CN103923017B - The basis set propylhomoserin of double; two dansyls and application thereof - Google Patents

Abstract

The present invention discloses a new compound bisdansyl histidine and its preparation method and application. Its chemical formula is shown in formula I: , can be used as a fluorescent probe for iron ion detection; the invention also discloses a method for recovering fluorescence after bisdansyl histidine recognizes iron ions; bisdansyl histidine has good water solubility, is easy to prepare an aqueous solution and has stable fluorescence, It can conveniently detect the concentration of iron ions in aqueous solution; bisdansyl histidine is a highly sensitive fluorescent probe for iron ions, with a detection limit of 2.0×10 ^-7 mol/L for iron ions. 10.35 can recognize iron ions, and common metal ions will not affect the specific recognition of bisdansylhistidine to iron ions; bisdansylhistidine is used as a fluorescent probe for iron ions under acidic conditions, The invention has the characteristics of wide application range, strong identification and good accuracy, and is suitable for popularization and application.

Description

Bisdansylhistidine and its application

technical field

The invention relates to a new compound bisdansyl histidine and its application.

Background technique

Iron is an extremely abundant transition metal element in biological systems and an essential element in biological systems, playing a very important role in the metabolism of organisms. In organisms, ferric iron has the function of transporting heme, and at the same time it is a cofactor for many enzyme reactions. The lack of iron in organisms can cause diseases such as anemia, liver and kidney damage, diabetes and neonatal failure. The detection methods of iron ions generally include atomic absorption spectroscopy, plasma emission spectroscopy, electrochemical methods, colorimetry, gas chromatography, biological and nano sensor means. A common disadvantage of these testing methods is that they require complex and diverse sample preparation and sophisticated experimental instruments, resulting in high analysis costs, and it is difficult to achieve real-time detection. The other is the fluorescent probe method to identify metal ions. In fluorescent probes, conjugated aromatic rings are often used as fluorescent groups, which can easily lead to poor water solubility of the probe and make it difficult to detect metal ions in aqueous solution.

Contents of the invention

One object of the present invention is to provide a new compound named bisdansylhistidine.

Another object of the present invention is to provide the application of bis-dansyl histidine as a fluorescent ion probe for iron ions.

Another object of the present invention is to provide a method for detecting iron ions based on bis-dansyl histidine.

The technical scheme that the present invention takes is:

Didansyl histidine, its chemical formula is shown in formula I:

.

The preparation method of bis-dansyl histidine uses dansyl chloride and histidine as raw materials for synthesis.

Further, the preparation method of bisdansyl histidine, its specific synthesis steps are:

1) Dissolve histidine with sodium bicarbonate solution, and place it in an ice-salt bath;

2) Dissolve dansyl chloride in acetonitrile, add it to the above solution containing histidine, and stir while adding dropwise. After the dropwise addition, continue to stir and react in an ice-salt bath for 30 minutes, and then place it at 18-25°C down to complete reaction;

3) Remove acetonitrile by distillation under reduced pressure, then wash with ether for 2 to 3 times, collect the water layer, adjust its pH value to 4.3 to 4.7, then extract with ethyl acetate, collect the ethyl acetate layer, and use vacuum distillation to extract It was evaporated to dryness to obtain a crude product, and then recrystallized with ether and dichloromethane to obtain bisdansyl histidine.

Further, the molar ratio of dansyl chloride to histidine in the above step 2) is 1: (5-7).

Application of bisdansylhistidine in the detection of iron ions.

A detection method for iron ion concentration, comprising the following steps:

1) Making a standard curve: adding a standard concentration of iron ion solution into the bisdansyl histidine solution according to claim 1, recording the amount of iron ion solution added and the fluorescence change of the bisdansyl histidine solution;

2) Add the solution to be tested into the bisdansyl histidine solution, and record the fluorescence change of the bis dansyl histidine solution;

3) Calculate the concentration of iron ions in the liquid to be tested.

A method for detecting the concentration of iron ions, comprising gradually adding the liquid to be tested into a bisdansyl histidine solution of known concentration until the fluorescence quenching of the bis dansyl histidine solution reaches the maximum value, according to the liquid to be tested The amount of addition and the concentration of the bisdansyl histidine solution, calculate the concentration of iron ions in the solution to be tested.

Further, the pH value of the bisdansyl histidine solution in the above iron ion concentration detection method is 2.5-10.35.

Further, the pH value of the bisdansyl histidine solution in the above iron ion concentration detection method is 4.5-5.5.

The beneficial effects of the present invention are:

1) The bisdansyl histidine of the present invention has good water solubility, can be easily formulated into an aqueous solution, and can conveniently detect the concentration of ferric ions in the aqueous solution. It is a sensitive fluorescent detector for identifying ferric ions. Needle.

2) In the present invention, when bisdansyl histidine is used to detect ferric ions, the pH range of the solution is 2.5-10.35, which can still show the recognition function of the probe, which has a wide application range and high accuracy.

3) The bisdansyl histidine of the present invention exhibits a fluorescent response signal to ferric ions: when the excitation wavelength is 330 nm, the fluorescence spectrum of bis dansyl histidine increases with the concentration of ferric ions Exhibits strong fluorescence quenching.

4) The present invention uses a fluorescent probe to directly detect iron ions in aqueous solution, and has the advantages of strong operability, high sensitivity, and good selectivity.

Description of drawings

The anion mass spectrometry test result of Fig. 1 bisdansyl histidine;

The NMR test result of Fig. 2 bisdansyl histidine;

Figure 3 is the change curve of fluorescence intensity difference before and after adding the same amount of Fe ³⁺ to bisdansyl histidine (concentration: 2.0×10 ^-6 mol/L) at different pH values (excitation wavelength: 330nm);

Figure 4 is a diagram of fluorescence changes when different concentrations of Fe ³⁺ are added to bisdansyl histidine solution (concentration: 2.0×10 ^-6 mol/L) at pH 5.0;

Figure 5 shows the fluorescence phenomenon of 2.0×10 ^-6 mol/L bisdansyl histidine solution containing iron ions (right picture) and no iron ion (left picture) under 365nm ultraviolet light;

Figure 6 shows when the pH value is 5.0 and the concentration of 2.0×10 ^-6 mol/L bisdansyl histidine solution contains 0.0, 2.0, 3.0, 5.0, 10.0 (×10 ^-7 mol/L) iron ions respectively Fluorescence intensity change situation, wherein I ₀ is the fluorescence intensity value when 0.0 μ M iron ion, and I represents the fluorescence intensity value when different concentrations of iron ion exist;

Figure 7 shows that when the pH is 5.0, 8.0×10 ^-5 mol/L of Ca ²⁺ , Cu ²⁺ , and Fe ² were added dropwise to the bisdansyl histidine solution (concentration: 2.0×10 ^-6 mol/L). ⁺ , Hg ²⁺ , K ⁺ , Mg ²⁺ , Pb ²⁺ , Zn ²⁺ , Ba ²⁺ , Co ²⁺ , Cd ²⁺ , Na ⁺ common metal ions, their fluorescence emission intensity at 501nm (excitation wavelength 330nm);

Fig. 8 is when the pH is 5.0, the bisdansyl histidine solution (0), the solution containing bisdansyl histidine and Fe ³⁺ (1), and the bisdansyl histidine and Fe ³⁺ solution respectively Contains Hg ²⁺ (2), Pb ²⁺ (3), Ba ²⁺ (4), K ⁺ (5), Mn ²⁺ (6), Ca ²⁺ (7), Zn ²⁺ (8), Fluorescence emission from Ni ²⁺ (9), Fe ²⁺ (10), Cu ²⁺ (11), Na ⁺ (12), Mg ²⁺ (13), Cd ²⁺ (14), Co ²⁺ (15) Intensity (excitation wavelength is 330nm);

Figure 9 shows the recovery of fluorescence after the concentration of bisdansyl histidine is 2.0×10 ^-6 mol/L and the concentration of iron ion is 2.0×10 ^-5 mol/L, and the concentration of sodium phosphate is 1.0×10 ^-4 mol/L Figure (excitation wavelength is 330nm); a represents the fluorescence spectrum of bisdansyl histidine solution, b represents the fluorescence spectrum after adding FeCl ₃ solution to a, c represents the fluorescence spectrum after adding sodium phosphate to b.

detailed description

The present invention will be further described below in conjunction with specific implementation, but it is not limited thereto.

Embodiment 1: the synthesis of bisdansyl histidine

The synthetic route of bis dansyl histidine is as follows:

1) Add 0.1245g histidine (0.65mmol) into a round bottom flask, take 15.0ml of sodium bicarbonate solution with a concentration of 0.10mol/L to dissolve it, and put it in an ice-salt bath to cool for 10min;

2) Dissolve 0.035g of dansyl chloride (0.13mmol) in 5.0mL of acetonitrile, and add the solution of dansyl in acetonitrile to the above histidine solution dropwise with a separatory funnel, and stir the solution while adding. The reaction system was stirred and reacted in the ice-salt bath for 30 minutes, then withdrawn from the ice-salt bath, and continued to react at room temperature for 3 hours. During the reaction, the reaction progress was monitored by thin layer chromatography (TLC) until the reaction was completed. ;

3) The reaction mixture was distilled out of acetonitrile by vacuum distillation, and then 20.0 mL of ether was added to the remaining liquid to wash three times to remove unreacted dansyl chloride. Then hydrochloric acid is added dropwise in the aqueous layer solution to adjust the pH value to about 4.5, then ethyl acetate is added to the solution for multiple extractions, the ethyl acetate layer is collected, and evaporated to dryness by vacuum distillation to obtain a crude product, which is then used Recrystallized from ether and dichloromethane. 23.1 mg of the product was obtained with a yield of 57.18%.

Mass spectrometry detection results of bisdansylhistidine: ESI-MSm/z is 620.64, as shown in Figure 1, the relative molecular mass of bisdansylhistidine (C ₃₀ H ₃₀ N ₅ O ₆ S ₂ ) is 621.73, The two basically match.

The chemical shifts and corresponding proton types in the ¹ HNMR spectrum (400MHz, CDCl ₃ ) of bis-dansylhistidine were tested by NMR: 8.66-8.68 (d, 1H), 8.51-8.53 (d, 1H), 8.16 -8.26(d, 3H), 8.01-8.07(d, 1H), 7.97(s, 1H), 7.52-7.61(m, 3H), 7.44-7.47(t, 1H), 7.18-7.21(t, 2H) , 6.87 (s, 1H), 4.08-4.14 (m, 1H, -CH ₂ -), 3.97-3.98 (s, 1H, -CH-), 2.87 (m, 13H, -CH ₃ ), as shown in Figure 2 Show. (wherein, s represents a singlet, d represents a doublet, t represents a triplet, m represents a multiplet, and the number in front of "H" represents the number of hydrogen protons).

Embodiment 2: iron ion concentration detection method

(1) Effect of pH value on recognition of iron ions by bisdansylhistidine

Add bisdansyl histidine into aqueous solutions with different pH values, prepare bisdansyl histidine solutions with different pH values but the concentration is 2.0×10 ^-6 mol/L, record the fluorescence intensity values respectively, and then add Add an equal amount of FeCl ₃ solution so that Fe ³⁺ is 2.0×10 ^-5 mol/L, calculate the difference in fluorescence intensity after adding Fe ³⁺ (excitation wavelength is 330nm, emission wavelength is 501nm), and draw a graph, such as As shown in Figure 3, Figure 3 is the change value of the fluorescence intensity of bisdansyl histidine before and after adding Fe ³⁺ in the pH value range from 2.5 to 10.35, where I ₀ represents bis dansyl histidine in the absence of Fe ³⁺ The fluorescence intensity value under the excitation wave (330nm), I represents the fluorescence intensity value of bisdansylhistidine in the presence of Fe ³⁺ .

It can be seen from Figure 3 that the fluorescence intensity of the bisdansyl histidine solution changes greatly when the pH value is 4.5 to 5.5, so the pH value of 4.5 to 5.5 is selected as the interaction between the bis dansyl histidine and iron ions. Experimental environment for spectral detection, and preferably pH 5.0.

(2) Sensitivity experiment

Add iron ions to the bisdansyl histidine aqueous solution with a pH value of 5.0 and a concentration of 2.0×10 ^-6 mol/L, so that the concentrations of iron ions are 2.0, 4.0, 6.0, 8.0, 10.0, 14.0, 16.0, 18.0, 20.0, 24.0, 28.0, 32.0, 36.0, 40.0, 44.0, 50.0, 56.0, 62.0, 68.0, 80.0 (×10 ^-6 mol/L), and respectively detect their fluorescence intensity at the excitation wavelength of 330nm, draw Fluorescence spectrum, the obtained fluorescence spectrum is shown in Figure 4, from which it can be seen that as the concentration of added iron ions increases, the fluorescence emission peak of bisdansyl histidine gradually weakens (as shown by the arrow in the figure) . When the concentration of added iron ions is 80×10 ^-6 mol/L, the fluorescence can reach the maximum quenching, which is about 95.2%. It can be seen from Figure 4 that bisdansyl histidine has a high sensitivity.

Add FeCl ₃ to the bisdansyl histidine solution with a pH value of 5.0 and a concentration of 2.0×10 ^-6 mol/L to make the iron ion concentration 80 μM, and observe the fluorescence phenomenon under 365nm ultraviolet light irradiation, and compare with 2.0×10 ^-6 mol/L bisdansyl histidine solution of iron ions for comparison, as shown in Figure 5, the left is the solution without iron ions, and the right is the solution with iron ions, as can be seen from Figure 5 After adding iron ions, the fluorescence intensity of bisdansyl histidine solution was obviously weakened.

Add iron ions to the bisdansyl histidine aqueous solution with a pH value of 5.0 and a concentration of 2.0×10 ^-6 mol/L, so that the concentrations of iron ions are 0.0, 2.0, 3.0, 5.0, 10.0 (×10 ^-7 mol/L), record the fluorescence intensity values before and after adding iron ions, calculate the difference in fluorescence intensity after adding Fe ³⁺ (excitation wavelength is 330nm, emission wavelength is 501nm), and draw Figure 6, where I ₀ represents the concentration of metal ions The intensity value of the fluorescence emission peak of bisdansyl histidine at 0.0 μM, I represents the intensity value of the fluorescence emission peak of bis dansyl histidine in the presence of different concentrations of metal ions. It can be seen from Figure 6 that when the iron ion in the bisdansylhistidine solution is 2.0×10 ^-7 mol/L, the fluorescence intensity is quenched obviously, and the bisdansylhistidine exhibits high sensitivity to iron ions , and the detection limit is about 2.0×10 ^-7 mol/L.

In summary, the bisdansyl histidine solution has a high sensitivity for the detection of iron ions.

(3) Detection of iron ion concentration:

1) Make a standard curve: Add the standard concentration of iron ion solution to the bisdansyl histidine solution (the operation method is the same as the related experiment in sensitivity detection), record the amount of iron ion solution added and the ratio of the bisdansyl histidine solution Fluorescence variation, make a standard curve;

2) Add the solution to be tested containing iron ions into the bisdansyl histidine solution, and record the fluorescence change of the solution;

3) Calculate the iron ion concentration in the solution to be tested according to the standard curve.

(4) Selective experiment

Repeat the above iron ion fluorescence measurement experiment, carry out the fluorescence test on 14 kinds of common heavy metal ions in a similar way, take the bisdansyl histidine fluorescence emission peak at 501nm when different concentrations of heavy metal ions exist, and draw the peak at 501nm, the results are shown in Figure 7 Show.

It can be seen from Figure 7 that, except that Hg ²⁺ slightly quenches Cd ²⁺ , Cu ²⁺ , and other 13 heavy metal ions have no effect on the fluorescence emission properties of bisdansyl histidine, only Fe ³⁺ can make bisdansyl histidine Acylhistidine exhibits a strong fluorescence quenching effect, and bisdansylhistidine exhibits a very high selective recognition of iron ions.

(5) Anti-interference experiment when bisdansylhistidine fluorescent probe recognizes iron ions

The fluorescence recognition of ^{bisdansylhistidine} to iron ions is hardly affected by metal ions. Take the following experiment as an example: 2.0×10 ^-5 mol/L iron ions were added respectively, and 2.0×10 ^-5 mol/L iron ions and 8.0×10 ^-5 mol/L other 14 common ions (Hg ²⁺ , Pb ²⁺ ,Ba ²⁺ ,K ⁺ ,Mn ²⁺ ,Ca ²⁺ ,Zn ²⁺ ,Ni ²⁺ ,Fe ²⁺ ,Cu ²⁺ ,Na ⁺ ,Mg ²⁺ ,Cd ²⁺ ,Co ²⁺ ), Using 330nm as the excitation wavelength, the fluorescence spectrum of the bisdansylhistidine solution was measured, and the intensity of the fluorescence emission peak at 501nm was plotted against different metal ions. The results are shown in Figure 8. It can be seen from the figure that bisdansylhistidine has a very high selective recognition effect on iron ions, and the presence of other ions will not affect the recognition ability of bisdansylhistidine on iron ions.

In Fig. 8, 0 is the fluorescence intensity value of the bisdansylhistidine solution when no metal ion is added, 1 is the fluorescence intensity after adding iron ions in the bisdansylhistidine solution, and 2 is adding Fe ³⁺ and Hg ²⁺ , 3 is for adding Fe ³⁺ and Pb ²⁺ , 4 is for adding Fe ³⁺ and Ba ²⁺ , 5 is for adding Fe ³⁺ and K ⁺ , 6 is for adding Fe ³⁺ and Mn ²⁺ , 7 is for adding Fe ³⁺ and Ca ²⁺ , 8 is for adding Fe ³⁺ and Zn ²⁺ , 9 is for adding Fe ³⁺ and Ni ²⁺ , 10 is for adding Fe ³⁺ and Fe ²⁺ , 11 is for adding Fe ³⁺ and Cu ^{2 +} , 12 is adding Fe ³⁺ and Na ⁺ , 13 is adding Fe ³⁺ and Mg ²⁺ , 14 is adding Fe ³⁺ and Cd ²⁺ , 15 is adding Fe ³⁺ and Co ²⁺ to bisdansyl histidine Fluorescence intensity value of acid solution, in which the concentration of bisdansyl histidine is 2.0×10 ^-6 mol/L, the concentration of Fe ³⁺ is 2.0×10 ^-5 mol/L, and the concentration of other metal ions is 8.0×10 ^-5 mol/L.

Example 3: Fluorescence recovery of bisdansylhistidine

After continuous experimental exploration, it was found that adding a certain amount of sodium phosphate to the system after the interaction between bisdansylhistidine and Fe ³⁺ can restore the fluorescence intensity of bisdansylhistidine to a large extent, as shown in Figure 9 , where a represents the fluorescence spectrum of bisdansyl histidine solution, b represents the fluorescence spectrum of adding FeCl ₃ solution to a to make the Fe ³⁺ concentration 2.0×10 ^-5 mol/L, c represents the fluorescence spectrum added to b The fluorescence spectrum after adding 1.0×10 ^-4 mol/L sodium phosphate.

As can be seen from Figure 9, when adding Fe ³⁺ 5 times the amount of sodium phosphate (multiple of the amount of substance), the fluorescence intensity of the dansylhistidine solution can be basically restored to the initial state. This shows that the fluorescent probe of dansylhistidine as Fe ³⁺ has good reversibility, which is very beneficial for the repeated use of fluorescent probes and is more conducive to practical applications.

The above embodiments are only preferred cases for introducing the present invention. For those skilled in the art, any obvious changes and improvements made within the scope of not departing from the spirit of the present invention should be regarded as a part of the present invention.

Claims (5)

1. The application of bisdansyl histidine in iron ion detection, the chemical formula of said bisdansyl histidine is shown in formula I: (I). 2. a detection method of iron ion concentration, is characterized in that: comprise the following steps: 1) Making a standard curve: adding a standard concentration of iron ion solution into the bisdansyl histidine solution according to claim 1, recording the amount of iron ion solution added and the fluorescence change of the bisdansyl histidine solution; 2) Add the solution to be tested into the bisdansyl histidine solution, and record the fluorescence change of the bis dansyl histidine solution; 3) Calculate the concentration of iron ions in the liquid to be tested. 3. A detection method for iron ion concentration, comprising gradually adding liquid to be tested in the bisdansyl histidine solution described in claim 1 of known concentration, until the fluorescence quenching amount of the bis dansyl histidine solution When the maximum value is reached, calculate the concentration of iron ions in the test solution according to the added amount of the test solution and the concentration of the bisdansyl histidine solution. 4. The method for detecting iron ion concentration according to claim 2 or 3, characterized in that: the pH value of the bisdansyl histidine solution is 2.5-10.35. 5 . The method for detecting iron ion concentration according to claim 4 , wherein the pH value of the bisdansyl histidine solution is 4.5 to 5.5.