CN105331358B - A bisrhodamine-based Sn4+ fluorescent probe molecule and its preparation method and application - Google Patents

Abstract

The present invention relates to a kind of high selectivity Sn based on double rhodamine ethanolamine derivants⁴⁺The preparation method of fluorescent probe.Condensation acylation reaction is carried out by rhodamine B and ethanolamine and obtains rhodamine ethanolamine derivant, rhodamine ethanolamine derivant is obtained into the Sn containing double rhodamines with m-phthaloyl chloride reaction⁴⁺Fluorescent probe SnP1.The probe is in Sn⁴⁺Show preferable selectivity in the system coexisted with other metal ions, sensitivity is high, response is fast, the features such as invertible operation, can be very good to be applied to Sn in environment⁴⁺Detection.

Description

A bisrhodamine-based Sn4+ fluorescent probe molecule and its preparation method and application

technical field

The invention relates to a tin ion (Sn ⁴⁺ ) detection agent, in particular to a preparation method of a bisrhodamine-based Sn ⁴⁺ fluorescent probe.

Background technique

Tin is widely present in air, soil and water, and is one of the common heavy metals in industry. Since the industrial revolution, more and more inorganic tin and organic tin have been introduced into the environment. Tin is an essential trace element for the human body, and it is most abundant in organs such as the adrenal gland, liver, brain, spleen and thyroid. Tin deficiency may cause poor growth and hearing loss, and there is some evidence that tin is involved in growth factors and cancer prevention. At the same time, since tin is widely used in agriculture, industry and other fields, tin has also become one of the common heavy metal pollutants in the environment. Excess tin absorption by the human body can also have important health consequences. The symptoms of tin poisoning are mostly limited to gastrointestinal reactions such as nausea, abdominal pain, and vomiting. Therefore, it is of great significance to study a method that can quickly detect Sn ⁴⁺ .

Fluorescent probes have the advantages of good selectivity, high sensitivity, simple and fast operation, and less damage to the detection object, and have been widely used in the detection of metal cations, anions, and active small molecules in vivo in the environment and biological systems. The development and utilization of fluorescent probe molecules is an interdisciplinary field of chemistry and biology, medicine, agriculture and other sciences. Fluorescent probe technology is an analytical method for studying research objects at the molecular level. Fluorescent probes have been widely used in the detection of heavy metals, bioactive small molecules and other substances in the environment and organisms.

Rhodamine dyes have the advantages of large molar absorptivity, high fluorescence quantum yield, good optical stability, wide wavelength range and wide pH stability range, etc., and have been widely used in the design and development of fluorescent probes. synthesis. In recent years, rhodamine derivatives have been used to design fluorescent probes to detect metal cations or anions in organisms or the environment. The mechanism of rhodamine-based fluorescent probes for recognizing metal ions is shown in the figure. The ring-closed rhodamine derivatives themselves have no color and fluorescence, but when the corresponding metal cations or anions are added to make the intramolecular lactam spiro ring open, the solution will change from colorless to pink, and at the same time release a strong of fluorescence.

Contents of the invention

The object of the present invention is to provide a kind of preparation method of Sn ⁴⁺ fluorescent probe based on bisrhodamine.

A kind of preparation method of the Sn ⁴⁺ fluorescent probe based on bisrhodamine proposed by the present invention, first carries out condensation acylation reaction by commercialized rhodamine B and ethanolamine, obtains a kind of rhodamine B-ethanolamine derivative; The rhodamine A compound containing bisrhodamine B can be obtained by acid chlorination reaction of rhodamine B-ethanolamine derivatives with isophthaloyl chloride, which can be used as a highly selective fluorescent probe for Sn ⁴⁺ in methanol-water solution.

A kind of preparation method of the Sn ⁴⁺ fluorescent probe based on bisrhodamine proposed by the present invention, its specific synthetic route is as follows:

(1) Stir rhodamine B and ethanolamine in the ethanol solution and reflux until fully reacted. After the reaction is complete, cool the reaction system to room temperature, remove the solvent under reduced pressure, redissolve with ethyl acetate, and use pure water and saturated sodium chloride solution After washing, the organic phase was dried with anhydrous sodium sulfate, filtered, the solvent was removed under reduced pressure, and the intermediate product RhB-OH was obtained by silica gel column chromatography.

(2) The intermediate RhB-OH and triethylamine are mixed in the methylene chloride solution, and the methylene chloride solution of isophthaloyl chloride is added dropwise under ice bath conditions. After the dropwise addition, it should be a room temperature reaction. After the reaction is complete , washed with saturated sodium chloride solution, the organic phase was dried with anhydrous sodium sulfate, filtered, the solvent was removed under reduced pressure, ethyl acetate and petroleum ether were used as detergents, and the product SnP1 was obtained by silica gel column chromatography.

In the step (1), the ratio of rhodamine B to ethanolamine is 1:4, the heating and reflux temperature is 80-90° C., and the reaction time is 8-15 hours.

In the step (1), the eluent separated by the silica gel column is CH ₂ Cl ₂ and CH ₃ OH, the volume ratio of the CH ₂ Cl ₂ to CH ₃ OH is 10-18:1, and the yield is 60- 85%.

In the step (2), the compound RhB-OH is 2-4 equivalents, isophthaloyl chloride is 1 equivalent, triethylamine is 2-5 equivalents, and the reaction time at room temperature is 6-10 h.

In the step (2), the eluent separated by the silica gel column is CH ₂ Cl ₂ and CH ₃ OH, the volume ratio of the CH ₂ Cl ₂ to CH ₃ OH is 10-20:1, and the yield is 60- 75%.

Application of bisrhodamine-based Sn ⁴⁺ fluorescent probe molecules in the detection of Sn ⁴⁺ .

Description of drawings

Fig. 1 is fluorescent probe SnP1 proton nuclear magnetic resonance spectrogram of the present invention;

Fig. 2 is fluorescent probe SnP1 carbon nuclear magnetic resonance spectrogram of the present invention;

Fig. 3 is the high-resolution mass spectrogram of fluorescent probe SnP1 of the present invention;

Fig. 4 is the fluorescent selectivity figure of fluorescent probe SnP1 of the present invention, excitation wavelength 520nm;

Fig. 5 is the anti-metal cation interference diagram of the fluorescent probe SnP1 of the present invention recognizing Sn ⁴⁺ , the excitation wavelength is 520nm, and the emission wavelength is 577nm;

Fig. 6 is a graph showing the anti-anion interference of the fluorescent probe SnP1 of the present invention in recognizing Sn ⁴⁺ , the excitation wavelength is 520nm, and the emission wavelength is 577nm.

detailed description

The chemical reagents, solvents, and metal ions used in the process of preparing the fluorescent probe SnP1 in the present invention were all purchased from Aladdin Reagent Company. In the confirmation and performance testing process of fluorescent probe SnP1, DTX-400 nuclear magnetic resonance spectrometer from Bruke Company was used, the solvent was deuterated chloroform, and TMS was used as internal standard to record H-NMR and C-NMR spectra. High-resolution mass spectrometry data were recorded using a Thermo Q-Exactive HR-MS mass spectrometer. Fluorescence spectra were recorded using a Hitachi F-7000 fluorescence spectrometer.

The structure of the final product SnP1 synthesized by the present invention is confirmed by nuclear magnetic resonance hydrogen spectrum, carbon spectrum and high-resolution mass spectrum.

1. Preparation of intermediate RhB-OH:

In a 100mL single-necked round bottom flask, add Rhodamine B hydrochloride (1.00g, 2.08mmol), ethanolamine (0.54mL, 8.32mmol) and 50mL ethanol, after the reflux reaction is complete (10h) the reaction solution is cooled to room temperature, The solid obtained after removing the solvent under reduced pressure was dissolved in 30 mL of ethyl acetate solution, washed with water (30 mL × 2) and saturated sodium chloride solution (30 mL × 2), the organic phase was dried with anhydrous sodium sulfate, filtered, and reduced After the solvent was removed under pressure, the intermediate product RhB-OH was obtained by column chromatography (eluent: CH ₂ Cl ₂ :CH ₃ OH=10:1, volume ratio) with a yield of 75%.

2. Preparation of probe SnP1:

In a 100mL single-necked round-bottom flask, add the intermediate RhB-OH (1.21g, 2.5mmol) and triethylamine (0.25g, 2.5mmol) and mix in anhydrous dichloromethane solution (40mL). Add anhydrous dichloromethane solution (15mL) of isophthaloyl chloride (0.2g, 1mmol). After the dropwise addition, it should be reacted at room temperature. After the reaction is complete (about 8h), saturated sodium chloride solution (30mL×2) Wash, dry the organic phase with anhydrous sodium sulfate, filter, remove the solvent under reduced pressure, and separate by silica gel column chromatography (eluent: CH ₂ Cl ₂ :CH ₃ OH=15:1, volume ratio) to obtain the product SnP1. The rate is 69%.

¹ H NMR (CDCl ₃ , 400MHz) δ1.15(t, J=6.0Hz, 24H), 3.30(q, J=6.7Hz, 16H), 3.36(s, 4H), 4.02( t, J=4Hz, 4H), 6.22(d, J=8Hz, 4H), 6.37(s, 4H), 6.46(d, J=8 Hz, 4H), 7.08(q, J=2.7Hz, 2H) , 7.44 (d, J = 4Hz, 5H), 7.94 (q, J = 2.7Hz, 2H), 8.08 (t, J = 4Hz, 2H), 8.46 (s, 1H).

¹³ C NMR (CDCl ₃ , 100MHz) δ168.53, 165.1, 153.9, 153.1, 148.8, 133.7, 132.6, 130.8, 130.6, 130.4, 128.7, 128.4, 128.0, 123.8, 122.9, 1058.2, 90 , 64.8, 62.3, 44.3, 38.6, 12.6.

High-resolution mass spectrometry: HR-ESI-MS calcd for C ₆₈ H ₇₃ N ₆ O ₈ ⁺ : 1101.5484, found 1101.5344.

3. Application example of probe SnP1

Solution preparation:

Metal inorganic salts: lead nitrate, silver nitrate, cadmium nitrate, others are chlorides (K ⁺ , Na ⁺ , Ca ²⁺ , Mg ²⁺ , Ba ²⁺ , Zn ^{2 +} , Fe ²⁺ , Fe ³⁺ , Mn ²⁺ , Cu ²⁺ , Co ²⁺ , Ni ²⁺ , Hg ²⁺ ) and the manufacturers are Aladdin Reagent Company or Tianjin Kemiou Reagent Company. Accurately weigh the corresponding metal salt, dissolve it in high-purity water to prepare a 10mM solution for later use.

Preparation of 1 mM probe solution: accurately weigh the corresponding probe (SnP1), dissolve SnP1 in methanol solution to prepare a 1 mM solution for use.

Fluorescence selectivity experiment:

The fluorescence selectivity was tested using a fluorescence spectrometer. As shown in Figure 4, the single probe SnP1 (10 μM) has a weak fluorescence emission intensity in CH ₃ OH-H ₂ O (99/1, v/v) solution, when Sn ⁴⁺ (10 equivalents) is added Afterwards, the fluorescence emission intensity was significantly enhanced, but when other metal ions (100 μM) were added, only the solution system with Fe ³⁺ , Sn ²⁺ , and Cr ³⁺ had a certain increase in fluorescence intensity, but the response intensity was relatively low, and the other metal ion systems The fluorescence intensity did not change significantly. The above experimental results show that the probe has good specificity and selectivity for mercury ions.

Fluorescence interference experiment:

In order to test the anti-interference ability of the probe molecule to Sn ⁴⁺ detection, its metal cation interference and anion interference were tested respectively in the fluorescence emission spectrum. As shown in Figure 5, add various metal cations (100 μM) to SnP1 (10 μM) in CH ₃ OH-H ₂ O (99/1, v/v) solution to test its fluorescence emission intensity (577nm) , and then add 100 μ M Sn ⁴⁺ solution to the solution containing various metal ions, as can be seen from Figure 5, the fluorescence intensity (577nm) obtained when adding mercury ions in the presence of other metal cations is basically the same as that obtained when mercury ions are added alone Similarly, this result indicates that the probe SnP1 has a strong ability to resist the interference of metal cations in the detection of Sn ⁴⁺ . According to a similar method, its anti-interference ability to common anions (Cl ^- , Br ^- , F ^- , I ^- , CO ₃ ^2- , NO ₃ ^- , PO ₄ ^3- , SO ₄ ^2- ) was tested, as shown in the attached As shown in Figure 6, the probe SnP1 has a strong ability to resist anion interference in the detection of Sn ⁴⁺ .

Claims (7)

1. a kind of Sn based on double rhodamines⁴⁺Fluorescent probe molecule, it is characterised in that the following institute of the fluorescent probe structural formula Show：

2. the Sn based on double rhodamines as described in right 1 is required⁴⁺The preparation method of fluorescent probe molecule, it is characterised in that：It The step of it is as follows：

(1) rhodamine B and ethanolamine are stirred in ethanol solution, is heated to reflux to fully reaction, question response is cooled down completely afterwards Reaction system is dissolved with ethyl acetate, is washed with pure water and saturated nacl aqueous solution, organic faciess to room temperature, removal of solvent under reduced pressure With anhydrous sodium sulfate drying, filter, removal of solvent under reduced pressure, the isolated intermediate product RhB-OH of silica gel column chromatography；

(2) intermediate product RhB-OH is mixed in dichloromethane solution with triethylamine, under condition of ice bath, is added dropwise to isophthalic diformazan The dichloromethane solution of acyl chlorides, is changed to room temperature reaction after completion of dropping, after reaction completely, saturated nacl aqueous solution washing is organic Anhydrous sodium sulfate drying is mutually used, is filtered, removal of solvent under reduced pressure, with dichloromethane and methanol as detergent, Jing silica gel column chromatographies The isolated Sn containing double rhodamines⁴⁺Fluorescent probe molecule SnP1.

3. the Sn based on double rhodamines as claimed in claim 2⁴⁺The preparation method of fluorescent probe molecule, it is characterised in that：Institute State in step (1), rhodamine B is 1 with the ratio of the amount of the material of ethanolamine:4, described to be heated to reflux temperature for 80-90 DEG C, reaction Time is 8-15 hours.

4. the Sn based on double rhodamines as claimed in claim 2⁴⁺The preparation method of fluorescent probe molecule, it is characterised in that：Institute State in step (1), the detached eluent of silicagel column is CH₂Cl₂And CH₃OH, the CH₂Cl₂With CH₃The volume ratio of OH is 10-18: 1, yield is 60-85%.

5. the Sn based on double rhodamines as claimed in claim 2⁴⁺The preparation method of fluorescent probe molecule, it is characterised in that：Institute State in step (2), compound R hB-OH is 2-4 equivalents, and m-phthaloyl chloride is 1 equivalent, and triethylamine is 2-5 equivalents, and room temperature is anti- It is 6-10h between seasonable.

6. the Sn based on double rhodamines as claimed in claim 2⁴⁺The preparation method of fluorescent probe molecule, it is characterised in that：Institute State in step (2), the detached eluent of silicagel column is CH₂Cl₂And CH₃OH, the CH₂Cl₂With CH₃The volume ratio of OH is 10-20: 1, yield is 60-75%.

7. the Sn based on double rhodamines as claimed in claim 1⁴⁺Fluorescent probe molecule is in detection Sn⁴⁺In application.