an application thereof. The preparation method includes: S1, synthesizing compound I; S2, dissolving compound I in methanol, dropwise adding concentrated sulfuric acid under stirring and cooling conditions, and refluxing for 20-25 h; removing the excess methanol, pouring the residual liquid into an ice-water mixture and adjusting the pH to neutral, then extracting with CH2Cl2, combining the organic phases, drying with anhydrous sodium sulfate, concentrating to obtain a crude product II; separating and purifying by silica gel column chromatography to obtain compound II; S3, dissolving compound II in methanol, dropping 2-hydrazinopyridine at room temperature, refluxing under nitrogen protection, centrifuging after cooling, and washing with methanol to obtain the rhodamine derivative fluorescent probe. The preparation method of the rhodamine derivative fluorescent probe in the present invention is simple, easy to operate and low in cost, realizing highly selective and highly sensitive detection of Cu 2+ in water, and the detection limit reaches 10 ‑9 M order of magnitude.

Description

Technical Field

[0001] The invention belongs to the technical field of optical sensing imaging detection, and in particular relates to a rhodamine derivative fluorescent probe, a preparation method and an application thereof. Background Art

[0002] Copper ions are essential heavy metal ions for the human body. Their content in the human body is second only to iron ions and zinc ions. They are important transition elements in biological systems. Insufficient or excessive copper ion levels in the cytoplasm of neurons may lead to some neurological diseases, such as gastrointestinal diseases, kidney damage, Alzheimer's disease and familial amyotrophic lateral sclerosis. In addition, copper ions are also an important environmental pollutant. Copper ions circulate in nature through the atmosphere, water, soil, rocks and organisms. Copper pollution is one of the important factors of environmental pollution, and its main sources are copper smoke, wastewater and waste residue.

[0003] Traditional analytical detection methods include chromatography, spectroscopy and electrochemical analysis, but they are not suitable for real-time detection due to expensive equipment, complicated pretreatment and long time consumption. Fluorescent probes have specific recognition functions for target substances and can be detected intuitively through changes in fluorescence after responding to the target. However, when applied to environmental and biological detection, they still have shortcomings, such as small Stokes shift, the "switch" change of fluorescent signals is greatly affected by the paramagnetism of copper, and the selectivity and water solubility are poor. We need to develop new fluorescent probes to solve these problems.

[0004] Therefore, the design and development of highly selective luminescent sensors for copper ion detection in organisms and environments is of great significance to life sciences and environmental sciences. Summary of the invention

[0005] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a rhodamine derivative fluorescent probe, a preparation method and its application for high selectivity and high sensitivity response to Cu 2+ Detection in water.

[0006] To achieve the above-mentioned object and other related objects, the present invention provides a rhodamine derivative fluorescent probe, wherein the fluorescent probe has the chemical structural formula shown below:

[0007]

[0008] The present invention also provides a method for preparing a rhodamine derivative fluorescent probe, the preparation method comprising the following steps:

[0009] S1, synthesis of compound Ⅰ;

[0010] S2, dissolving the compound I in methanol, adding concentrated sulfuric acid dropwise under stirring and cooling conditions, and reflux reaction for 20 to 25 hours; after the reaction, removing excess methanol, pouring the residual liquid into an ice-water mixture, and adjusting the pH to neutral, then extracting with CH2Cl2, combining the organic phases, drying with anhydrous sodium sulfate, and concentrating to obtain a crude product II; separating and purifying the crude product II by silica gel column chromatography to obtain compound II;

[0011] S3. Dissolve the compound II in methanol, add a certain amount of 2-hydrazinepyridine dropwise at room temperature, reflux under nitrogen protection, centrifuge after cooling, and wash with methanol to obtain compound III, which is a rhodamine derivative fluorescent probe.

[0012] Preferably, the synthesis method of compound I in step S1 comprises the following steps:

[0013] S11, mixing compound A and compound B according to a ratio, adding concentrated sulfuric acid dropwise under cooling conditions, reacting at 80-100° C. for 24-48 hours to obtain a reaction solution;

[0014] Wherein, the chemical structural formula of compound A is: The chemical structure of compound B is

[0015] S12, pouring the reaction solution into an ice-water mixture, adjusting the pH to neutral, and then extracting with CH2Cl2, combining the organic phases, drying with anhydrous sodium sulfate, and concentrating to obtain a crude product I;

[0016] S13. Separate and purify the crude product I by silica gel column chromatography to obtain compound I.

[0017] Preferably, in step S11, the molar ratio of the compound A to the compound B is 1:1 to 1:1.2.

[0018] Preferably, in step S11, the ratio of the total mass of the compound A and the compound B to the mass of the concentrated sulfuric acid is 1:2 to 1:2.5.

[0019] Preferably, the eluent used for separation and purification by silica gel column chromatography in step S13 is PE / EtOAc.

[0020] Preferably, the volume ratio of PE to EtOAc in the eluent is 50:1.

[0021] Preferably, in step S2, the mass ratio of the compound I to the concentrated sulfuric acid is 1:1.5 to 1:2.

[0022] Preferably, the eluent used for separation and purification by silica gel column chromatography in step S2 is CH2Cl2 / CH3OH.

[0023] Preferably, the volume ratio of CH2Cl2 to CH3OH in the eluent is 10:1.

[0024] Preferably, the molar ratio of the 2-hydrazinepyridine added in step S3 to the compound II is 2:1 to 2.5:1.

[0025] Preferably, the reflux reaction time in step S3 is 6 to 8 hours.

[0026] The present invention also provides an application of a rhodamine derivative fluorescent probe, wherein the rhodamine derivative fluorescent probe is used for Cu 2+ Detection.

[0027] As described above, the rhodamine derivative fluorescent probe, preparation method and application thereof of the present invention have the following beneficial effects:

[0028] The preparation method of the rhodamine derivative fluorescent probe of the present invention is simple and easy to implement with low cost. 2+ After the reaction, Cu 2+ Inducing the ring opening of spirolactam and further hydrolysis, triggering the bond energy transfer process, enhancing the fluorescence, and realizing the Cu 2+ The detection is highly selective and sensitive, and the detection limit can reach 10 -9 The order of magnitude of M. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Shown is the mass spectrum of compound I prepared in Example 1 of the present invention.

[0030] Figure 2 It is shown that the compound I prepared in Example 1 of the present invention 1 H NMR (400MHz) nuclear magnetic spectrum.

[0031] Figure 3 It is shown that the compound I prepared in Example 1 of the present invention 13 C NMR (100MHz) nuclear magnetic spectrum.

[0032] Figure 4 Shown is the mass spectrum of compound II prepared in Example 1 of the present invention.

[0033] Figure 5 It is shown that the compound II prepared in Example 1 of the present invention 1 H NMR (400MHz) nuclear magnetic spectrum.

[0034] Figure 6It is shown that the compound II prepared in Example 1 of the present invention 13 C NMR (100MHz) nuclear magnetic spectrum.

[0035] Figure 7 Shown is the mass spectrum of the rhodamine derivative fluorescent probe prepared in Example 1 of the present invention.

[0036] Figure 8 The rhodamine derivative fluorescent probe prepared in Example 1 of the present invention is shown 1 H NMR (400MHz) nuclear magnetic spectrum.

[0037] Fig. 9 The rhodamine derivative fluorescent probe prepared in Example 1 of the present invention is shown 13 C NMR (100MHz) nuclear magnetic spectrum.

[0038] Fig.10 The fluorescence probe of the rhodamine derivative prepared in Example 1 of the present invention is shown to be Cu 2+ Detected fluorescence emission spectra.

[0039] Fig.11 The fluorescence probe of the rhodamine derivative prepared in Example 1 of the present invention is shown to be Cu 2+ Detection limit data plot for the assay. DETAILED DESCRIPTION

[0040] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0041] The present invention provides a rhodamine derivative fluorescent probe, which has the following chemical structural formula:

[0042] The present invention also provides a method for preparing a rhodamine derivative fluorescent probe, which comprises the following steps:

[0043] S1, synthesis of compound Ⅰ;

[0044] S2. Dissolve compound I in methanol, add concentrated sulfuric acid dropwise under stirring and cooling conditions, and reflux for 20 to 25 hours (e.g., 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, etc.); after the reaction, remove excess methanol, pour the residual liquid into an ice-water mixture, and adjust the pH to neutral (using NaHCO3, Na2CO3, KHCO3, K2CO3, etc.), then extract with CH2Cl2 (the number of extractions is determined according to actual operations, usually 3 to 5 times), combine the organic phases, dry with anhydrous sodium sulfate, and concentrate to obtain a crude product II; separate and purify the crude product II by silica gel column chromatography to obtain compound II;

[0045] S3. Dissolve compound II in methanol, add a certain amount of 2-hydrazinepyridine dropwise at room temperature, reflux under nitrogen protection, centrifuge after cooling, and wash with methanol to obtain compound III, which is a rhodamine derivative fluorescent probe.

[0046] Specifically, in step S2, compound I is dissolved in methanol, and the amount of methanol only needs to be sufficient to dissolve compound I, or slightly excessive, and the specific value is not limited here; after the reaction is completed, excess methanol is removed, and the excess methanol is specifically removed by rotary evaporation; silica gel column chromatography separation and purification refers to a column chromatography separation technology based on silica gel filler, which uses silica gel stationary phase to separate samples, and according to different chemical properties and interaction forces, the samples experience different retention times in the silica gel stationary phase, thereby achieving sample separation, and then using the mobile phase (eluent) for purification; Regarding the step of using silica gel column chromatography for separation and purification, the method familiar to those skilled in the art can be used, and no excessive restrictions are made here. The amount of methanol in step S3 is also not specifically limited, and it can be sufficient to dissolve compound II.

[0047] As an example, the synthesis method of compound I in step S1 includes the following steps:

[0048] S11. Compound A and compound B are mixed according to a ratio, concentrated sulfuric acid is added dropwise under cooling conditions, and the mixture is reacted at 80 to 100° C. (e.g., 80° C., 85° C., 90° C., 95° C., 100° C., etc.) for 24 to 48 hours (e.g., 24 hours, 30 hours, 36 hours, 42 hours, 48 ​​hours, etc.) to obtain a reaction solution; wherein the chemical structural formula of compound A (2-(4-diethylamino-2-hydroxy-benzoyl)-benzoic acid) is: The chemical structure of compound B (5-hydroxyindazole) is

[0049] S12, pouring the reaction solution into an ice-water mixture, and adjusting the pH to neutral, then extracting with CH2Cl2 (the number of extractions is determined according to the actual operation, usually 3 to 5 times), combining the organic phases, drying with anhydrous sodium sulfate, and concentrating to obtain a crude product I; wherein, in this embodiment, NaHCO3, Na2CO3, KHCO3, K2CO3, etc. can be used to adjust the pH value;

[0050] S13. Separate and purify the crude product I by silica gel column chromatography to obtain compound I (rhodamine-indazole derivative).

[0051] As an example, the molar ratio of compound A to compound B in step S11 is 1:1 to 1:1.2.

[0052] Specifically, the molar ratio of compound A to compound B may include values ​​within any range such as 1:1, 1:1.05, 1:1.1, 1:1.15, 1:2, etc., and may be adjusted according to actual conditions.

[0053] As an example, in step S11 , the ratio between the total mass of compound A and compound B and the mass of concentrated sulfuric acid is 1:2 to 1:2.5.

[0054] Specifically, the ratio between the total mass of compound A and compound B and the mass of concentrated sulfuric acid may include values ​​within any range such as 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, etc., and may be adjusted according to actual conditions.

[0055] As an example, the eluent used for separation and purification by silica gel column chromatography in step S13 is PE / EtOAc (petroleum ether / ethyl acetate).

[0056] As an example, the volume ratio of PE (petroleum ether) to EtOAc (ethyl acetate) in the eluent is 50:1.

[0057] As an example, in step S2, the mass ratio of compound I to concentrated sulfuric acid is 1:1.5 to 1:2.

[0058] Specifically, the mass ratio of compound I to concentrated sulfuric acid may include values ​​within any range of 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, etc., and may be adjusted according to actual conditions.

[0059] As an example, the eluent used for separation and purification by silica gel column chromatography in step S2 is CH2Cl2 / CH3OH (dichloromethane / methanol).

[0060] As an example, the volume ratio of CH2Cl2 to CH3OH in the eluent is 10:1.

[0061] As an example, the molar ratio of 2-hydrazinepyridine added in step S3 to compound II is 2:1 to 2.5:1.

[0062] Specifically, the molar ratio of 2-hydrazinepyridine added in step S3 to compound II may include values ​​within any range such as 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, etc., and may be adjusted according to actual conditions.

[0063] As an example, the reflux reaction time in step S3 is 6 to 8 hours.

[0064] Specifically, the reflux reaction time may include values ​​within any range such as 6 h, 7 h, 8 h, etc., and may be adjusted according to actual conditions.

[0065] In addition, the temperature of the reflux reaction is 80 to 100° C. (eg, 80° C., 85° C., 90° C., 95° C., 100° C., etc.).

[0066] Specifically, the chemical reaction equation during the synthesis of compound I is:

[0067]

[0068] The chemical reaction equations of step S2 and step S3 are:

[0069]

[0070] The present invention also provides an application of a rhodamine derivative fluorescent probe, wherein the rhodamine derivative fluorescent probe is used to detect Cu in water. 2+ Detection.

[0071] Specifically, the rhodamine derivative fluorescent probe was first prepared to a concentration of 5×10 -3 mol / L DMF solution and save it for later use; then -3 mol / L DMF solution was dissolved in water to prepare multiple 3 mL groups with a concentration of 1×10 -5 mol / L of the test solution, and add different concentrations of Cu 2+ (0~150μM), and its fluorescence emission spectrum was measured using 340nm as the excitation wavelength.

[0072] Fluorescent probe for Cu 2+ The detection principle is that the rhodamine derivative fluorescent probe is combined with Cu 2+ After the reaction, Cu 2+ The spirolactam ring opening and further hydrolysis are induced, triggering the bond energy transfer process and enhancing the fluorescence.

[0073] In order to better understand the rhodamine derivative fluorescent probe, preparation method and application thereof in the present invention, the rhodamine derivative fluorescent probe, preparation method and application thereof in the present invention are described below with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0074] Example 1

[0075] This embodiment provides a rhodamine derivative fluorescent probe and a preparation method thereof. The rhodamine derivative fluorescent probe has the following chemical structural formula:

[0076]

[0077] The preparation method comprises the following steps:

[0078] S1, synthesis of compound Ⅰ;

[0079] S11. A mixture of compound A (3.13 g, 10 mmol) and compound B (1.34 g, 10 mmol) was added to a 50 mL round-bottom flask, concentrated sulfuric acid (6 mL, 98 wt %) was added dropwise under cooling, and the mixture was reacted at 90° C. for 36 h to obtain a reaction solution;

[0080] Wherein, the chemical structural formula of compound A is: The chemical structure of compound B is

[0081]

[0082] S12, pouring the reaction solution into an ice-water mixture, adding NaHCO3 to adjust the pH of the mixture to neutral, then extracting with CH2Cl2 three times, combining the organic phases, drying with anhydrous sodium sulfate, and concentrating to obtain a crude product I;

[0083] S13, separating and purifying the crude product I by silica gel column chromatography (eluent: PE / EtOAc=50 / 1, v / v), to obtain 2.66 g of pink solid, i.e., compound I, with a yield of 64.7%;

[0084] After testing, the yield of compound I was 64.7%, Mp: 247-249°C; Figure 1 , Figure 2 , Figure 3 The mass spectra of compound Ⅰ, 1 H NMR (400MHz) 13 C NMR (100MHz) nuclear magnetic spectrum:

[0085] HRMS(ESI)calcd.m / z 412.1655[M+H] +,found.m / z 412.1653[M+H] + .] + .

[0086] The H NMR spectrum is 1 H NMR (400MHz, DMSO-d6) δ13.36(s,1H),8.14(d,J=6.4Hz,1H),7.82-7.76(m,2H),7.72(d,J=9.2Hz,1H),7.42(d,J=9.2Hz,1H), 7.32(d,J=6.8Hz,1H),6.56(d,J=9.2Hz,1H),6.50(d,J=7.6Hz,2H),6.31(s,1H),3.36(q,J=6.8Hz,4H),1.09(t,J=6.8Hz,6H).

[0087] The NMR carbon spectrum is 13 C NMR(100MHz,DMSO-d6)δ168.7,152.6,152.0,149.2,146.5,137.2,135.6,130.5,130.2,128 .3,126.7,124.9,124.4,118.9,117.6,113.9,108.9,107.4,104.3,96.8,83.8,43.8,12.3.

[0088] S2. Dissolve compound Ⅰ (2.05 g, 5 mmol) in methanol (30 mL), add concentrated sulfuric acid (2 mL) dropwise under stirring and cooling, and reflux for 24 h; after the reaction, remove excess methanol by rotation, pour the residual liquid into an ice-water mixture, add NaHCO3 to adjust the pH to neutral, and then extract with CH2Cl2 for 3 times. After combining the organic phases, dry with anhydrous sodium sulfate, and concentrate to obtain a crude product Ⅱ; separate and purify the crude product Ⅱ by silica gel column chromatography (eluent: CH2Cl2 / CH3OH=10 / 1, v / v) to obtain 1.08 g of purple solid, namely compound Ⅱ, with a yield of 50.7%;

[0089] After testing, the yield of compound II was 50.7%, Mp: 239~241℃; Figures 4 to 6 The mass spectra of compound II are respectively 1 H NMR (400MHz) 13 C NMR (100MHz) nuclear magnetic spectrum:

[0090] HRMS(ESI)calcd.m / z 426.1812[M],found.m / z 426.1810[M].

[0091] The H NMR spectrum is 1 H NMR (400MHz, DMSO-d6) δ14.22(s,1H),8.43(d,J=7.2Hz,1H),8.35(d,J=8.8Hz,1H),8.0 7(dd,J=7.6,6.4Hz,1H),8.02(dd,J=7.6,6.4Hz,1H),7.96(d,J=8.8Hz,1H),7.63(d,J=7 .2Hz,1H),7.49(dd,J=9.6,1.6Hz,1H),7.37(d,J=2.0Hz,1H),7.32(d,J=9.6Hz,1H),6. 14(s,1H),3.84-3.78(m,4H),3.53(s,3H),1.31(t,J=6.8Hz,3H),1.24(t,J=6.8Hz,3H).

[0092] The NMR carbon spectrum is 13 C NMR(100MHz,DMSO-d6)δ164.9,158.6,158.2,157.0,151.7,137.4,135.2,134.5,131.9,131.4,13 1.3,130.9,129.6,128.3,122.3,119.5,118.2,116.9,114.3,95.8,52.5,46.4,46.1,13.2,12.1.

[0093] S3. Dissolve compound II (0.43 g, 1 mmol) in methanol (5 mL), add 2-hydrazinepyridine (0.22 g, 2 mmol) dropwise at room temperature, reflux for 6 h under nitrogen protection, centrifuge after cooling, and wash with methanol to obtain a white solid, namely compound III, which is a rhodamine derivative fluorescent probe, with a yield of 41.9%.

[0094] After testing, the yield of compound III was 41.9%, Mp:>300℃; see Figure 7 to Figure 9 They are the mass spectra of compound III, 1 H NMR (400MHz) 13 C NMR (100MHz) nuclear magnetic spectrum:

[0095] MS calcd.m / z 503.2190[M+H] + ,found.m / z 503.2192[M+H] + .

[0096] The H NMR spectrum is 1H NMR (400 MHz, DMSO-d6) δ13.13 (s, 1H), 8.14 (s, 1H), 8.02 (d, J = 7.2 Hz, 1H), 7.74-7.70 (m, 2H), 7.66 (t, J = 7.2 Hz, 1H), 7.53 (d, J = 8.8 Hz, 1H), 7.23 (dd, J = 11.6, 7.6 Hz, 2H), 7.03 (s, 1H), 6.59 (s, 1H), 6.49-6.46 (m, 1H), 6.42 (d, J = 7.2 Hz, 1H), 6.31 (d, J = 16.4 Hz, 2H), 5.92 (s, 1H), 3.32 (d, J = 7.2 Hz, 4H), 1.07 (t, J = 6.8 Hz, 6H).

[0097] The nuclear magnetic carbon spectrum is 13C NMR (100 MHz, DMSO-d6) δ 164.6, 158.6, 153.2, 149.8, 148.4, 147.6, 146.8, 137.2, 136.5, 133.5, 131.0, 130.5, 129.4, 124.8, 123.0, 119.7, 117.4, 114.6, 112.5, 108.2, 108.0, 106.0, 104.3, 96.9, 65.3, 43.7, 12.4.

[0098] Application Example 1

[0099] This application example provides an application of a rhodamine derivative fluorescent probe, using the rhodamine derivative fluorescent probe in Example 1 to detect Cu in water. 2+ The detection includes the following steps:

[0100] The rhodamine derivative fluorescent probe in Example 1 was prepared to a concentration of 5×10 -3 mol / L DMF solution and save it for later use; then prepare multiple groups of DMF solutions with a concentration of 1×10 -5 mol / L fluorescent probe solution to be tested 3mL; add different concentrations of Cu 2+ (0~150μM), and its fluorescence emission spectrum was measured using 340nm as the excitation wavelength.

[0101] See also Fig.10 The rhodamine derivative fluorescent probe prepared in Example 1 is a fluorescent probe for Cu 2+ The fluorescence emission spectrum of the detection shows that with the increase of Cu 2+ As the concentration of the fluorescent probe solution gradually increases, the fluorescence intensity of the fluorescent probe solution gradually increases.

[0102] In addition, the rhodamine derivative fluorescent probe in Example 1 was prepared to a concentration of 5×10 -3 mol / L DMF solution and save it for later use; then prepare 7 groups of DMF solutions with a concentration of 1×10 -5 mol / L fluorescent probe solution to be tested 3mL; in 6 3mL solutions with a concentration of 1×10 -5 mol / L of the fluorescent probe solution to be tested was added with 0μM, 1μM, 2μM, 3μM, 4μM, 5μM, and 6μM Cu 2+ ions, the fluorescence emission spectrum was measured using an excitation wavelength of 340 nm, and then the fluorescence emission intensity of the fluorescent probe solution at an emission wavelength of 600 nm was compared with that of Cu 2+ ion concentrations, see Fig.11 The detection limit was calculated to be 1.96 nM, indicating that the fluorescent probe is effective for Cu in aqueous solution. 2+ It has high selectivity and high sensitivity response.

[0103] In summary, the preparation method of the rhodamine derivative fluorescent probe in the present invention is simple and easy, low in cost, and the rhodamine derivative fluorescent probe is 2+ After the reaction, Cu 2+ Inducing the ring opening of spirolactam and further hydrolysis, triggering the bond energy transfer process, enhancing the fluorescence, and realizing the Cu 2+ The detection is highly selective and sensitive, and the detection limit can reach 10 -9 The order of magnitude of M. Therefore, the present invention effectively overcomes various shortcomings in the prior art and has high industrial utilization value.

[0104] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. Use of a rhodamine derivative in preparing a fluorescent probe, characterized in that: The fluorescent probe is used to detect Cu in water 2+ Detection; the fluorescent probe has the chemical structural formula shown below:

Images