Application of lanthanide series metal complex as 31P CEST high-field contrast agent in ATP (adenosine triphosphate) recognition

Abstract

The invention discloses an application of lanthanide metal complexes as a 31P CEST high-field contrast agent in ATP (adenosine triphosphate) recognition, which is characterized in that a chemical exchange saturation transfer (CEST) technology is utilized, the lanthanide metal complexes (such as Eu-DO3A, Tb-DO3A, Pr-DO3A and Yb-DO3A) are used as the 31P CEST high-field contrast agent, phosphate radicals of ATP are used as large-cell signals of CEST, and the chemical exchange saturation transfer (CEST) technology is utilized to identify the ATP. According to the present invention, the phosphate radical of ATP coordinated with the lanthanide series metal complex is adopted as the CEST small pool signal, such that the 31P CEST signals of the alpha-P, the beta-P and the gamma-P three exchange sites of ATP can be obtained, the 31P CEST signals of the ATP analogues ADP (alpha-P and beta-P) and AMP can be significantly distinguished, and the problem that the ATP and the ATP analogues are difficult to distinguish by using the traditional optical and electrochemical means is solved.

Description

Technical Field

[0001] The present invention belongs to the field of biological analysis and detection technology, and specifically relates to a method for 31 Application of P CEST high-field contrast agent in identifying ATP. Background Art

[0002] Adenosine triphosphate (ATP) is a key biomolecule synthesized by mitochondria, and abnormalities in its metabolism serve as a clinical diagnostic indicator. For example, in neurodegenerative diseases (Alzheimer's disease, Parkinson's disease) and cardiovascular diseases (myocarditis, atherosclerosis), ATP synthesis is reduced relative to normal tissue. Conversely, in tumor tissues with hypermetabolism, ATP synthesis is relatively increased.

[0003] The technologies currently used for ATP content detection mainly include bioluminescence, fluorescence, electrochemistry, 1 H NMR and 31 P NMR, etc. For optical detection technology, it is difficult to detect ATP in deep tissues of organisms due to the limitation of light penetration depth (<1cm). For electrochemical methods, electrodes need to be inserted for detection, which is an invasive technology. In addition, both optical and electrochemical technologies have difficulty in distinguishing ATP and its analogs ADP and AMP, making it difficult to accurately identify ATP. NMR technology has the advantages of no tissue penetration depth limitation, no ionizing radiation and non-invasiveness, but 1 ATP signals in the complex environment of H NMR are prone to stacking, which is not conducive to the accurate identification of ATP. 31 P NMR technology has the advantage of a wide chemical shift (>400ppm), which can avoid NMR signal stacking of target molecules, but the ATP content in living bodies is low, making accurate quantification difficult.

[0004] Chemical exchange saturation transfer (CEST) is a technique that uses the NMR signal of a selectively saturated small cell to indirectly detect the signal intensity of the small cell by observing the change of the signal of the large cell. CEST technology can increase the intensity of magnetic resonance signals by about 100 times, and is suitable for complex environments with low ATP content and 31 The problem of PNMR signal stacking has a better advantage. Therefore, the development of new 31 PCRest detection technology is necessary for the accurate identification of ATP. Summary of the Invention

[0005] Based on the above prior art, the present invention provides a lanthanide metal complex as a 31The application of PCR high field contrast agent in identifying ATP, the present invention uses lanthanide metal complexes (such as Eu-DO3A, etc.) as 31 P CEST high field contrast agent, for ATP and its analogs ADP and AMP 31 P CEST test, obtain the three 31 The PCEST signal has a large chemical shift difference, which is used for the precise identification of ATP, ADP and AMP, thereby achieving accurate detection of ATP.

[0006] The technical solution adopted to achieve the above-mentioned purpose of the present invention is:

[0007] Based on lanthanide metal complexes as 31 Application of P CEST high-field contrast agent in identifying ATP.

[0008] Furthermore, the lanthanide metal complex is at least one of Eu-DO3A, Tb-DO3A, Pr-DO3A and Yb-DO3A.

[0009] The preparation method of lanthanide metal complexes refers to the literature "Separation of Intra-and Extracellular Lactate NMR Signals Using a Lanthanide Shift Reagent, Silvio Aime,1*Mauro Botta,2Valentina Mainero,3and Enzo Terreno1".

[0010] The present invention utilizes the chemical exchange saturation transfer (CEST) technique, using the phosphate of ATP as the large pool signal of CEST and the phosphate of ATP coordinated with the lanthanide metal complex as the small pool signal of CEST, thereby obtaining the three exchange sites of ATP: α-P, β-P and γ-P. 31 PCEST signal can clearly distinguish between ATP analogs ADP (α-P and β-P) and AMP 31 The P CEST signal can identify ATP in a complex environment, overcoming the difficulty of traditional optical and electrochemical methods in distinguishing ATP and its analogs.

[0011] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0012] 1. The present invention utilizes lanthanide metal complexes (such as Eu-DO3A, Tb-DO3A, Pr-DO3A and Yb-DO3A, etc.) as 31P CEST high field contrast agent, ATP 31 PCEST detection can accurately distinguish ATP and its analogs ADP and AMP, thereby achieving precise identification of ATP.

[0013] 2. Compared with optical methods, the method of the present invention has no limitation on tissue penetration depth and is more suitable for the detection of ATP in deep tissues of living bodies.

[0014] 3. Compared with the electrochemical method, the method of the present invention does not require the insertion of the detection equipment into the living tissue, and is a non-destructive test, which is more suitable for the detection of ATP in the deep tissue of the living body.

[0015] 4. With 1 H and 31 Compared with the P NMR method, the ATP, ADP and AMP obtained by the method of the present invention are 31 The PCEST signal has a larger chemical shift difference and does not have the problem of signal stacking, which is more conducive to the accurate identification of ATP and its analogs. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Quantitative analysis of Eu-DO3A prepared in Example 1 1 H NMR spectrum.

[0017] Figure 2 Quantitative analysis of Pr-DO3A prepared in Example 1 1 H NMR spectrum.

[0018] Figure 3 The quantitative analysis of Tb-DO3A prepared in Example 1 1 H NMR spectrum.

[0019] Figure 4 The quantitative analysis of Yb-DO3A prepared in Example 1 1 H NMR spectrum.

[0020] Figure 5 is the ATP at different Eu-DO3A concentrations 31 P NMR spectrum.

[0021] Figure 6 is the ADP at different Eu-DO3A concentrations 31 P NMR spectrum.

[0022] Figure 7 is the AMP at different Eu-DO3A concentrations 31 P NMR spectrum.

[0023] Figure 8 Eu-DO3A as 31ATP under P CEST contrast agent 31 P CEST map.

[0024] Figure 9 Eu-DO3A as 31 P CEST contrast agent under ADP 31 P CEST map.

[0025] Figure 10 Eu-DO3A as 31 AMP under P CEST contrast agent 31 P CEST map.

[0026] Figure 11 Pr-DO3A as 31 ATP, ADP and AMP under PCEST contrast agent 31 P CEST map.

[0027] Figure 12 Tb-DO3A as 31 ATP, ADP and AMP under PCEST contrast agent 31 P CEST map.

[0028] Figure 13 Yb-DO3A as 31 ATP, ADP and AMP under PCEST contrast agent 31 P CEST map.

[0029] Figure 14 Eu-DO3A as 31 The α-P NMR signal site of ATP under the contrast agent P CEST was determined as the spectral center at different saturation pulse powers. 31 P CEST test results. Among them, 14 (a) ATP α-P NMR signal site is set as the spectrum center 31 PCEST spectrum, 14(b) shows the α-P NMR signal of ATP when the center of the spectrum is determined. 31 The linear relationship between the P CEST signal intensity and the saturation pulse power; 14(c) is the CEST exchange rate between Eu-DO3A-bound P and ATP β-P.

[0030] Figure 15 Eu-DO3A as 31 The β-P NMR signal site of ATP under the contrast agent P CEST was determined as the spectral center at different saturation pulse powers. 31 P CEST test results. Among them, 15 (a) ATP β-P NMR signal site is set as the spectrum center 31PCEST spectrum, 15(b) is the β-P NMR signal site of ATP when it is determined as the spectrum center 31 The linear relationship between the P CEST signal intensity and the saturation pulse power; 15(c) is the CEST exchange rate between Eu-DO3A-bound P and ATP β-P.

[0031] Figure 16 Eu-DO3A as 31 The γ-P NMR signal site of ATP under the contrast agent P CEST was determined as the spectral center at different saturation pulse powers. 31 P CEST test results. Among them, 16 (a) ATP γ-P NMR signal site is set as the spectrum center 31 PCEST spectrum, 16(b) is the γ-P NMR signal of ATP when the center of the spectrum is determined 31 The linear relationship between the P CEST signal intensity and the saturation pulse power; 16(c) is the CEST exchange rate between Eu-DO3A-bound P and γ-P of ATP.

[0032] Figure 17 Eu-DO3A as 31 The α-P NMR signal site of ADP under the contrast agent P CEST was determined as the spectral center at different saturation pulse powers. 31 P CEST test results. Among them, 17 (a) ADP α-P NMR signal site is set as the spectrum center 31 PCEST spectrum, 17(b) is the α-P NMR signal of ADP when the center of the spectrum is determined 31 The linear relationship between the P CEST signal intensity and the saturation pulse power; 17(c) is the CEST exchange rate between P bound to Eu-DO3A and α-P of ADP.

[0033] Figure 18 Eu-DO3A as 31 The β-P NMR signal site of ADP under the contrast agent P CEST was determined as the spectral center at different saturation pulse powers. 31 P CEST test results. Among them, 18 (a) ADP β-P NMR signal site is set as the spectrum center 31 PCEST spectrum, 18(b) is the β-P NMR signal of ADP when the spectral center is determined 31 The linear relationship between the P CEST signal intensity and the saturation pulse power; 18(c) is the CEST exchange rate between Eu-DO3A-bound P and ADP β-P.

[0034] Figure 19 Eu-DO3A as 31The NMR signal site of AMP under the contrast agent P CEST was determined as the spectral center at different saturation pulse powers. 31 P CEST test results. Among them, the NMR signal site of 19(a)AMP is set as the center of the spectrum. 31 P CEST spectrum, 19(b) is the NMR signal site of AMP when it is set as the spectrum center 31 The linear relationship between the P CEST signal intensity and the saturation pulse power; 19(c) is the CEST exchange rate between Eu-DO3A-bound P and AMP. DETAILED DESCRIPTION

[0035] In order to facilitate those skilled in the art to understand and implement the present invention, the present invention is further described in detail below in conjunction with embodiments. It should be understood that the embodiments described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.

[0036] Example 1

[0037] 1. Prepare 10 mM aqueous solutions of europium trifluoromethanesulfonate (Eu(CF3SO3)3), praseodymium trifluoromethanesulfonate (Pr(CF3SO3)3), terbium trifluoromethanesulfonate (Tb(CF3SO3)3) and ytterbium trifluoromethanesulfonate (Yb(CF3SO3)3) as solutions A1-A4, and prepare 11.76 mM aqueous solution of 1,4,7,10-tetraazacyclododecane-1,4,7-triacetate (DO3A) as solution B.

[0038] 2. Take 5 mL of A1 solution and 5 mL of B solution and add them to 50 mL round-bottom centrifuge tubes respectively. Mix them evenly with magnetic stirring. At the same time, use 1 M HCl and 1 M NaOH to adjust the pH value of the mixed solution to 5.5-6.0. Continue magnetic stirring. After the pH value stabilizes at 5.5-6.0, transfer the mixed solution to a 45°C constant temperature water bath and keep it for 10 minutes. Then test the pH value of the reaction system. If the pH value of the reaction system exceeds the range of 5.5-6.0, continue to use 1 M HCl and 1 M NaOH to adjust the pH value of the reaction system until the pH value of the reaction system remains stable in the range of 5.5-6.0, indicating that the reaction of metal Eu and DO3A is complete, and Eu-DO3A solution is obtained.

[0039] 3. Treat solution A2 and solution B according to the method of step 2 to obtain a Pr-DO3A solution. Treat solution A3 and solution B according to the method of step 2 to obtain a Tb-DO3A solution. Treat solution A4 and solution B according to the method of step 2 to obtain a Yb-DO3A solution.

[0040] 4. Take 198 μL of each Eu-DO3A, Pr-DO3A, Tb-DO3A, and Yb-DO3A complex solution and mix them with 2 μL of isopropanol to obtain solutions C1-C4. Mix 495 μL of D2O with 5 μL of isopropanol to obtain solution D.

[0041] 4. Quantify the concentrations of Eu-DO3A, Pr-DO3A, Tb-DO3A and Yb-DO3A using magnetic resonance method. The specific operation is as follows: add 200 μL of C1, C2, C3 and C4 solutions to the inner tubes of four NMR tubes respectively, add 500 μL of D2O solution to the outer tubes of four NMR tubes respectively, and use 500M magnetic resonance spectrometer to measure the solutions in the inner and outer tubes of each NMR tube. 1 H NMR test, the isopropanol in two environments was measured 1 H NMR signals and calculate the chemical shift difference (δ x ), the concentrations c of Eu-DO3A, Pr-DO3A, Tb-DO3A and Yb-DO3A were calculated by the following formula (1):

[0042]

[0043] Where, T is 295K, s=1 / 3, and the μ of Eu is eff =3.455, μ of Pr eff =3.620, μ of Tb eff =9.700, μ of Yb eff =4.500.

[0044] 1 H NMR test results show that the δ x =0.088ppm, δ of Pr-DO3A x =0.093ppm, δ of Tb-DO3A x =0.800ppm, δ of Yb-DO3A x =0.154ppm (see Figure 1-4 ), δ x Substituting into the formula, the concentration c values ​​of Eu-DO3A, Pr-DO3A, Tb-DO3A and Yb-DO3A are calculated to be 4.23mM, 4.07mM, 4.88mM and 4.36mM respectively.

[0045] Experiment 1: Effects of different concentrations of Eu-DO3A on ATP, ADP and AMP 31 P NMR signal influence

[0046] Test method:

[0047] 1. The ATP standard and the Eu-DO3A solution prepared in Example 1 were dissolved in a mixed solvent consisting of ultrapure water and heavy water to prepare a series of mixed solutions with Eu-DO3A gradient concentrations of 0, 50, 100, 200, and 400 μM. In each mixed solution, the ATP concentration was 50 mM, and the volume proportion of heavy water in the mixed solvent was 15%.

[0048] 2. Adjust the concentration of each mixed solution to 7 with 1M HCl and 1M NaOH to obtain a sample solution. Take 500 μL of the sample solution and add it to a 5mm NMR tube. Use a 500MHz magnetic resonance spectrometer to analyze the sample solution. 31 P NMR test and analysis of ATP 31 P NMR signal changes with the concentration of added Eu-DO3A.

[0049] 3. Treat the ADP and AMP standards according to steps 1-2.

[0050] Test results:

[0051] Effects of different concentrations of Eu-DO3A on ATP 31 P NMR signal influences such as Figure 5 As shown in Figure 2, the effect of different concentrations of Eu-DO3A on ADP 31 P NMR signal influences such as Figure 6 As shown in the figure, the effect of different concentrations of Eu-DO3A on AMP 31 P NMR signal influences such as Figure 7 shown.

[0052] ATP, ADP and AMP at different Eu-DO3A concentrations 31 The chemical shift changes of the P NMR signals are shown in Table 1 below:

[0053] Table 1 ATP, ADP and AMP at different Eu-DO3A concentrations 31 Chemical shift of P NMR signal

[0054]

[0055] ATP, ADP and AMP at different Eu-DO3A concentrations 31 The changes in P NMR signal intensity are shown in Table 2 below:

[0056] Table 2 ATP, ADP and AMP at different Eu-DO3A concentrations 31 P NMR signal intensity

[0057]

[0058] ATP has three 31 P NMR signals are attributed to α-P, β-P and γ-P respectively; ADP has two 31 P NMR signals are attributed to α-P and β-P respectively; AMP has only one 31 P NMR signal. Figure 5-7 As shown in Table 1, with the increase of Eu-DO3A concentration, the concentrations of ATP (α-P, β-P and γ-P), ADP (α-P and β-P) and AMP increased. 31 The chemical shift of the P NMR signal was almost unchanged (<0.1 ppm).

[0059] As shown in Table 2, the increase of Eu-DO3A concentration can affect the activity of ATP (α-P, β-P and γ-P), ADP (α-P and β-P) and AMP. 31 The intensity of the P NMR signal is weakened. It is speculated that the increase in Eu-DO3A concentration leads to a shortening of the relaxation time of ATP (α-P, β-P and γ-P), ADP (α-P and β-P) and AMP, and an acceleration of the signal decay rate, thereby reducing the 31 P NMR signal intensity.

[0060] Example 2

[0061] 1. The ATP standard and the Eu-DO3A solution prepared in Example 1 were dissolved in a mixed solvent consisting of ultrapure water and heavy water to prepare a mixed solution. In the mixed solution, the concentration of ATP was 50 mM, the concentration of Eu-DO3A was 50 μM, and the volume proportion of heavy water in the mixed solvent was 15%.

[0062] 2. The mixed solution was adjusted to 7 with 1M HCl and 1M NaOH to obtain a sample solution. 500 μL of the sample solution was added to a 5 mm NMR tube and analyzed using an 11.75 T magnetic resonance spectrometer. 31 P CEST test. 31 In the P CEST test, ATP 31 The corresponding α-P, β-P and γ-P NMR signals in the P NMR spectrum are set as the spectral center, and the sites (chemical shifts) of the α-P, β-P and γ-P NMR signals are used as the large pool signals of CEST. 31 P CEST test, ATP 31 The PCEST saturation point was set to -100 to 50 ppm.

[0063] 3. Treat the ADP standard and AMP standard according to steps 1-2. 31The PNMR spectrum corresponds to the two NMR signals of α-P and β-P. The sites of the two NMR signals of α-P and β-P are set as the spectrum centers respectively and used as the large pool signals of CEST. 31 P CEST test, ADP 31 The PCEST saturation point is set to -200~80ppm. 31 The P NMR spectrum corresponds to an NMR signal, and the site of the AMP NMR signal is set as the spectrum center, which is used as the large pool signal of CEST. 31 P CEST test, AMP 31 The PCEST saturation point was set to -200 to 80 ppm.

[0064] 4. ATP 31 P CEST spectrum Figure 8 As shown, Figure 8 It shows that ATP is observed at -64ppm, -20ppm and -60ppm respectively. 31 P CEST signal. ADP 31 P CEST spectrum Figure 9 As shown, Figure 9 It shows that ADP was observed at -30ppm and -128ppm, respectively. 31 P CEST signal. AMP 31 P CEST spectrum Figure 10 As shown, Figure 10 It shows that AMP is observed at -144ppm 31 PCREST signal.

[0065] From the above analysis, we can see that the relationship between ATP, ADP and AMP is 31 The minimum shift difference of PCEST signal is 10ppm (β-P of ATP is the center of the spectrum). 31 The PCEST signal is centered on the α-P of ADP. 31 The maximum chemical shift difference is 124 ppm (β-P of ATP is the center of the spectrum). 31 P CEST signal and AMP 31 The displacement difference of PCEST signal can clearly distinguish ATP, ADP and AMP. 31 P CEST signal, thereby achieving accurate recognition of ATP and its analogs.

[0066] Example 3

[0067] 1. The ATP standard and the Pr-DO3A solution prepared in Example 1 were dissolved in a mixed solvent consisting of ultrapure water and heavy water to prepare a mixed solution E1. In the mixed solution E1, the concentration of ATP was 50 mM, the concentration of Pr-DO3A was 50 μM, and the volume proportion of heavy water in the mixed solvent was 15%.

[0068] The ADP standard and the Pr-DO3A solution prepared in Example 1 were dissolved in a mixed solvent consisting of ultrapure water and heavy water to prepare a mixed solution E2. In the mixed solution E2, the concentration of ADP was 50 mM, the concentration of Pr-DO3A was 50 μM, and the volume proportion of heavy water in the mixed solvent was 15%.

[0069] The AMP standard and the Pr-DO3A solution prepared in Example 1 were dissolved in a mixed solvent consisting of ultrapure water and heavy water to prepare a mixed solution E3. In the mixed solution E3, the concentration of AMP was 50 mM, the concentration of Pr-DO3A was 50 μM, and the volume proportion of heavy water in the mixed solvent was 15%.

[0070] 2. Prepare mixed solutions E4-E6 by mixing ATP standard, ADP standard, and AMP standard with Tb-DO3A solution according to the method in step 1.

[0071] 3. Prepare mixed solutions E7-E9 by mixing ATP standard, ADP standard, and AMP standard with Yb-DO3A solution according to the method in step 1.

[0072] 4. Mix the solutions E1-E9 according to the method of steps 2-3 of Example 2. 31 P CEST test.

[0073] 5. Using Pr-DO3A as 31 ATP, ADP and AMP under P CEST high field contrast agent 31 P CEST spectrum Figure 11 As shown, ATP was observed at 24 ppm, 22 ppm and 54 ppm respectively. 31 The P CEST signal of ADP β-P was observed at 90 ppm. 31 P CEST signal, AMP was observed at 108 ppm 31 PCREST signal.

[0074] Using Tb-DO3A as 31 ATP, ADP and AMP under P CEST high field contrast agent 31 P CEST spectrum Figure 12 As shown, ATP was observed at -188 ppm and -86 ppm, respectively.31 The P CEST signals of ADP were observed at -200ppm and -126ppm respectively. 31 The P CEST signal of AMP was observed at -170 ppm. 31 PCREST signal.

[0075] Yb-DO3A as 31 ATP, ADP and AMP under P CEST high field contrast agent 31 P CEST spectrum Figure 13 As shown, ATP was observed at -56ppm, -20ppm and -42ppm respectively. 31 The P CEST signals and ADP were observed at -44ppm and -46ppm, respectively. 31 The P CEST signal of AMP was observed at -70 ppm. 31 PCREST signal.

[0076] From the above analysis, it can be seen that the synthesized lanthanide metal-DO3A complexes can obtain ATP, ADP and AMP. 31 The PCEST signal confirmed the universality of Ln-DO3A in recognizing ATP.

[0077] Experiment 2: Effect of Eu-DO3A on ATP, ADP and AMP 31 Impact of PCEST Exchange Rate

[0078] Test method:

[0079] 1. The ATP standard and the Eu-DO3A solution prepared in Example 1 were dissolved in a mixed solvent consisting of ultrapure water and heavy water to prepare a mixed solution. In the mixed solution, the concentration of ATP was 50 mM, the concentration of Eu-DO3A was 50 μM, and the volume proportion of heavy water in the mixed solvent was 15%.

[0080] 2. The mixed solution was adjusted to 7 with 1M HCl and 1M NaOH to obtain a sample solution. 500 μL of the sample solution was added to a 5mm NMR tube and analyzed using a 500MHz magnetic resonance spectrometer under different saturation pulse power conditions. 31 P CEST test, calculate the exchange rate between CEST signal site and CEST large pool signal. 31 In the P CEST test, the ATP 31 The corresponding α-P, β-P and γ-P NMR signals in the P NMR spectrum were set as the spectral center and used as the large pool signal of CEST. 31 P CEST test, ATP31 The PCEST saturation point was set to -100 to 50 ppm.

[0081] 3. Treat the ADP standard and AMP standard according to steps 1-2. 31 The PNMR spectrum corresponds to the two NMR signals of α-P and β-P. The sites of the two NMR signals of α-P and β-P are set as the spectrum centers respectively and used as the large pool signals of CEST. 31 P CEST test, ADP 31 The PCEST saturation point is set to -200~80ppm. 31 The P NMR spectrum corresponds to an NMR signal, and the site of the AMP NMR signal is set as the spectrum center, which is used as the large pool signal of CEST. 31 P CEST test, AMP 31 The PCEST saturation point was set to -200 to 80 ppm.

[0082] Test results:

[0083] The α-P NMR signal site of ATP was determined as the spectral center under different saturation pulse powers. 31 P CEST test results are as follows Figure 14 As shown, the α-P NMR signal of ATP is located at the center of the spectrum. 31 The P CEST spectrum was observed at -64 ppm 31 P CEST signal ( Figure 14 a), and the signal strength is proportional to the saturation pulse power ( Figure 14 b) Further calculations were performed to determine the exchange rate k between the phosphorus coordinated to Eu-DO3A and the α-P of ATP. b is 22269 Hz, and the residence time of phosphorus coordinated with Eu-DO3A is 45 μs ( Figure 14 c).

[0084] The β-P NMR signal site of ATP was determined as the spectral center under different saturation pulse powers. 31 P CEST test results are as follows Figure 15 As shown, the β-P NMR signal of ATP is located at the center of the spectrum. 31 The P CEST spectrum shows that the observed 31 PCEST signal ( Figure 15 a), and the signal strength is proportional to the saturation pulse power ( Figure 15 b) Further calculations were performed to determine the exchange rate k between the phosphorus coordinated to Eu-DO3A and the β-P of ATP. bis 7561Hz, and the residence time of phosphorus coordinated with Eu-DO3A is 132μs ( Figure 15 c).

[0085] The γ-P NMR signal site of ATP was determined as the spectral center under different saturation pulse powers. 31 P CEST test results are as follows Figure 16 As shown, the γ-P NMR signal of ATP is located at the center of the spectrum. 31 The P CEST spectrum shows that the observed 31 PCEST signal ( Figure 16 a), and the signal strength is proportional to the saturation pulse power ( Figure 16 b) Further calculations were performed to determine the exchange rate k between the phosphorus coordinated to Eu-DO3A and the β-P of ATP. b is 13228 Hz, and the residence time of phosphorus coordinated with Eu-DO3A is 75 μs ( Figure 16 c).

[0086] The α-P NMR signal site of ADP was determined as the spectral center at different saturation pulse powers. 31 P CEST test results are as follows Figure 17 As shown, the α-P NMR signal of ADP is located at the center of the spectrum. 31 The P CEST spectrum was observed at -30 ppm 31 P CEST signal ( Figure 17 a), and the signal strength is proportional to the saturation pulse power ( Figure 17 b) Further calculations were performed to determine the exchange rate k between the phosphorus coordinated with Eu-DO3A and the α-P of ADP. b is 6250Hz, and the residence time of phosphorus coordinated with Eu-DO3A is 160μs ( Figure 17 c).

[0087] The β-P NMR signal site of ADP was determined as the spectral center at different saturation pulse powers. 31 P CEST test results are as follows Figure 18 As shown, the β-P NMR signal of ADP is located at the center of the spectrum. 31 The P CEST spectrum was observed at -126 ppm 31 PCEST signal ( Figure 18 a), and the signal strength is proportional to the saturation pulse power ( Figure 18 b) Further calculations were performed to determine the exchange rate k between the phosphorus coordinated with Eu-DO3A and ADP. b is 6899 Hz, and the residence time of phosphorus coordinated with Eu-DO3A is 145 μs ( Figure 18 c).

[0088] The NMR signal site of AMP is determined as the spectral center at different saturation pulse powers. 31 P CEST test results are as follows Figure 19 As shown, the NMR signal site of ATP is determined as the center of the spectrum. 31 The P CEST spectrum was observed at -142 ppm 31 P CEST signal ( Figure 19 a), and the signal strength is proportional to the saturation pulse power ( Figure 19 b) Further calculations were performed to determine the exchange rate k between the phosphorus coordinated with Eu-DO3A and the phosphorus of AMP. b is 34561Hz, and the residence time of phosphorus coordinated with Eu-DO3A is 29μs ( Figure 19 c).

Claims

1. Based on lanthanide metal complexes as 31 Application of P CEST high-field contrast agent in identifying ATP.

2. The use of lanthanide metal complex according to claim 1, characterized in that: The lanthanide metal complex is at least one of Eu-DO3A, Tb-DO3A, Pr-DO3A and Yb-DO3A.

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