A potential SABRE-enhanced screening method for P-31-containing compounds based on P-31 CEST

Abstract

The present invention provides a potential SABRE-enhanced P-31 compound screening method based on P-31CEST, the method comprising the following steps: (1) preparing a test solution composed of a test compound, a catalyst and a solvent; (2) performing hydrogen spectrum and phosphorus spectrum magnetic resonance detection of the test solution to determine the position of the free test compound P-31 magnetic resonance signal; (3) selecting a P-31CEST pulse sequence, setting experimental parameters, and collecting the CEST Z spectrum of the test solution; (4) using the CEST Z spectrum collected in step (2) to calculate the asymmetric magnetization transfer rate spectrum of the test solution; (5) using a quantitative method to analyze the CEST Z spectrum collected in step (2), obtaining exchangeable signal quantitative information, determining whether the test compound is combined with the catalyst, and screening potential SABRE-enhanced test compounds. The method can simply, quickly and reproducibly screen out P-31 compounds that can be reversibly bound to the catalyst, and provide technical support for SABRE-enhanced P-31 compound condition selection and its potential application.

Description

Technical Field

[0001] The invention belongs to the field of phosphorus (P-31) magnetic resonance technology and analysis and detection technology, and particularly relates to a method for screening potential SABRE-enhanced P-31-containing compounds based on P-31CEST. Background Art

[0002] Phosphorus (P-31) is an essential element for the human body, playing a crucial role in cellular structure, energy metabolism, pH balance, and nutrient metabolism. P-31 magnetic resonance spectroscopy (MRS) can non-invasively detect phosphorus-containing molecules in the human body and has been reported to be used to study brain diseases (such as brain tumors, cerebral ischemia, Alzheimer's disease, Parkinson's disease, epilepsy, and depression) and energy metabolism-related brain function. However, the inherent low sensitivity of MR and the low concentration levels of phosphorus-containing molecules in the body limit its widespread application in vivo.

[0003] Nardi-Schreiber et al. proposed using dynamic nuclear polarization (DNP) technology to significantly improve the magnetic resonance sensitivity of inorganic phosphate P-31 (Nardi-Schreiber A, et al. Nat. Commun., 2017, 8(1):341), but the instrument system is expensive. Based on the signal amplification by reversible exchange (SABRE) technology, the hyperpolarized state of parahydrogen is transferred to the polynuclear atoms of the substrate molecule through reversible exchange, thereby significantly enhancing the magnetic resonance sensitivity. SABRE includes the following steps: dissolving a transition metal catalyst and a target molecule in a solvent, introducing parahydrogen-rich gas, so that the parahydrogen-rich gas forms a reversible coordination complex with the transition metal catalyst and the target molecule, promoting the spin polarization and polarization transfer of the parahydrogen-rich gas, and using the transition metal catalyst to transfer the polarization of the parahydrogen-rich gas to the target molecule, thereby enhancing the magnetic resonance signal of the target nucleus and detecting the enhanced signal of the target molecule. However, the only P-31-containing compound that has been reported to be enhanced by SABRE is triphenylphosphine (Zhivonitko VV, et al. Chem. Commun., 2015, 51(13): 2506-2509). Other SABRE-enhanced P-31-containing compounds are still lacking. In this report, the catalyst Ir(IMes)(COD)Cl and triphenylphosphine were dissolved in deuterated methanol.

[0004] Currently, researchers screen for SABRE-enhancing compounds by comparing changes in nuclear magnetic resonance signals observed in the spectra before and after the introduction of parahydrogen. On the one hand, the testing process involves the construction of ventilation gas lines, gas line pressure increase and decrease, sample transfer, etc., which is inefficient; on the other hand, continuous or multiple ventilation during the testing process may cause the volume of the solvent deuterated methanol to decrease, thereby affecting the accuracy and repeatability of the measurement. Summary of the Invention

[0005] Based on the above, the present invention provides a method for screening P-31-containing compounds for potential SABRE enhancement based on P-31CEST. Under non-ventilated conditions, the P-31CEST Z spectrum is used to determine whether the P-31-containing compound under test can reversibly bind to the catalyst, thereby screening for P-31-containing compounds with potential SABRE enhancement. This method can simply, rapidly, and reproducibly screen for P-31-containing compounds that can reversibly bind to the catalyst, providing technical support for the selection of conditions for SABRE enhancement of P-31-containing compounds under test and their potential applications.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] A method for screening potential SABRE-enhanced P-31-containing compounds based on P-31CEST, characterized by comprising the following steps:

[0008] S1. The test compound containing P-31, a catalyst and a solvent are mixed to prepare a test solution;

[0009] S2. Set the test temperature, perform hydrogen and phosphorus magnetic resonance detection of the test solution, and determine the chemical shift position of the magnetic resonance signal of the test compound P-31;

[0010] S3. The CEST.XP.uT_1 sequence with continuous wave irradiation was selected to collect the P-31CEST Z spectrum;

[0011] S4. Fitting the CEST Z spectrum and calculating the asymmetric magnetization transfer rate spectrum of the test solution according to the formula;

[0012] S5. If an exchangeable signal is observed in step S4, it is determined that the test compound containing P-31 can reversibly bind to the catalyst and is a potential SABRE-enhanced compound; if no exchangeable signal is observed in step S4, steps S2-S5 are repeated after changing the test temperature.

[0013] The catalyst in step S1 is a catalyst for SABRE enhancement, including an iridium-based catalyst; the solvent in step S1 is one of deuterated methanol, toluene, and water.

[0014] In step S1, the molar concentration ratio of the compound to be tested containing P-31 to the catalyst is ≥2:1.

[0015] The test temperature setting range in step S2 is 263K~353K. In step S2, the Bruker zg sequence is used to measure the hydrogen spectrum and phosphorus spectrum of the solution to be tested. The spectrum width SW when measuring the hydrogen spectrum is greater than the chemical shift range of hydrogen, and the spectrum width SW when measuring the phosphorus spectrum is greater than the chemical shift range of P-31. The relaxation recovery time when measuring the hydrogen spectrum and the phosphorus spectrum matches the relaxation time T1 of hydrogen and P-31, respectively.

[0016] The test temperature in step S2 is 310K.

[0017] The number of sampling points when measuring hydrogen spectrum and phosphorus spectrum is 32768 and 65536 respectively.

[0018] The parameters set when the continuous wave irradiation CEST.XP.uT_1 sequence is selected in step S3 to collect the P-31 CEST Z spectrum include: a relaxation recovery time range of 3s-12s, different saturation irradiation intensities, a saturation irradiation intensity range of 1μT to 20μT, a saturation irradiation time range of 1s to 24s, a frequency offset range of -6480Hz to 6480Hz for the saturation radio frequency pulse, a center frequency of the CEST Z spectrum acquisition being the chemical shift position of the magnetic resonance signal of the test compound P-31 measured by the phosphorus spectrum in step S2, and an integration range of the CEST Z spectrum acquisition being: the chemical shift position of the magnetic resonance signal of the test compound P-31 -0.2ppm to the chemical shift position of the magnetic resonance signal of the test compound P-31 +0.2ppm;

[0019] In step S3, the CEST Z spectra under different saturation radio frequency pulse irradiation intensities are obtained by integrating the P-31 magnetic resonance signals of the free test compound.

[0020] In step S3, the relaxation recovery time range is 10 s, the different saturation irradiation intensities range are 6 μT, 7 μT, 8 μT, and 9 μT, the saturation irradiation time is 20 s, and the frequency offset range of the saturation RF pulse is -1652.4 Hz to 4860 Hz, with a step of 48.6 Hz.

[0021] The formula in step S4 is MTR asym =(I off ―I on ) / I0, where I on The signal of the compound P-31 to be tested is measured when the saturated radio frequency pulse irradiation frequency bias is greater than or equal to 0 Hz, I off The saturation RF pulse irradiation frequency bias is I on The signal of the test compound P-31 is measured at a symmetrical position relative to the magnetic resonance signal of the test compound P-31, and I0 is the signal of the test compound P-31 when the saturation radio frequency pulse irradiation intensity is 0.

[0022] If there is a peak with an amplitude ≥ 0.03 in the asymmetric magnetization transfer rate spectrum obtained in step S4, it indicates that there is an exchangeable signal.

[0023] The present invention provides a P-31CEST-based screening method for potential SABRE-enhanced P-31-containing compounds. Compared with existing methods: ① This method adopts a P-31CEST-based method and utilizes the advantages of CEST signal amplification to determine whether the test compound can reversibly bind to the catalyst, providing technical support for SABRE-enhanced P-31 signals and its potential applications; ② This method performs relevant tests under non-ventilated conditions. On the one hand, it avoids continuous or multiple ventilation that leads to a reduction in the solvent volume of the test sample, thereby improving the accuracy and repeatability of the test. On the other hand, researchers who are not equipped with SABRE enhancement instruments can also carry out relevant research to screen for potential SABRE-enhanced P-31-containing compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is a flow chart of the method of the present invention;

[0025] Figure 2 Phosphorus spectra of NaH2PO4 and catalyst deuterated methanol solutions at 310K;

[0026] Figure 3 Schematic diagram of the hydrogen and phosphorus spectrum sampling sequence (zg) and the continuous wave irradiation P-31CEST Z spectrum sampling sequence (CEST.XP.uT_1);

[0027] Figure 4 P-31CEST Z spectra and fitting results of NaH2PO4 and catalyst deuterated methanol solutions at different saturated irradiation intensities at 310K;

[0028] Figure 5 is the asymmetric magnetization transfer rate MTR of the test solution at different saturation irradiation intensities asym Score. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is described in detail below with reference to the accompanying drawings. It should be understood that the embodiments described herein are only used to illustrate and explain the present invention, and are not intended to limit the present invention.

[0030] The main reagents and materials used in the following examples are described as follows: 10 mm NMR tubes were purchased from Wilmad, USA, 400M NMR spectrometer (Bruker, Switzerland), NaH2PO4 and DMSO were purchased from Shanghai Hushi, deuterated methanol was purchased from Adamas, and Ir(IMes)(COD)Cl was purchased from Anhui Dunmao New Materials Technology Co., Ltd.

[0031] All magnetic resonance experiments in the examples were performed on a Bruker 400M nuclear magnetic resonance spectrometer, and the data were processed using Matlab software.

[0032] "Free test compound P-31" refers to a compound containing the phosphorus-31 (P-31) isotope, wherein the P-31 nucleus is located in a molecule or molecule portion that is not irreversibly bound to the catalyst.

[0033] Example 1

[0034] In this example, NaH2PO4 is used as the P-31-containing compound to be tested, Ir(IMes)(COD)Cl is used as a catalyst, where Ir represents iridium, COD is 1,5-cyclooctadiene, IMes is 1,3-bis(2,4,6-trimethylphenyl)imidazol-2-methylene, Cl represents chlorine, Ir(IMes)(COD)Cl is an organometallic complex, and deuterated methanol is used as a solvent.

[0035] S1. Prepare the test solution consisting of the test compound, catalyst and solvent:

[0036] Using NaH2PO4 as the P-31 compound to be tested, Ir(IMes)(COD)Cl as the catalyst, deuterated methanol as the solvent, DMSO and H2O as cosolvents, 2 mL of deuterated methanol solution containing 15 mM NaH2PO4, 6 mM Ir(IMes)(COD)Cl, 20 mM DMSO and 0.5 M H2O was prepared as the test solution.

[0037] S2. Set the test temperature and perform H-spectrum and P-spectrum magnetic resonance detection on the test solution to determine the position of the magnetic resonance signal of the free test compound P-31:

[0038] The test temperature was set to 310K and the Bruker zg sequence (such as Figure 3 The sequence shown on the upper side of , each cycle includes a 90° pulse, and a signal detection window after the 90° pulse, which is used to collect nuclear magnetic resonance signals, as well as the relaxation recovery time before the 90° pulse) for hydrogen spectrum and phosphorus spectrum measurement. When sampling hydrogen spectrum, the Bruker zg sequence parameters are set as follows: spectrum width SW = 20ppm, sampling points TD = 32768, relaxation recovery time D1 = 5s, 90° pulse width is 26μs, 90° pulse power is -13.42dB, sampling number NS = 1. When sampling phosphorus spectrum, the Bruker zg sequence parameters are set as follows: spectrum width SW = 503ppm, sampling points TD = 65536, relaxation recovery time D1 = 10s, 90° pulse width is 30μs, 90° pulse power is -13.62dB, sampling number NS = 4, and the hydrogen spectrum and phosphorus spectrum of the solution to be tested are obtained by sampling ( Figure 2), and determined that the chemical shift of the P-31 magnetic resonance signal of free NaH2PO4 in the phosphorus spectrum of the test solution was -1.62 ppm.

[0039] S3. Select the P-31CEST pulse sequence, set the experimental parameters, and acquire the CEST Z spectrum of the test solution:

[0040] In this step, the P-31 magnetic resonance signal of the free P-31 compound NaH2PO4 to be tested is used as the observation signal, and the CEST.XP.uT_1 (such as Figure 3 The sequence shown on the lower side of the ) is used as the P-31CEST Z spectrum sampling sequence, using a continuous wave saturation radio frequency pulse to irradiate the solution to be tested, and then using a 90° pulse excitation signal to sample, CEST The Z spectrum sampling sequence parameters are set as follows: relaxation recovery time is 10 s, saturation irradiation intensities are set to 6 μT, 7 μT, 8 μT and 9 μT respectively, saturation irradiation time is 20 s, the P-31 signal (-1.62 ppm) of free NaH2PO4 in the phosphorus spectrum of step S2 is set as the signal acquisition frequency f2 (corresponding to the resonance frequency of P-31 in the test solution), the frequency f1 of the saturation RF pulse is set to -1652.4 Hz~4860 Hz, with a step of 48.6 Hz, and CEST data of the test solution at different saturation RF pulse irradiation intensities (6 μT, 7 μT, 8 μT and 9 μT) are collected. By integrating the P-31 magnetic resonance signal of free NaH2PO4 in the integration range of -1.82 ppm to -1.42 ppm, the CEST Z spectra of the test solution at different saturation RF pulse irradiation intensities (6 μT, 7 μT, 8 μT and 9 μT) are obtained, as shown in FIG. Figure 4 The scattered points in exp are shown.

[0041] S4. Fit the CEST Z spectrum and calculate the asymmetric magnetization transfer rate spectrum of the test solution according to the formula:

[0042] The CEST Z spectrum was fitted using the Lorentz function. The fitting results are shown in the figure below. Figure 4 As shown in the fitting line, according to the formula MTR asym =(I off ―I on ) / I0 to calculate the asymmetric magnetization transfer rate MTR asym , where I off is the fitted I / I0 value when the saturated RF pulse irradiation frequency bias is -4860Hz~0Hz with a step of 48.6Hz, I on The saturation RF pulse irradiation frequency bias is I offThe symmetrical position of the P-31 magnetic resonance signal (-1.62ppm) relative to free NaH2PO4, that is, the I / I0 value fitted when the frequency bias is 4860Hz~0Hz and the step is 48.6Hz, I0 is the P-31 signal of the test compound when the saturation irradiation intensity is 0. Since the P-31 signal when the saturation irradiation intensity is 0 is used as the reference signal, I0=1 is taken. The asymmetric magnetization transfer rate spectra under different saturation irradiation intensities (6μT, 7μT, 8μT and 9μT) are calculated as follows Figure 5 shown.

[0043] S5. If an exchangeable signal is observed in step S4, it is determined that the P-31-containing compound to be tested can reversibly bind to the catalyst and is a potential SABRE-enhanced compound; if no exchangeable signal is observed in step S4, steps S2-S5 are repeated after changing the test temperature:

[0044] The asymmetric magnetization transfer rate spectrum ( Figure 5 ), the signal peaks at chemical shifts of 3ppm (1.38ppm in phosphorus spectrum) and 10.5ppm (8.88ppm in phosphorus spectrum) can be clearly observed, and the MTR at 3ppm and 10.5ppm is significantly different when the saturation irradiation intensity is different (6μT, 7μT, 8μT and 9μT). aysm They are 0.2344, 0.2439, 0.2537, 0.2813 and 0.1574, 0.1828, 0.2003, 0.2368, respectively, all of which are greater than 0.03, that is, the P-31-containing compound NaH2PO4 to be tested can reversibly combine with the catalyst Ir(IMes)(COD)Cl, and is a potential P-31-containing compound that can be enhanced by SABRE.

[0045] It should be noted that the specific embodiments described herein are merely illustrative of the present invention. Persons skilled in the art may make various modifications, additions, or substitutions to the described embodiments without departing from the spirit of the present invention or exceeding the scope of the appended claims.

Claims

1. A method for screening potential SABRE-enhanced P-31-containing compounds based on P-31CEST, characterized in that: The following steps are involved: S1. The test compound containing P-31, a catalyst and a solvent are mixed to prepare a test solution; S2. Set the test temperature, perform hydrogen and phosphorus magnetic resonance detection of the test solution, and determine the chemical shift position of the magnetic resonance signal of the test compound P-31; S3. The CEST.XP.uT_1 sequence with continuous wave irradiation was selected to collect the P-31CEST Z spectrum; S4. Fitting the CEST Z spectrum and calculating the asymmetric magnetization transfer rate spectrum of the test solution according to the formula; S5. If an exchangeable signal is observed in step S4, it is determined that the test compound containing P-31 can reversibly bind to the catalyst and is a potential SABRE-enhanced compound; if no exchangeable signal is observed in step S4, steps S2-S5 are repeated after changing the test temperature.

2. The method according to claim 1, characterized in that The catalyst in step S1 is a catalyst for SABRE enhancement, including an iridium-based catalyst; the solvent in step S1 is one of deuterated methanol, toluene, and water.

3. The method according to claim 1, characterized in that In step S1, the molar concentration ratio of the compound to be tested containing P-31 to the catalyst is ≥2:

1.

4. The method according to claim 1, wherein The test temperature setting range in step S2 is 263K~353K. In step S2, the Bruker zg sequence is used to measure the hydrogen spectrum and phosphorus spectrum of the solution to be tested. The spectrum width SW when measuring the hydrogen spectrum is greater than the chemical shift range of hydrogen, and the spectrum width SW when measuring the phosphorus spectrum is greater than the chemical shift range of P-31. The relaxation recovery time when measuring the hydrogen spectrum and the phosphorus spectrum matches the relaxation time T1 of hydrogen and P-31, respectively.

5. The method according to claim 1, wherein The test temperature in step S2 is 310K.

6. The method according to claim 1, characterized in that The number of sampling points when measuring hydrogen spectrum and phosphorus spectrum is 32768 and 65536 respectively.

7. The method according to claim 1, characterized in that The parameters set when the continuous wave irradiation CEST.XP.uT_1 sequence is selected in step S3 to collect the P-31 CEST Z spectrum include: a relaxation recovery time range of 3s-12s, different saturation irradiation intensities, a saturation irradiation intensity range of 1μT to 20μT, a saturation irradiation time range of 1s to 24s, a frequency offset range of -6480Hz to 6480Hz for the saturation radio frequency pulse, a center frequency of the CEST Z spectrum acquisition being the chemical shift position of the magnetic resonance signal of the test compound P-31 measured by the phosphorus spectrum in step S2, and an integration range of the CEST Z spectrum acquisition being: the chemical shift position of the magnetic resonance signal of the test compound P-31 -0.2ppm to the chemical shift position of the magnetic resonance signal of the test compound P-31 +0.2ppm; In step S3, the CEST Z spectra under different saturation radio frequency pulse irradiation intensities are obtained by integrating the P-31 magnetic resonance signals of the free test compound.

8. The method according to claim 7, characterized in that In step S3, the relaxation recovery time range is 10 s, the different saturation irradiation intensities range are 6 μT, 7 μT, 8 μT, and 9 μT, the saturation irradiation time is 20 s, and the frequency offset range of the saturation RF pulse is -1652.4 Hz to 4860 Hz, with a step of 48.6 Hz.

9. The method according to claim 1, characterized in that The formula in step S4 is MTR asym =(I off ―I on ) / I0, where I on The signal of the compound P-31 to be tested is measured when the saturated radio frequency pulse irradiation frequency bias is greater than or equal to 0 Hz, I off The saturation RF pulse irradiation frequency bias is I on The signal of the test compound P-31 is measured at a symmetrical position relative to the magnetic resonance signal of the test compound P-31, and I0 is the signal of the test compound P-31 when the saturation radio frequency pulse irradiation intensity is 0.

10. The method according to claim 1, characterized in that If there is a peak with an amplitude ≥ 0.03 in the asymmetric magnetization transfer rate spectrum obtained in step S4, it indicates that there is an exchangeable signal.

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