Preparation method of chiral ultra-sensitive magnetic resonance probe and application of chiral ultra-sensitive magnetic resonance probe in chiral amino acid identification

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a preparation method of a chiral ultra-sensitive magnetic resonance probe and application of the chiral ultra-sensitive magnetic resonance probe in chiral amino acid identification. The chiral ultra-sensitive magnetic resonance probe is obtained by carrying out post-synthesis modification on a 129Xe molecular cage NiZn-ZIF-8 by virtue of a chiral histidine ligand. The < 129 > Xe molecular cage NiZn-ZIF-8 is synthesized from 2-methylimidazole, zinc acetate dihydrate and nickel acetate tetrahydrate, and the modified < 129 > Xe molecular cage NiZn-ZIF-8H, namely the chiral ultra-sensitive magnetic resonance probe, is further obtained through post-synthesis modification of a histidine ligand. After the novel NiZn-ZIF-8H probe adsorbs chiral amino acid, chemical shift different from that of NiZn-ZIF-8H appears in a hyperpolarized 129Xe CEST MRI spectrogram, and the novel NiZn-ZIF-8H probe can be used for identifying the chiral amino acid.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for preparing a chiral ultrasensitive magnetic resonance probe and an application thereof in identification of chiral amino acids. Background Art

[0002] 129 Xe is a non-toxic inert gas that is very sensitive to the chemical environment, has large chemical shift changes, has good lipid solubility, and can interact with a variety of proteins, phospholipids, spores, etc., making it very suitable for use in the biomedical field. 129 After Xe is hyperpolarized, 129 The nuclear magnetic resonance signal of Xe is greatly enhanced, with a sensitivity 60,000 times higher than that of the thermally polarized state, making it useful as a contrast agent for magnetic resonance imaging of the lungs or brain in animals and humans. 129 Xe technology not only inherits the advantage of non-destructive magnetic resonance detection, but also makes up for the disadvantage of low sensitivity of traditional magnetic resonance, providing a very promising technology for detecting ultra-low concentration chemical or biological molecules. 129 Xe directly used as a molecular probe has the disadvantages of being non-targeted and having an exchange rate that is too fast to be detected. In order to overcome these shortcomings and make full use of the advantages of Xe, it is necessary to design specific Xe cage molecules that have a good affinity for Xe and capture free Xe through molecular cages to achieve specific sites. 129 Xe MRI. Considering that in 2020, Zhou Xin's group found that the metal organic framework ZIF-8 can be used as a widely used cage molecule, it also has a certain affinity for Xe, and confirmed that ZIF-8 can also be used as 129 The main molecule of Xe. The current identification of chiral amino acids mainly relies on high-resolution liquid chromatography-mass spectrometry, which has a certain effect on chiral detection, but its use process requires the use of multiple solvents for mobile phase elution, the test process is cumbersome, and it is easy to cause environmental pollution. Traditional optical techniques such as circular dichroism have the characteristics of short optical path, and the experimental results are very sensitive to sample concentration, temperature and buffer conditions, and the signal is easily affected. Therefore, the present invention selects a better signal 129 Xe magnetic resonance probe NiZn-ZIF-8 molecular cage, on this basis, further through the exchange of chiral histidine ligands, to prepare NiZn-ZIF-8H probes of different chirality, through the Hyper-CEST technology for the study of chiral amino acid identification. Summary of the invention

[0003] The existing NiZn-ZIF-8 itself is composed of 2-methylimidazole coordinated with zinc ions and nickel ions. After forming Ni-doped ZIF-8, 129 Xe signal strength is improved and129 Xe is relatively stable in a continuous ventilation environment and has been used in in vitro and cell studies. Based on previous research, the applicant used NiZn-ZIF-8 as 129 The main molecule of Xe was exchanged with the chiral histidine ligand to obtain a chiral molecular probe, which was then tested with Hyper-CEST technology and found to have good 129 Xe NMR signal, and 129 The Xe signal intensity shows different changing trends due to the influence of the chirality of histidine and can be used to distinguish chiral amino acids.

[0004] In order to achieve the above invention object, the present invention adopts the following technical scheme:

[0005] A chiral ultra-sensitive magnetic resonance probe, the chiral ultra-sensitive magnetic resonance probe is composed of 129 The Xe molecular cage NiZn-ZIF-8 is obtained by post-synthesis modification of a histidine ligand, wherein the histidine is D-histidine or L-histidine.

[0006] The present invention also provides a method for preparing the chiral ultrasensitive magnetic resonance probe, which specifically comprises the following steps:

[0007] 1) Synthesis of NiZn-ZIF-8:

[0008] Under stirring, a methanol solution of 2-methylimidazole is added to a methanol solution of zinc acetate dihydrate and nickel acetate tetrahydrate, and the reaction is completed by continuous stirring at room temperature (preferably the reaction time is 3 hours). After the reaction is completed, the mixture is allowed to stand at room temperature (preferably the standing time is 24 hours), and then centrifuged and washed (preferably washed three times with methanol), and the obtained precipitate is vacuum dried to obtain NiZn-ZIF-8;

[0009] 2) Synthesis of NiZn-ZIF-8H probe:

[0010] The dried NiZn-ZIF-8 obtained in step 1) is uniformly dispersed in water to obtain a NiZn-ZIF-8 suspension. Under stirring, an aqueous solution of histidine is added to the NiZn-ZIF-8 suspension, and the reaction is continued at room temperature overnight, followed by centrifugation and washing (preferably three times with water). Finally, the obtained solid is dispersed in ultrapure water to obtain a NiZn-ZIF-8H probe aqueous solution, i.e., an aqueous solution of the chiral ultrasensitive magnetic resonance probe.

[0011] Furthermore, in the step 1), the molar ratio of zinc acetate dihydrate to nickel acetate tetrahydrate is 5:1, and the molar ratio of 2-methylimidazole to zinc acetate dihydrate is 8:1.

[0012] Furthermore, in the step 2), the mass ratio of the dried NiZn-ZIF-8 to the added amount of histidine is (2.76-5.52):1, and the preferred mass ratio is 2.76:1.

[0013] Furthermore, the vacuum drying condition in step 1) is vacuum drying at 50° C. for 12 h.

[0014] The present invention also provides application of the chiral ultrasensitive magnetic resonance probe in identification of D-proline and / or L-proline.

[0015] Further, the application is based on the chiral ultrasensitive magnetic resonance probe after adsorbing different chiral prolines. 129 The chemical shift of Xe CEST MRI signal is used to identify different chiral prolines: the chiral proline aqueous solution to be detected is added to the chiral ultrasensitive magnetic resonance probe aqueous solution for adsorption, and then 129 Xe CEST MRI spectrum detection, based on the fact that after the chiral ultrasensitive magnetic resonance probe adsorbs heterochiral proline, the chemical shift of the probe signal peak shifts more toward the low field than after the adsorption of homochiral proline, different chiral amino acids can be identified.

[0016] Furthermore, the specific operations of the application are:

[0017] When it is necessary to determine whether a chiral proline sample is D-proline or L-proline, first prepare two chiral ultrasensitive magnetic resonance probes corresponding to histidine D-histidine and L-histidine; add the chiral proline aqueous solution to be identified to the aqueous solutions of the two chiral ultrasensitive magnetic resonance probes respectively, shake on a shaker until evenly distributed, and then perform 129 Xe CESTMRI spectrum detection, according to the chemical shift of the signal peak after adsorption of heterochiral proline, is more shifted to the low field than after adsorption of homochiral proline, so as to judge whether the chiral proline sample is D-proline or L-proline;

[0018] When two samples are known to be D-proline and L-proline, and they need to be accurately identified, firstly, a chiral ultrasensitive magnetic resonance probe corresponding to histidine D-histidine or L-histidine is prepared according to the preparation method of the chiral ultrasensitive magnetic resonance probe; the chiral proline aqueous solution to be identified is added to the chiral ultrasensitive magnetic resonance probe aqueous solution, and the mixture is shaken on a shaker for uniformity. 129 Xe CEST MRI spectrum detection determines whether the chiral proline sample is D-proline or L-proline based on the fact that the chemical shift of the signal peak after adsorption of heterochiral proline is more offset to the low field than that after adsorption of homochiral proline.

[0019] Furthermore, the concentration of the chiral proline aqueous solution to be detected is not less than 10 mM; and the shaking time of the shaking table in the above application is not less than 1 hour.

[0020] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0021] The NiZn-ZIF-8 selected in the present invention has good chiral discrimination ability after chiral modification. The probe material is doped with metal Ni. 129 The Xe signal intensity is further improved, and combined with Hyper-CEST technology, the chiral probe adsorbs chiral amino acids to obtain 129 Xe NMR signal, the probe signal peak has obvious chemical shift changes, which can be used to identify D-proline and L-proline. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 TEM images of NiZn-ZIF-8H probes of different chirality in Example 1;

[0023] Figure 2 Circular dichroism spectra of NiZn-ZIF-8H probes of different chirality in Example 1;

[0024] Figure 3 The NiZn-ZIF-8H probes with different chirality in Example 2 in aqueous solution 129 Xe spectrum;

[0025] Figure 4 This is the Hyper-CEST test spectrum of the NiZn-ZIF-8H probes with different chirality in Example 2 after adsorbing chiral proline for 1 hour. DETAILED DESCRIPTION

[0026] The following is a detailed description of the embodiments and the attached Figure 1 to Figure 4 , the technical solution of the present invention is described in detail.

[0027] The main reagents and material sources used in the following Examples 1-3 are as follows:

[0028] Zinc acetate dihydrate, nickel acetate tetrahydrate, 2-methylimidazole, D-histidine, L-histidine, D-proline, L-proline, and methanol were all purchased from Sinopharm Group and were of analytical grade.

[0029] Unless otherwise specified, the above reagents were used directly without further purification.

[0030] All water used was ultrapure water.

[0031] Example 1 Synthesis of chiral ultrasensitive magnetic resonance probe, the specific steps are as follows:

[0032] 1) Synthesis of NiZn-ZIF-8:

[0033] Zinc acetate dihydrate (1 mmol) and nickel acetate tetrahydrate (0.2 mmol) were placed in a 250 mL conical flask, and 50 mL of methanol was added thereto, and the mixture was sonicated until clear and transparent to obtain an acetate solution; 2-methylimidazole (8 mmol) was placed in another 250 mL conical flask, and 50 mL of methanol was added thereto, and the mixture was sonicated until clear and transparent to obtain a 2-methylimidazole solution. Then, the 2-methylimidazole solution was added to the acetate solution while stirring, and after being evenly mixed and stirred at room temperature (25°C, the same below) for 3 hours, the magnet was taken out, the conical flask was sealed and allowed to stand at room temperature for 24 hours; after standing, it was centrifuged at a speed of 12000 rpm in a high-speed centrifuge, and the supernatant was removed to obtain a lavender precipitate; the lavender precipitate was then washed with methanol to remove the unreacted ligand 2-methylimidazole, and the supernatant was removed by centrifugation after washing; the lavender precipitate was centrifuged and washed three times under the same conditions, and finally the lavender precipitate was placed in a double-row tube and vacuum dried at 50°C for 12 hours to remove the residual organic solvent inside the pores to obtain NiZn-ZIF-8.

[0034] 2) Post-synthesis modification method for synthesis of NiZn-ZIF-8H probe:

[0035] 60 mg of NiZn-ZIF-8 obtained in step 1) was uniformly dispersed in 10 mL of water, and ultrasonically treated at 30 W for 30 min to obtain a 6 mg / mL NiZn-ZIF-8 suspension. After adding 10 mL of 2.17 mg / mL D-histidine aqueous solution under stirring at 1500 rpm, the reaction was continued at room temperature overnight (the overnight time in the present invention is 12 h). After the reaction was completed, the supernatant was removed by centrifugation at 12,000 rpm in a high-speed centrifuge, and the lower white solid was retained. The white solid was washed three times by centrifugation with water (centrifuged at 12,000 rpm, the same below), and finally the obtained white precipitate was dispersed in ultrapure water to obtain a D-type NiZn-ZIF-8H probe aqueous solution.

[0036] The above D-histidine aqueous solution was replaced with an L-histidine aqueous solution of the same volume and concentration, and the L-type NiZn-ZIF-8H probe aqueous solution was prepared in the same way.

[0037] The NiZn-ZIF-8H probe aqueous solution prepared in this example (NiZn-ZIF-8H concentration 0.5 mg / mL) was scanned with a high-resolution transmission electron microscope. The obtained high-resolution transmission electron microscope image is as follows: Figure 1 As shown, from Figure 1 It can be seen that the synthesis of D-type NiZn-ZIF-8H ( Figure 1Left) and L-type NiZn-ZIF-8H ( Figure 1 The particle sizes of D-type NiZn-ZIF-8H and L-type NiZn-ZIF-8H are 171.95±5.84nm and 165.32±7.78nm respectively. The D-type NiZn-ZIF-8H is angular, while the morphology of L-type NiZn-ZIF-8H has changed to a certain extent. It is speculated that the exchange degree between L-histidine and 2-methylimidazole ligand is high, so the morphology and pore size of the probe surface have changed to a certain extent.

[0038] Figure 2 The circular dichroism spectrum of the NiZn-ZIF-8H probe prepared in this example is Figure 2 It can be seen that in the range of 165-175 nm, the optical rotation of the D-NiZn-ZIF-8H probe is positive, and the optical rotation of the L-NiZn-ZIF-8H is negative, indicating that two chiral NiZn-ZIF-8H probes were successfully prepared.

[0039] Example 2 Chiral ultrasensitive magnetic resonance probe in aqueous solution 129 The specific steps for testing the Xe spectrum are as follows:

[0040] 129 Xe NMR experiments were performed on a 400 MHz (9.4 T) Bruker AV400 wide-aperture spectrometer (Bruker Biospin, Ettlingen, Germany) equipped with a micro-imaging gradient coil, with the RF pulse frequency of the Xe nuclei being 110.7 MHz. 129 Xe NMR spectroscopy was performed using a 10 mm double resonance probe ( 129 Xe and 1 H, PA BBO 400W1 / S2 BB-HD-10Z), rectangular pulses with flip angle (90°). Hyperpolarized light was generated by spin exchange optical pumping using a continuous flow polarization device. 129 Xe gas. The nuclear spin polarization is about 20%. It is composed of 10% N2, 88% He and 2% Xe (Xe is 86% enriched) by volume. 129 Xe or natural abundance 129 Xe, the natural abundance used in this example 129 The mixed gas consisting of Xe) was directly passed into a 10 mm NMR tube for 20 seconds, and then waited for 3 seconds to ensure that the generated bubbles were completely broken before collecting the signal. The sample temperature was set to 298K on the NMR spectrometer.

[0041] According to the preparation method of Example 1, the mass ratio of NiZn-ZIF-8 to histidine was changed to 2.76:1, 4.15:1, and 5.52:1, respectively, to prepare 2 mL of 5 mg / mL D / L NiZn-ZIF-8H aqueous solution, vortexed for 5 min, and then the above129 Xe NMR experiment: The chemical shift range of continuous (CW) saturation pulse scanning is δ=30-250 ppm.

[0042] In this embodiment, the probe is measured in aqueous solution. 129 The Xe spectrum results are as follows Figure 3 As shown, the mass ratio of NiZn-ZIF-8 to histidine added from top to bottom is 2.76:1, 4.15:1, 5.52:1. The integrated area is normalized and the integrated ratio is found to be D-NiZn-ZIF-8H:L-NiZn-ZIF-8H=2.3, 1.75, 1.61. In view of the greater difference between the two chiral probes, the more conducive to the subsequent identification of different chiral prolines, the preferred mass ratio of NiZn-ZIF-8 to histidine added is 2.76:1, and this mass ratio is selected for subsequent research.

[0043] Example 3 Probe prepared in Example 2 after adsorption of chiral proline 129 The specific steps for testing the Xe CEST spectrum are as follows:

[0044] 129 Xe NMR experiments were performed on a 400 MHz (9.4 T) Bruker AV400 wide-aperture spectrometer (Bruker Biospin, Ettlingen, Germany) equipped with a micro-imaging gradient coil, with the RF pulse frequency of the Xe nuclei being 110.7 MHz. 129 Xe NMR spectroscopy was performed using a 10 mm double resonance probe ( 129 Xe and 1H, PA BBO 400W1 / S2 BB-HD-10Z), rectangular pulses with flip angle (90°). Hyperpolarized light was generated by spin exchange optical pumping using a continuous flow polarization device. 129 Xe gas. The nuclear spin polarization is about 20%. It is composed of 10% N2, 88% He and 2% Xe (Xe is 86% enriched) by volume. 129 Xe or natural abundance 129 Xe, the natural abundance used in this example 129 The mixed gas consisting of Xe) was directly passed into a 10 mm NMR tube for 20 seconds, and then waited for 3 seconds to ensure that the generated bubbles were completely broken before collecting the signal. The sample temperature was set to 298K on the NMR spectrometer.

[0045] The D / L NiZn-ZIF-8H aqueous solution with a mass ratio of NiZn-ZIF-8 to histidine of 2.76:1 prepared in Example 2 was prepared into 2 mL of D-NiZn-ZIF-8H aqueous solution with a test concentration of 25 μg / mL and L-NiZn-ZIF-8H aqueous solution with a test concentration of 50 μg / mL, and 10 μL of 10 mM D / L proline solution was added to each of them. After shaking on a shaker at room temperature for 1 h, the mixture was stirred for 2 h. 129 XeCEST spectrum test: The chemical shift range of continuous (CW) saturation pulse scanning is δ=71-101 ppm.

[0046] The measured value in this embodiment 129 The Xe spectrum results are as follows Figure 4 After the NiZn-ZIF-8H probe adsorbed homochiral proline, the signal weakened and the chemical shift shifted from 84ppm to 85ppm. After the NiZn-ZIF-8H probe adsorbed heterochiral proline, the signal weakened and the chemical shift shifted from 84ppm to 88ppm, which showed obvious differences, indicating that the NiZn-ZIF-8H probe prepared in Example 1 can be used according to 129 The chemical shift of Xe CEST MRI signal is used to identify different chiral prolines. The possible principle is that NiZn-ZIF-8 itself is a porous structure. After modification with chiral groups, the adsorption of homochiral amino acids is weaker, while the adsorption of heterochiral amino acids is stronger. Proline itself is a cyclic amino acid. After adsorbing heterochiral proline, it will further bind to the pores of NiZn-ZIF-8, thereby affecting the exchange of Xe. This can be detected by ultrasensitive 129 Xe magnetic resonance is used for characterization. In practical applications, Hyper-CEST is mainly judged by the strength of the CEST signal and its chemical shift offset. After the probe is adsorbed with different chiral prolines, the signal is weakened to a certain extent, so it is judged based on its chemical shift. Figure 4 It can be seen that after the NiZn-ZIF-8H probe adsorbs heterochiral proline, its chemical shift shifts to the low field more obviously than after the adsorption of homochiral proline, thereby distinguishing different chiral amino acids.

[0047] Conclusion: The chiral-modified NiZn-ZIF-8H has obvious chiral distinction. The nuclear magnetic resonance experiment test shows that the two chiral probes have good 129 The Hyper-CEST technique further demonstrated that chiral NiZn-ZIF-8H has a good ability to distinguish chiral proline and has application prospects in the chiral identification of amino acids.

Claims

1. A chiral ultrasensitive magnetic resonance probe, the chiral ultrasensitive magnetic resonance probe is composed of 129 The Xe molecular cage NiZn-ZIF-8 is obtained by post-synthesis modification of a histidine ligand, wherein the histidine is D-histidine or L-histidine.

2. A method for preparing the chiral ultrasensitive magnetic resonance probe according to claim 1, characterized in that: The preparation method specifically comprises the following steps: 1) Synthesis of NiZn-ZIF-8: Under stirring, a methanol solution of 2-methylimidazole is added to a methanol solution of zinc acetate dihydrate and nickel acetate tetrahydrate, and the mixture is continuously stirred at room temperature until the reaction is complete. After the reaction is completed, the mixture is allowed to stand at room temperature, and then centrifuged, washed, and vacuum dried to obtain NiZn-ZIF-8; 2) Synthesis of NiZn-ZIF-8H probe: The dried NiZn-ZIF-8 obtained in step 1) is uniformly dispersed in water to obtain a NiZn-ZIF-8 suspension. Under stirring, an aqueous solution of histidine is added to the NiZn-ZIF-8 suspension, and the reaction is continued at room temperature overnight, followed by centrifugation and washing. Finally, the obtained solid is dispersed in ultrapure water to obtain a NiZn-ZIF-8H probe aqueous solution, i.e., an aqueous solution of the chiral ultrasensitive magnetic resonance probe.

3. The preparation method according to claim 2, characterized in that: In the step 1), the molar ratio of zinc acetate dihydrate to nickel acetate tetrahydrate is 5:1, and the molar ratio of 2-methylimidazole to zinc acetate dihydrate is 8:

1.

4. The preparation method according to claim 2, characterized in that: In the step 2), the mass ratio of the dried NiZn-ZIF-8 to the added amount of histidine is (2.76-5.52):

1.

5. The preparation method according to claim 2, characterized in that: The vacuum drying condition in step 1) is vacuum drying at 50° C. for 12 h.

6. Use of the chiral ultrasensitive magnetic resonance probe according to claim 1 or the chiral ultrasensitive magnetic resonance probe obtained by the preparation method according to any one of claims 2 to 5 in the identification of D-proline and / or L-proline.

7. The use according to claim 6, characterized in that: The application is to detect the chiral ultrasensitive magnetic resonance probe after adsorbing different chiral prolines. 129 The chemical shift of Xe CEST MRI signal is used to identify different chiral prolines: the chiral proline aqueous solution to be detected is added to the chiral ultrasensitive magnetic resonance probe aqueous solution for adsorption, and then 129 Xe CEST MRI spectrum detection can identify different chiral amino acids based on the fact that the chemical shift of the signal peak after the chiral ultrasensitive magnetic resonance probe adsorbs heterochiral proline is more offset to the low field than that after adsorbing homochiral proline.

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