Lanthanide metal complexes as 31 Application of P CEST high-field contrast agent in ATP identification

Abstract

This invention discloses a method based on lanthanide metal complexes as... 31 The application of P CEST high-field contrast agents in ATP recognition: This invention utilizes Chemical Exchange Saturation Transfer (CEST) technology, using lanthanide metal complexes (such as Eu-DO3A, Tb-DO3A, Pr-DO3A, and Yb-DO3A) as... 31 P CEST high-field contrast agent uses ATP phosphate as the macrocell signal and ATP phosphate coordinated with lanthanide metal complexes as the microcell signal, thus obtaining the three ATP exchange sites: α-P, β-P, and γ-P. 31 P CEST signal, and can clearly distinguish ATP analogs ADP (α-P and β-P) and AMP. 31 The P CEST signal overcomes the difficulty of distinguishing ATP and its analogues using traditional optical and electrochemical methods.

Description

Technical Field

[0001] This invention belongs to the field of bioanalytical detection technology, specifically relating to lanthanide metal complexes as... 31 Application of P CEST high-field contrast agent in ATP identification. Background Technology

[0002] Adenosine triphosphate (ATP) is a crucial biomolecule synthesized in mitochondria, and its metabolic abnormalities 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 relatively reduced compared to normal tissues. Conversely, in tumor tissues with abnormally high metabolic activity, ATP synthesis is relatively increased.

[0003] Currently, the main technologies used for ATP content detection include bioluminescence, fluorescence, and electrochemistry. 1 H NMR and 31 P NMR, etc. Optical detection techniques are limited by the tissue penetration depth (<1 cm), making it difficult to detect ATP in deep biological tissues. Electrochemical methods require electrode insertion, making them invasive techniques. Furthermore, both optical and electrochemical techniques struggle to distinguish ATP from its analogues ADP and AMP, hindering accurate ATP identification. NMR technology offers advantages such as no tissue penetration depth limitations, no ionizing radiation, and non-invasiveness, but... 1 ATP signals are prone to stacking in complex H NMR environments, which makes it difficult to accurately identify ATP. 31 P NMR technology has the advantage of a wide chemical shift (>400ppm) and can avoid the stacking of NMR signals of target molecules, but the low ATP content in living organisms makes accurate quantification difficult.

[0004] Chemical exchange saturation transfer (CEST) is a technique that utilizes the NMR signal from a selectively saturated small-cell NMR image to indirectly detect the signal intensity of the small-cell image by observing changes in the signal from the large-cell image. CEST can increase the intensity of the magnetic resonance signal by approximately 100 times, and is particularly useful in complex environments with low ATP content. 31 The problem of PNMR signal stacking has significant advantages. Therefore, developing new... 31 P CEST detection technology is essential for the accurate identification of ATP. Summary of the Invention

[0005] Based on the above-mentioned prior art, the present invention provides a lanthanide metal complex as... 31The application of P CEST high-field contrast agents in ATP recognition: This invention utilizes lanthanide metal complexes (such as Eu-DO3A) as... 31 P CEST high-field contrast agent, used to detect ATP and its analogues ADP and AMP. 31 P CEST test, obtaining the three 31 The P CEST signal has a large chemical shift difference, which is used for the accurate identification of ATP, ADP and AMP, thereby enabling the accurate detection of ATP.

[0006] The technical solution adopted to achieve the above-mentioned objectives of this invention is as follows:

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

[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 is based on 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] This invention utilizes Chemical Exchange Saturation Transfer (CEST) technology, using the phosphate group of ATP as the macro-pool signal and the phosphate group of ATP coordinated with lanthanide metal complexes as the micro-pool signal, to obtain the α-P, β-P, and γ-P exchange sites of ATP. 31 P CEST signal, and can clearly distinguish ATP analogs ADP (α-P and β-P) and AMP. 31 The P CEST signal can identify ATP in complex environments, overcoming the difficulty of distinguishing ATP and its analogues by traditional optical and electrochemical methods.

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

[0012] 1. This invention utilizes lanthanide metal complexes (such as Eu-DO3A, Tb-DO3A, Pr-DO3A, and Yb-DO3A) as... 31P CEST high-field contrast agent, for ATP... 31 P CEST assay can accurately distinguish ATP from its analogues ADP and AMP, thus enabling precise identification of ATP.

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

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

[0015] 4. with 1 H and 31 Compared to 1NMR methods, the method of this invention obtains ATP, ADP, and AMP. 31 The P CEST 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 analogues. Attached Figure Description

[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 Quantitative analysis of Tb-DO3A prepared in Example 1 1 H NMR spectrum.

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

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

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

[0022] Figure 7 For 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 chart.

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

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

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

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

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

[0029] Figure 14 Eu-DO3A as 31 α-P NMR signal sites of ATP under P CEST contrast agent were determined as spectral centers under different saturation pulse powers. 31 P CEST test results. Among them, the α-P NMR signal site of 14(a)ATP was determined to be at the spectral center. 31 PCEST spectrum, 14(b) shows the α-P NMR signal site of ATP, which was determined as the spectral center. 31 The linear relationship between P CEST signal intensity and 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 β-P NMR signal sites of ATP under P CEST contrast agent were determined as spectral centers under different saturation pulse powers. 31 P CEST test results. Among them, the β-P NMR signal site of 15(a)ATP was determined to be at the spectral center. 31PCEST spectrum, 15(b) shows the β-P NMR signal site of ATP, which was determined as the spectral center. 31 The linear relationship between P CEST signal intensity and saturation pulse power; 15(c) represents the CEST exchange rate between Eu-DO3A-bound P and ATP β-P.

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

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

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

[0034] Figure 19 Eu-DO3A as 31The NMR signal sites of AMP under P CEST contrast agent were determined by the spectral centers under different saturation pulse powers. 31 P CEST test results. Among them, the NMR signal sites of 19(a)AMP were determined to be at the spectral center. 31 P CEST spectrum, 19(b) shows the NMR signal sites of AMP when the spectrum center is determined. 31 The linear relationship between P CEST signal strength and saturation pulse power; 19(c) is the CEST exchange rate of P and AMP combined with Eu-DO3A. Detailed Implementation

[0035] To facilitate understanding and implementation of the present invention by those skilled in the art, the present invention will be further described in detail below with reference to embodiments. It should be understood that the embodiments described herein are for illustration and explanation only and are not intended 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. Prepare 11.76 mM aqueous solution of sodium 1,4,7,10-tetraazacyclododecane-1,4,7-triacetate (DO3A) as solution B.

[0038] 2. Add 5 mL of solution A1 and 5 mL of solution B to separate 50 mL round-bottom centrifuge tubes and mix thoroughly with magnetic stirring. Simultaneously, adjust the pH of the mixed solution to 5.5-6.0 using 1 M HCl and 1 M NaOH. Continue magnetic stirring until the pH stabilizes at 5.5-6.0. Then, transfer the mixed solution to a 45°C constant temperature water bath and maintain it for 10 minutes. Test the pH of the reaction system again. If the pH of the reaction system exceeds the range of 5.5-6.0, continue to adjust the pH of the reaction system with 1 M HCl and 1 M NaOH until the pH of the reaction system remains stable within the range of 5.5-6.0. This indicates that the metal Eu has completely reacted with DO3A, yielding an Eu-DO3A solution.

[0039] 3. Process solutions A2 and B according to the method in step 2 to obtain Pr-DO3A solution. Process solutions A3 and B according to the method in step 2 to obtain Tb-DO3A solution. Process solutions A4 and B according to the method in step 2 to obtain Yb-DO3A solution.

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

[0041] 4. The concentrations of Eu-DO3A, Pr-DO3A, Tb-DO3A, and Yb-DO3A were quantified using magnetic resonance spectroscopy. Specifically, 200 μL of solutions of C1, C2, C3, and C4 were added to the inner tubes of four NMR tubes, respectively, and 500 μL of D2O solution was added to the outer tubes of the same tubes. The solutions in both the inner and outer tubes of each NMR tube were then analyzed using a 500 M magnetic resonance spectrometer. 1 ¹H NMR analysis revealed that isopropanol was detected under two different conditions. 1 H NMR signal and calculation of chemical shift difference (δ) x The concentrations c of Eu-DO3A, Pr-DO3A, Tb-DO3A, and Yb-DO3A are calculated using the following formula (1):

[0042]

[0043] In the formula, T is 295K, s = 1 / 3, and 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 ¹H NMR test results showed that Eu-DO3A had a δ¹⁻¹ 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 concentrations c of Eu-DO3A, Pr-DO3A, Tb-DO3A, and Yb-DO3A are calculated to be 4.23 mM, 4.07 mM, 4.88 mM, and 4.36 mM, respectively.

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

[0046] Test method:

[0047] 1. Dissolve the ATP standard and the Eu-DO3A solution prepared in Example 1 in a mixed solvent composed 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 concentration of ATP is 50 mM and the volume percentage of heavy water in the mixed solvent is 15%.

[0048] 2. Adjust the concentration of each mixed solution to pH 7 using 1M HCl and 1M NaOH to obtain the sample solution. Add 500 μL of the sample solution to a 5 mm NMR tube and perform NMR spectroscopy using a 500 MHz magnetic resonance spectrometer. 31 p NMR assay and analysis of ATP 31 The P NMR signal varies with the concentration of added Eu-DO3A.

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

[0050] Experimental results:

[0051] The effects of different concentrations of Eu-DO3A on ATP 31 The influence of P NMR signal, such as Figure 5 As shown, different concentrations of Eu-DO3A have an effect on ADP. 31 The influence of P NMR signal, such as Figure 6 As shown, different concentrations of Eu-DO3A affect AMP. 31 The influence of P NMR signal, such as Figure 7 As shown.

[0052] ATP, ADP and AMP at different Eu-DO3A concentrations 31 The chemical shift changes of the pNMR signal 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 The P NMR signals were assigned to α-P, β-P, and γ-P, respectively; ADP had two 31 The P NMR signals were assigned to α-P and β-P, respectively; there was only one AMP. 31 P NMR signal. (From) Figure 5-7 As shown in Table 1, with increasing Eu-DO3A concentration, the levels of ATP (α-P, β-P, and γ-P), ADP (α-P and β-P), and AMP... 31 The chemical shift of the p NMR signal remained almost unchanged (<0.1 ppm).

[0059] Table 2 shows that increasing Eu-DO3A concentration can affect ATP (α-P, β-P, and γ-P), ADP (α-P and β-P), and AMP. 31 The weakening effect of P NMR signal intensity is presumably due to the increased Eu-DO3A concentration leading to a shortened relaxation time of ATP (α-P, β-P, and γ-P), ADP (α-P and β-P), and AMP, resulting in a faster signal decay rate and thus reducing the intensity of the signal. 31 P NMR signal intensity.

[0060] Example 2

[0061] 1. Dissolve the ATP standard and the Eu-DO3A solution prepared in Example 1 in a mixed solvent composed of ultrapure water and heavy water to prepare a mixed solution. The concentration of ATP in the mixed solution is 50 mM, the concentration of Eu-DO3A is 50 μM, and the volume percentage of heavy water in the mixed solvent is 15%.

[0062] 2. Adjust the mixture to pH 7 using 1M HCl and 1M NaOH to obtain the sample solution. Add 500 μL of the sample solution to a 5 mm NMR tube and perform NMR analysis using an 11.75T magnetic resonance spectrometer. 31 P CEST test. In 31 In the P CEST test, ATP... 31 The three NMR signals corresponding to α-P, β-P, and γ-P in the P NMR spectrum were used as the spectral centers, with the sites (chemical shifts) of the α-P, β-P, and γ-P NMR signals respectively set as the large cell signals for CEST. 31 P CEST test, ATP 31 The P CEST saturation site was set to -100 to 50 ppm.

[0063] 3. Process the ADP and AMP standards according to steps 1-2. For ADP, ADP's... 31The PNMR spectrum corresponds to two NMR signals, α-P and β-P. The sites of these two NMR signals are set as spectral centers, and these centers are used as the big cell signals for CEST analysis. 31 P CEST test, ADP 31 The P CEST saturation site is set to -200 to 80 ppm. For AMP, AMP's 31 The AMP NMR spectrum corresponds to one NMR signal. The spectral center of the AMP NMR signal is set as the big cell signal for CEST. 31 P CEST test, AMP 31 The P CEST saturation site was set to -200 to 80 ppm.

[0064] 4. ATP 31 P CEST chart as follows Figure 8 As shown, Figure 8 The results showed that ATP was observed at -64 ppm, -20 ppm, and -60 ppm, respectively. 31 P CEST signal. ADP 31 P CEST chart as follows Figure 9 As shown, Figure 9 The results showed that ADP was observed at -30 ppm and -128 ppm, respectively. 31 P CEST signal. AMP's 31 P CEST chart as follows Figure 10 As shown, Figure 10 The results showed that AMP was observed at -144 ppm. 31 P CEST signal.

[0065] The above analysis shows that there is a relationship between ATP, ADP, and AMP. 31 The minimum shift difference of the P CEST signal is 10 ppm (the β-P of ATP is the spectral center). 31 The P CEST signal and the ADP signal have α-P as their spectral centers. 31 The shift difference of the P CEST signal), the maximum chemical shift difference is 124 ppm (the β-P of ATP is the spectral center). 31 P CEST signal and AMP 31 The shift difference of the P CEST signal can clearly distinguish ATP, ADP, and AMP. 31 P CEST signal, thereby enabling accurate identification of ATP and its analogues.

[0066] Example 3

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

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

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

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

[0074] Using Tb-DO3A as 31 ATP, ADP, and AMP levels under high-field contrast in P CEST 31 P CEST chart as follows Figure 12 As shown, ATP was observed at -188 ppm and -86 ppm, respectively.31 The P CEST signal was observed to contain ADP at -200 ppm and -126 ppm, respectively. 31 The P CEST signal was observed at -170 ppm, and AMP was detected. 31 P CEST signal.

[0075] Using Yb-DO3A as 31 ATP, ADP, and AMP levels under high-field contrast in P CEST 31 P CEST chart as follows Figure 13 As shown, ATP was observed at -56 ppm, -20 ppm and -42 ppm, respectively. 31 The P CEST signal was observed to contain ADP at -44 ppm and -46 ppm. 31 The P CEST signal was observed with AMP at -70 ppm. 31 P CEST signal.

[0076] The above analysis shows that the synthesized lanthanide metal-DO3A complexes can all obtain ATP, ADP, and AMP. 31 The P CEST signal confirmed the universality of Ln-DO3A in recognizing ATP.

[0077] Experiment 2: Effects of Eu-DO3A on ATP, ADP, and AMP 31 Impact of P CEST switching rate

[0078] Test method:

[0079] 1. Dissolve the ATP standard and the Eu-DO3A solution prepared in Example 1 in a mixed solvent composed of ultrapure water and heavy water to prepare a mixed solution. The concentration of ATP in the mixed solution is 50 mM, the concentration of Eu-DO3A is 50 μM, and the volume percentage of heavy water in the mixed solvent is 15%.

[0080] 2. Adjust the mixture to pH 7 using 1M HCl and 1M NaOH to obtain the sample solution. Add 500 μL of the sample solution to a 5 mm NMR tube and perform NMR spectroscopy using a 500 MHz magnetic resonance spectrometer under different saturation pulse power conditions. 31 The P CEST test calculates the exchange rate between the CEST signal point and the CEST large pool signal. 31 In the P CEST test, ATP will be... 31 The three NMR signals corresponding to α-P, β-P, and γ-P in the P NMR spectrum were used as the spectral centers, and their locations were set as the big cell signals for CEST analysis. 31 P CEST test, ATP31 The P CEST saturation site was set to -100 to 50 ppm.

[0081] 3. Process the ADP and AMP standards according to steps 1-2. For ADP, ADP's... 31 The PNMR spectrum corresponds to two NMR signals, α-P and β-P. The sites of these two NMR signals are set as spectral centers, and these centers are used as the big cell signals for CEST analysis. 31 P CEST test, ADP 31 The P CEST saturation site is set to -200 to 80 ppm. For AMP, AMP's 31 The AMP NMR spectrum corresponds to one NMR signal. The spectral center of the AMP NMR signal is set as the big cell signal for CEST. 31 P CEST test, AMP 31 The P CEST saturation site was set to -200 to 80 ppm.

[0082] Experimental results:

[0083] The α-P NMR signal sites of ATP were determined by the spectral center under different saturation pulse powers. 31 The P CEST test results are as follows Figure 14 As shown, the α-P NMR signal site of ATP was located at the spectral center. 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 α-P exchange rate k between phosphorus coordinated to Eu-DO3A and ATP. b The Hz frequency was 22269 Hz, and the residence time of phosphorus coordinated with Eu-DO3A was 45 μs. Figure 14 c).

[0084] The β-P NMR signal sites of ATP were determined by the spectral center under different saturation pulse powers. 31 The P CEST test results are as follows Figure 15 As shown, the β-P NMR signal site of ATP was located at the spectral center. 31 The P CEST spectrum shows observations at -20 ppm. 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 of β-P between phosphorus coordinated with Eu-DO3A and ATP. bThe Hz frequency was 7561 Hz, and the residence time of phosphorus coordinated with Eu-DO3A was 132 μs. Figure 15 c).

[0085] The γ-P NMR signal sites of ATP were determined by the spectral center under different saturation pulse powers. 31 The P CEST test results are as follows Figure 16 As shown, the γ-P NMR signal site of ATP was located at the spectral center. 31 The P CEST spectrum shows an observation at -60 ppm. 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 of β-P between phosphorus coordinated with Eu-DO3A and ATP. b The Hz frequency was 13228 Hz, and the residence time of phosphorus coordinated with Eu-DO3A was 75 μs. Figure 16 c).

[0086] The α-P NMR signal sites of ADP were determined by the spectral centers under different saturation pulse powers. 31 The P CEST test results are as follows Figure 17 As shown, the α-P NMR signal sites of ADP are located at the spectral center. 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 α-P exchange rate k between phosphorus coordinated with Eu-DO3A and ADP. b The Hz frequency was 6250 Hz, and the residence time of phosphorus coordinated with Eu-DO3A was 160 μs. Figure 17 c).

[0087] The β-P NMR signal sites of ADP were determined by the spectral centers under different saturation pulse powers. 31 The P CEST test results are as follows Figure 18 As shown, the β-P NMR signal site of ADP is located at the spectral center. 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 β-P exchange rate k between phosphorus coordinated to Eu-DO3A and ADP. b The Hz frequency was 6899 Hz, and the residence time of phosphorus coordinated with Eu-DO3A was 145 μs. Figure 18 c).

[0088] The NMR signal loci of the AMP were determined by the spectral center under different saturation pulse powers. 31 The P CEST test results are as follows Figure 19 As shown, the NMR signal site of ATP is located at the spectral center. 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 phosphorus coordinated to Eu-DO3A and phosphorus in AMP. b The Hz frequency was 34561 Hz, and the residence time of phosphorus coordinated with Eu-DO3A was 29 μs. Figure 19 c).

Claims

1. A lanthanide-based metal complex as 31 Use of a PCEST high-field contrast agent in the identification of ATP, characterized in that: The phosphate of ATP is used as a CEST large pool signal, and the phosphate of ATP coordinated with a lanthanide metal complex is used as a CEST small pool signal, so that three exchange sites of the alpha-P, beta-P and gamma-P of ATP can be obtained 31 P CEST signals, and the ATP, ADP and AMP can be distinguished 31 P CEST signals, so that the ATP can be distinguished and identified. The lanthanide metal complex is at least one of Eu-DO3A, Tb-DO3A, Pr-DO3A and Yb-DO3A.

Images