CN109696451B - NMR determination method of PAMAM (polyamidoamine) on guest small molecule adsorption quantity - Google Patents
NMR determination method of PAMAM (polyamidoamine) on guest small molecule adsorption quantity Download PDFInfo
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Abstract
The invention belongs to the field of a method for measuring the adsorption quantity of a host molecule to a guest micromolecule in host-guest chemistry, and particularly relates to a nuclear magnetic resonance method for measuring the adsorption quantity of PAMAM to the guest micromolecule. The determination result is a specific numerical value, is intuitive, has broad spectrum, is simple to operate, accurate in result and high in accuracy, and is suitable for determining the adsorption quantity of PAMAM (polyamidoamine) to various types of guest small molecules.
Description
Technical Field
The invention belongs to the field of a method for measuring the adsorption quantity of a host molecule to a guest small molecule in host-guest chemistry, and particularly relates to a nuclear magnetic resonance method for measuring the adsorption quantity of PAMAM to the guest small molecule.
Background
The PAMAM has the characteristics of precise molecular structure, a large number of surface active functional groups, a nonpolar internal hydrophobic structure, high geometric symmetry, relatively controllable molecular mass and nanometer size, and has wide application prospect. The PAMAM can be used in the application of sustained-release drug carriers, high-efficiency catalysts, information storage materials and nano materials, the interaction between the PAMAM serving as a host molecule and various guest small molecules is widely researched, and at present, the DOSY can be used for discovering the interaction between the PAMAM and the guest small molecules by combining with other nuclear magnetic resonance methods such as chemical shift titration, NOE determination, relaxation time, saturation transfer difference spectrum and the like. Although the technical means clearly prove that the PAMAM and the guest small molecules have interaction and can indicate the specific action mode, the method for calculating the specific adsorption quantity of the PAMAM adsorbed guest small molecules by using a nuclear magnetic method has no corresponding method step.
The PAMAM can be used for measuring the adsorption quantity of the guest small molecules by combining methods such as an ultraviolet spectrophotometry method, an equilibrium dialysis method and a calorimetric titration method. However, the measurement process of these methods is complicated, the amount of the sample required is large, the measurement result of the adsorption amount is a relative range, and the accuracy is not high. In addition, the determination process of the methods is complicated, the required sample amount is large, and the method has certain limitation on the discovery of the action mechanism between the PAMAM and the guest small molecules.
Disclosure of Invention
The invention provides an NMR (nuclear magnetic resonance) measuring method for the adsorption quantity of PAMAM (polyamidoamine) to small molecules of an object, aiming at the defects that the existing methods for measuring the adsorption quantity of PAMAM to small molecules of the object, such as an ultraviolet spectrophotometry, equilibrium dialysis, calorimetric titration and the like, are relatively complex in process, relatively large in required sample quantity, large in error of a test result of the adsorption quantity, low in accuracy and the like.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
the NMR determination method of the PAMAM on the adsorption quantity of the guest small molecules is based on a nuclear magnetic resonance diffusion sequencing spectrum, and comprises the steps of determining the relative diffusion coefficients of different generations of PAMAM in a solution, drawing a standard curve of a correlation relation between the relative diffusion coefficients of different generations of PAMAM in the solution and the molecular weight of the PAMAM, determining the relative diffusion coefficients of PAMAM after adsorbing the guest small molecules in the solution, and calculating the molecular weight of PAMAM after adsorbing the guest small molecules in the solution according to the standard curve of the correlation relation between the relative diffusion coefficients of different generations of PAMAM in the solution and the molecular weight of the PAMAM, so that the adsorption quantity of the PAMAM on the guest small molecules is calculated. The method for measuring the adsorption quantity of the PAMAM adsorbed guest micromolecules based on the diffusion sequencing spectrum does not damage the sample in the whole test process, needs a small amount of sample, can directly measure the diffusion coefficient of the PAMAM before and after adsorbing the guest micromolecules without processing the sample, and further performs data analysis to directly obtain the value of the PAMAM on the specific adsorption quantity of the guest micromolecules. The result obtained by the method is a specific numerical value, is intuitive, has broad spectrum, is simple to operate, accurate in result and high in accuracy, and is suitable for determining the adsorption quantity of PAMAM (polyamidoamine) to various types of guest small molecules.
Further, the NMR measurement method is based on nuclear magnetic resonance diffusion sequencing of a two-way gradient longitudinal eddy current delay, BPPLED, pulse sequence or stimulated echo STE pulse sequence. The pulse is developed on the basis of a classical test method for measuring the molecular self-diffusion coefficient by nuclear magnetic resonance, the position of the molecule is changed due to self-diffusion movement in actual operation, the magnetization intensity cannot be completely reunited, and therefore the attenuation of signals is caused.
Further, the method for NMR measurement of the amount of adsorption of a guest small molecule by PAMAM according to claim 2, wherein: the testing steps of the nuclear magnetic resonance diffusion sequencing spectrum are as follows: 1) adjusting the temperature of the nuclear magnetic resonance spectrometer to be 273K-323K, the airflow to be 200-500 lph, and the sample tube does not rotate; 2) the sample is kept constant for 10-30 minutes at a set temperature and under a set airflow; 3) measuring a one-dimensional hydrogen nuclear magnetic spectrum of the sample; 4) calling out diffusion sequencing spectrum pulses, optimizing parameters and collecting a two-dimensional spectrum; 5) the resulting data were processed by the brook Topspin 3.1 or Dynamics Center 2.2.4 software to obtain the self-diffusion coefficient values of the samples. The temperature is 273K to 323K, the air flow is 200-500 lph, and the influence of the temperature gradient and the air flow gradient on the test result in the test process of the sample can be ensured; the sample is kept constant for 10-30 minutes under the set temperature and airflow, and parameters are optimized, so that the temperature of the sample can be kept stable in the testing process, and a more accurate testing result can be obtained; if the air flow and the temperature are not controlled, and the test parameters are not optimized, the error of the obtained test result is large; the data obtained by the software processing of the Bruk Topspin 3.1 or the dynamic center 2.2.4 can accurately fit the self-diffusion coefficient of the sample in the minimum error range, and the diffusion rate value of the molecule in the solution can be directly obtained.
Further, the nuclear magnetic resonance spectrometer in the step 1) is a 400-600 MHz liquid nuclear magnetic resonance spectrometer with a gradient field, and the nuclear magnetic resonance spectrometer with the gradient field is used for measuring the diffusion coefficient of molecules, wherein a series of different spectrums are obtained by gradually changing the intensity of the pulse gradient field, and then fitting each signal.
Furthermore, in the process of calling out diffusion sequencing spectrum pulses, optimizing parameters and acquiring a two-dimensional map in the step 4), the value range of gradient field intensity GPZ6 adopted by each nuclear magnetic resonance diffusion sequencing spectrum is between 2% and 98%; the used diffusion time delta is 40-300 ms; the pulse width/2 of the used pulse field gradient is 600-2500 mu s; the number of pulse scans used is a multiple of 8, i.e., NS-8 × n; the number of empty sweeps used is a multiple of 4, i.e., DS 4 × n; the dimension of the used two-dimensional map sampling time TD F1 is 8-128 times, and the dimension of the used sampling point number TD F2 is 16-128 k; so as to obtain the one-dimensional hydrogen spectrum with the matching proportion range of 2 to 10 percent under the maximum gradient field and the minimum gradient field. And optimizing parameters to more accurately obtain the value of the diffusion rate of the molecules in the solution.
The method comprises the following specific operation steps of measuring the relative diffusion coefficients of different generations of PAMAM in a solution, and drawing a standard curve of the correlation relationship between the relative diffusion coefficients of different generations of PAMAM in the solution and the molecular weight of the PAMAM, wherein the specific operation steps comprise: measuring diffusion rates of different generations of PAMAM and an internal standard substance in the solution, calculating the relative diffusion coefficients of the PAMAM in the solution relative to the diffusion rate of the added internal standard substance by fitting, taking the logarithm of the relative diffusion coefficients of the different generations of PAMAM in the solution as the abscissa, taking the logarithm of the molecular weights of the different generations of PAMAM as the ordinate, and fitting to obtain a standard curve of the correlation relationship between the relative diffusion coefficients of the different generations of PAMAM in the solution and the molecular weights of the PAMAM. The method adopts the correlation between the relative diffusion coefficients of different generations of PAMAM in a solution and the molecular weight relationship to make a standard curve, is simple, convenient and representative, and is suitable for the determination of PAMAM on the adsorption quantity of various object small molecules, such as homogeneous catalysts, anticancer drug small molecules, amino acids, vitamins, surfactants and the like.
Still further, the method for determining the relative diffusion coefficient of the PAMAM after the adsorption of the guest small molecules in the solution comprises the following specific operation steps: the diffusion rates of PAMAM after the adsorption of guest micromolecules and an internal standard substance in the solution are measured, fitting is carried out to respectively obtain the self-diffusion coefficients of the PAMAM and the internal standard substance in the solution, then the self-diffusion coefficient of the PAMAM after the adsorption of the guest micromolecules is divided by the self-diffusion coefficient of the internal standard substance to obtain the relative diffusion coefficient of the PAMAM after the adsorption of the guest micromolecules in the solution, the influence of the PAMAM in the solution environment and the test environment can be greatly reduced based on the relative diffusion coefficient of the PAMAM in the solution obtained by adding the internal standard substance, and the influence of environmental factors on the PAMAM molecule diffusion rate test value.
Further, the molecular weight of the PAMAM in the solution after the adsorption of the guest small molecules is calculated according to a standard curve of a correlation relationship between the relative diffusion coefficients of the PAMAM in the solution of different generations and the molecular weight of the PAMAM, so as to calculate the adsorption amount of the PAMAM to the guest small molecules, and the specific operation steps are as follows: substituting the relative diffusion coefficient of PAMAM after adsorbing the guest micromolecule into a standard curve of correlation between the relative diffusion coefficient of PAMAM in different generations in the solution and the molecular weight of the PAMAM to calculate the molecular weight of the PAMAM, subtracting the molecular weight of the PAMAM from the molecular weight, and dividing the molecular weight of the PAMAM by the molecular weight of the guest micromolecule to obtain the adsorption quantity of the PAMAM to the guest micromolecule. The method overcomes the defects of more complicated process, larger required sample amount, large error, inaccuracy, low accuracy and the like of the test result of the adsorption amount of the ultraviolet spectrophotometry, the equilibrium dialysis, the calorimetric titration and the like, and ensures that the obtained result is more accurate.
Still further, the preparation process of the sample in the step 2) comprises the following specific steps: dissolving PAMAM of different generations, PAMAM samples after adsorbing guest micromolecules and an internal standard substance into the same solvent, preparing 350-450 mu L of solution into a nuclear magnetic resonance sample tube, and placing the sample tube into a nuclear magnetic resonance spectrometer for detection, wherein the used internal standard substance and solvent are the same as the internal standard substance and solvent selected when a standard curve is drawn, and based on the fact that the internal standard substance and the samples are dissolved into the same solvent, the sample preparation can be simpler, the experimental error is reduced, and the measurement results of the diffusion rates of the samples and the internal standard substance are more accurate.
Still further, the internal standard substance is one or more of 3- (trimethylsilyl) -1-propanesulfonic acid sodium, tetramethylsilane or 1, 4-dioxane; the solvent is one or more of heavy water, deuterated methanol or deuterated chloroform. The internal standard selected is one that does not interact with PAMAM, and1the signals of H NMR chemical shift do not overlap with the PAMAM molecular signals, the relative diffusion coefficients of different PAMAMs in the solution are obtained by using the diffusion rate ratio of the PAMAM and the internal standard substance, the internal standard substance and the solute molecule are in the same environment, the influence of the environment on the diffusion rate test value can be completely eliminated, the relative diffusion coefficients of the PAMAM in the solution and the molecular weight have a definite relation, the molecular weight can be estimated through the relative diffusion rate of the PAMAM after adsorbing the guest micromolecules, and the adsorption quantity is further calculated, so that the method has accurate results.
Drawings
FIG. 1 is a standard curve of the correlation between the relative diffusion coefficient of PAMAM of different generations in heavy water and its molecular weight according to example 1 of the present invention.
FIG. 2 is a standard curve of the correlation between the relative diffusion coefficient of PAMAM of different generations in deuterated chloroform and its molecular weight in example 2 of the present invention.
FIG. 3 is a standard curve of the correlation between the relative diffusion coefficient of PAMAM of different generations in deuterated methanol and its molecular weight in example 3 of the present invention.
FIG. 4 is a standard curve of the correlation between the relative diffusion coefficient and the molecular weight of PAMAM of different generations in a mixed solution of heavy water and deuterated methanol in example 4 of the present invention.
Detailed Description
Example 1:
in this embodiment, an NMR measurement method of PAMAM on 5-Fluorouracil (FU) adsorption capacity is based on a bi-directional gradient longitudinal eddy delay, that is, a BPPLED pulse sequence nuclear magnetic resonance diffusion sequencing spectrum, by measuring relative diffusion coefficients of PAMAM of different generations in a solution, drawing a standard curve of a correlation between the relative diffusion coefficients of PAMAM of different generations in the solution and molecular weights thereof, and then measuring the relative diffusion coefficients of PAMAM after adsorbing FU molecules in the solution, and according to the standard curve of the correlation between the relative diffusion coefficients of PAMAM of different generations in the solution and molecular weights thereof, calculating the molecular weights of PAMAM after adsorbing FU molecules in the solution, thereby calculating the adsorption capacity of PAMAM on FU molecules, and specifically including the following steps:
the method comprises the following steps: the nuclear magnetic resonance diffusion sequencing spectrum test specifically comprises the following steps:
1) adjusting the temperature of the nuclear magnetic resonance spectrometer to 273K, the airflow to 200lph and the sample tube not to rotate; the nuclear magnetic resonance spectrometer is a 400MHz liquid nuclear magnetic resonance spectrometer with a gradient field.
2) The sample is constant for 10 minutes under the set temperature and airflow; the preparation process of the sample comprises the following specific steps: dissolving PAMAM samples (FU: G3PAMAM is 50:1,25:1, FU: G5PAMAM is 100:1, namely G3+50FU, G3+25FU, G5+100FU) which are 4mg of each PAMAM with different generations of G0, G2, G3, G3.5 and G5 and adsorb three FU molecules in a molar ratio, 5 microliter of an internal standard substance 1, 4-dioxane (10 microliter of 1, 4-dioxane is diluted by 20 times with heavy water) in heavy water to prepare 400 microliter of solution in a nuclear magnetic resonance sample tube, and putting the sample tube into a nuclear magnetic resonance spectrometer to be tested, wherein the internal standard substance and the solvent are 1, 4-dioxane and heavy water together with the internal standard substance and the solvent selected when a standard curve is drawn.
3) Measuring a one-dimensional hydrogen nuclear magnetic spectrum of the sample;
4) calling out a diffusion sequencing spectrum pulse and optimizing parameters, wherein in the process of acquiring a two-dimensional map, the value of gradient field strength GPZ6 is set to be 2%, and diffusion time delta (G0, G2, G3, G3.5, G5, G3+50FU, G3+25FU and G5+100FU systems) are respectively 40ms, 80ms, 100ms, 100ms, 200ms, 200ms, 200ms and 200 ms; gradient application time/2 is 1000 mus, and a first one-dimensional hydrogen spectrum is collected; then copying a first pulse sequence, setting the value of gradient field strength GPZ6 as 95%, changing the gradient application time/2 as (the setting values of G0, G2, G3, G3.5, G5, G3+50FU, G3+25FU and G5+100FU systems are respectively 600 mus, 750 mus, 1300 mus, 1700 mus, 2200 mus, 1550 mus, 1800 mus and 2000 mus), and acquiring a second hydrogen spectrum under the condition; then copying a second hydrogen spectrum pulse sequence, changing the one-dimensional spectrum into a two-dimensional spectrum, wherein the used pulse scanning times are 16, namely NS is 8 x 2; the number of empty sweeps used was 8, i.e. DS 4 x 2; the used two-dimensional map sampling times TD F1 are 16 times, and the used sampling point number TD F2 is 16 k; so as to obtain a one-dimensional hydrogen spectrum with the matching proportion range of 5 percent under the maximum gradient field and the minimum gradient field; collecting a two-dimensional map;
5) the resulting data were processed by the bruker Topspin 3.1 software to obtain the self-diffusion coefficient values of the samples.
Step two: the method comprises the following steps of measuring the relative diffusion coefficients of PAMAM of different generations in a solution, and drawing a standard curve of the correlation relationship between the relative diffusion coefficients of the PAMAM of different generations in the solution and the molecular weight of the PAMAM, wherein the standard curve specifically comprises the following steps: measuring the diffusion rates of different generations of PAMAM and internal standard substance 1, 4-dioxane in the solution, calculating the relative diffusion coefficients of the PAMAM in the solution relative to the diffusion rate of the added 1, 4-dioxane by fitting (table 1), taking the logarithm of the relative diffusion coefficients of different generations of PAMAM in the solution as the abscissa and the logarithm of the molecular weights of different generations of PAMAM as the ordinate (table 2), and fitting to obtain a standard curve of the correlation between the relative diffusion coefficients of different generations of PAMAM in heavy water and the molecular weights (figure 1).
Step three: measuring the relative diffusion coefficient of the PAMAM after adsorbing FU molecules in the solution, specifically: the diffusion rates of PAMAM and 1, 4-dioxane in the solution after adsorption of FU molecules are measured, the self-diffusion coefficients of the PAMAM and the 1, 4-dioxane in the solution are respectively obtained through fitting, and then the relative diffusion coefficient of the PAMAM in the solution after adsorption of FU molecules is obtained by dividing the self-diffusion coefficient of the PAMAM by the self-diffusion coefficient of the 1, 4-dioxane (Table 3).
Step four: according to a standard curve of a correlation relation between the relative diffusion coefficient of PAMAM of different generations in heavy water and the molecular weight of the PAMAM, the molecular weight of the PAMAM after adsorbing FU molecules in a solution is calculated, so that the adsorption capacity of the PAMAM to the FU molecules is calculated (table 4), and the method specifically comprises the following steps: substituting the relative diffusion coefficient of the PAMAM after adsorbing FU molecules into a standard curve of correlation between the relative diffusion coefficient of different generations of PAMAM in the solution and the molecular weight of the PAMAM, calculating the molecular weight of the PAMAM, subtracting the molecular weight of the PAMAM from the molecular weight, and dividing the molecular weight of the FU molecules by the molecular weight of the FU molecules to obtain the adsorption quantity of the PAMAM to the FU molecules.
Table 1 shows the relative diffusion coefficient values of PAMAM in heavy water and the related values.
Table 2 shows the logarithmic values of the relative diffusion coefficients of PAMAM of different generations and the relative values of the logarithms of the corresponding molecular weights.
FIG. 1 is a standard curve showing the relationship between the relative diffusion coefficient of PAMAM of different generations in heavy water and its molecular weight.
Table 3 shows the relative diffusion coefficient values and the related values of the PAMAM adsorbing FU molecules with different molar ratios in the invention.
Table 4 shows the calculated correlation values of the adsorption amounts of FU by PAMAM in the present invention.
TABLE 1
TABLE 2
TABLE 3
TABLE 4
Example 2:
in this embodiment, an NMR measurement method for determining an adsorption amount of PAMAM to vitamin B7 is based on a stimulated echo STE pulse sequence nuclear magnetic resonance diffusion sequencing spectrum, and includes the steps of measuring relative diffusion coefficients of PAMAM of different generations in a solution, drawing a standard curve of a correlation relationship between the relative diffusion coefficients of PAMAM of different generations in the solution and molecular weights thereof, then measuring the relative diffusion coefficients of PAMAM of different generations after adsorbing vitamin B7 molecules in the solution, and calculating the molecular weights of PAMAM after adsorbing vitamin B7 molecules in the solution according to the standard curve of the correlation relationship between the relative diffusion coefficients of PAMAM of different generations in the solution and molecular weights thereof, thereby calculating the adsorption amount of PAMAM to vitamin B7 molecules, and specifically including the following steps:
the method comprises the following steps: the nuclear magnetic resonance diffusion sequencing spectrum test specifically comprises the following steps:
1) adjusting the temperature of the nuclear magnetic resonance spectrometer to 323K, the airflow to 500lph, and the sample tube does not rotate; the nuclear magnetic resonance spectrometer is a 500MHz liquid nuclear magnetic resonance spectrometer with a gradient field.
2) The sample is constant for 30 minutes under the set temperature and airflow; the preparation process of the sample comprises the following specific steps: taking 4mg of PAMAM of G0, G2, G3, G3.5 and G5 in different generations and PAMAM samples (vitamin B7: G5PAMAM is 10:1,25:1,50:1,70:1, namely G5+10VB, G5+25VB, G5+50VB, G5+70VB) after absorbing four molecules of vitamin B7 in molar ratio and dissolving 1mg of internal standard substance tetramethylsilane in deuterated chloroform to prepare 400 mu L of solution in a nuclear magnetic resonance sample tube, and putting the sample tube into a nuclear magnetic resonance spectrometer to be tested, wherein the internal standard substance and the solvent used in the nuclear magnetic resonance spectrometer and the internal standard substance and the solvent used in the process of drawing a standard curve are tetramethylsilane and deuterated chloroform.
3) Measuring a one-dimensional hydrogen nuclear magnetic spectrum of the sample;
4) calling out a diffusion sequencing spectrum pulse, optimizing parameters, wherein in the process of acquiring a two-dimensional map, the value of gradient field strength GPZ6 is set to be 2%, and diffusion time delta (G0, G2, G3, G3.5, G5, G5+10VB, G5+25VB, G5+50VB, and G5+70VB systems) is respectively 40ms, 80ms, 100ms, 100ms, 200ms, 200ms, 200ms, 300ms and 300 ms; gradient application time/2 is 1000 mus, and a first one-dimensional hydrogen spectrum is collected; then copying a first pulse sequence, setting the value of gradient field strength GPZ6 as 98%, changing the gradient application time/2 as (the setting values of G5+10VB, G5+25VB, G5+50VB and G5+70VB systems are respectively 600 mus, 750 mus, 1300 mus, 1700 mus, 2200 mus, 2000 mus, 2200 mus, 1200 mus and 1750 mus), and acquiring a second hydrogen spectrum under the condition; then copying a second hydrogen spectrum pulse sequence, changing the one-dimensional spectrum into a two-dimensional spectrum, wherein the used pulse scanning times are 32, namely NS is 8 x 4; the number of empty sweeps used was 16, i.e., DS 4 x 4; the used two-dimensional map sampling times TD F1 are 128 times, and the used sampling point number TD F2 is 128 k; so as to obtain a one-dimensional hydrogen spectrum with the matching proportion range of 2 percent under the maximum gradient field and the minimum gradient field; collecting a two-dimensional map;
5) the resulting data were processed by the brook Dynamics Center 2.2.4 software to obtain the self-diffusion coefficient values of the samples.
Step two: the method comprises the following steps of measuring the relative diffusion coefficients of PAMAM of different generations in a solution, and drawing a standard curve of the correlation relationship between the relative diffusion coefficients of the PAMAM of different generations in the solution and the molecular weight of the PAMAM, wherein the standard curve specifically comprises the following steps: the diffusion rates of PAMAM in different generations in G0, G2, G3, G3.5 and G5 and tetramethylsilane as an internal standard substance in the solution are measured, the relative diffusion coefficients of the PAMAM in the solution relative to the diffusion rate of tetramethylsilane added are calculated by fitting (Table 5), then the logarithm of the relative diffusion coefficients of the PAMAM in different generations in the solution is used as an abscissa, the logarithm of the molecular weight of the PAMAM in different generations is used as an ordinate (Table 6), and a standard curve of the correlation between the relative diffusion coefficients of the PAMAM in different generations in the deuterated chloroform and the molecular weight thereof is obtained by fitting (FIG. 2).
Step three: determining the relative diffusion coefficient of the PAMAM after the vitamin B7 molecules are adsorbed in the solution, and specifically: the diffusion rates of PAMAM and tetramethylsilane in the solution after adsorption of vitamin B7 molecules were measured, and fitting was performed to obtain the self-diffusion coefficients of PAMAM and tetramethylsilane in the solution, respectively, and then the relative diffusion coefficient of PAMAM in the solution after adsorption of vitamin B7 molecules was obtained by dividing the self-diffusion coefficient of PAMAM by the self-diffusion coefficient of 1, 4-dioxane (table 7).
Step four: according to a standard curve of a correlation relation between the relative diffusion coefficient of different generations of PAMAM in deuterated chloroform and the molecular weight of the PAMAM, the molecular weight of the PAMAM in the solution after the adsorption of vitamin B7 molecules is calculated, so that the adsorption amount of the PAMAM on vitamin B7 molecules is calculated (table 8), and the specific steps are as follows: substituting the relative diffusion coefficient of the PAMAM after absorbing the vitamin B7 molecules into a standard curve of correlation between the relative diffusion coefficient of different generations of PAMAM in the solution and the molecular weight of the PAMAM to calculate the molecular weight of the PAMAM, subtracting the molecular weight of the PAMAM from the molecular weight, and dividing the molecular weight by the molecular weight of the vitamin B7 molecule to obtain the adsorption quantity of the PAMAM to the vitamin B7 molecule.
Table 5 shows the relative diffusivity values of PAMAM in deuterated chloroform and the related values thereof in the present invention.
Table 6 shows the logarithmic values of the relative diffusion coefficients of PAMAM of different generations and the relative values of the logarithms of the corresponding molecular weights.
FIG. 2 is a standard curve of the correlation between the relative diffusion coefficient of PAMAM of different generations in deuterated chloroform and its molecular weight.
Table 7 shows the relative diffusion coefficient values and the related values of the PAMAM adsorbed vitamin B7 molecules in different molar ratios.
Table 8 shows the calculated values of the amount of vitamin B7 adsorbed by PAMAM in the present invention.
TABLE 5
TABLE 6
TABLE 7
TABLE 8
Example 3
In this embodiment, an NMR measurement method for PAMAM to phenylbutazone adsorption amount is based on a bi-directional gradient longitudinal eddy delay, that is, a BPPLED pulse sequence nuclear magnetic resonance diffusion sequencing spectrum, and includes the steps of measuring relative diffusion coefficients of different generations of PAMAM in a solution, drawing a standard curve of a correlation between the relative diffusion coefficients of different generations of PAMAM in the solution and molecular weights thereof, then measuring the relative diffusion coefficients of PAMAM after adsorbing phenylbutazone molecules in the solution, and calculating the molecular weights of PAMAM after adsorbing phenylbutazone molecules in the solution according to the standard curve of the correlation between the relative diffusion coefficients of different generations of PAMAM in the solution and molecular weights thereof, thereby calculating the adsorption amount of PAMAM to phenylbutazone molecules, and specifically including the following steps:
the method comprises the following steps: the nuclear magnetic resonance diffusion sequencing spectrum test specifically comprises the following steps:
1) adjusting the temperature of the nuclear magnetic resonance spectrometer to 298K, the airflow to 400lph, and the sample tube does not rotate; the nuclear magnetic resonance spectrometer is a 600MHz liquid nuclear magnetic resonance spectrometer with a gradient field.
2) The sample is constant for 20 minutes under the set temperature and airflow; the preparation process of the sample comprises the following specific steps: respectively taking 4mg of PAMAM of G0, G2, G3, G3.5 and G5 in different generations, and a PAMAM sample (phenylbutazone: G5PAMAM ═ 5:1,10:1,20:1,30:1) after adsorbing four phenylbutazone molecules in a molar ratio and 1mg of an internal standard substance 3- (trimethylsilyl) -1-propanesulfonic acid sodium are dissolved in heavy water, preparing 450 mu L of solution in a nuclear magnetic resonance sample tube, putting the sample tube into a nuclear magnetic resonance spectrometer for testing, wherein the used internal standard substance and solvent and the internal standard substance and solvent selected when a standard curve is drawn are both 3- (trimethylsilyl) -1-propanesulfonic acid sodium and heavy water.
3) Measuring a one-dimensional hydrogen nuclear magnetic spectrum of the sample;
4) and (2) calling out a diffusion sequencing spectrum pulse, optimizing parameters, wherein in the process of acquiring a two-dimensional map, the value of gradient field strength GPZ6 is set to be 2%, and the diffusion time delta (G0, G2, G3, G3.5, G5, phenyl butyrone: g5PAMAM ═ 5:1,10:1,20:1,30:1 systems) for 40ms, 80ms, 100ms, 100ms, 200ms, 200ms, 300ms, 300ms, 300ms, respectively; gradient application time/2 is 1000 mus, and a first one-dimensional hydrogen spectrum is collected; then, a first pulse sequence is copied, the value of the gradient field strength GPZ6 is set to be 95%, and the gradient application time/2 is changed to be (the set values of systems of phenylbutazone: G5PAMAM ═ 5:1,10:1,20:1 and 30:1 are respectively 600 mus, 750 mus, 1300 mus, 1700 mus, 2200 mus, 2000 mus, 1200 mus, 1750 mus and 2500 mus), and a second hydrogen spectrum is collected under the condition; then copying a second hydrogen spectrum pulse sequence, changing the one-dimensional spectrum into a two-dimensional spectrum, wherein the used pulse scanning times are 8, namely NS is 8 x 1; the number of empty sweeps used was 4, i.e. DS 4 x 1; the dimension of the used two-dimensional map sampling frequency TD F1 is 8, and the dimension of the used sampling point number TD F2 is 64 k; so as to obtain a one-dimensional hydrogen spectrum with the matching proportion range of 10 percent under the maximum gradient field and the minimum gradient field; collecting a two-dimensional map;
5) the resulting data were processed by the brook Dynamics Center 2.2.4 software to obtain the self-diffusion coefficient values of the samples.
Step two: the method comprises the following steps of measuring the relative diffusion coefficients of PAMAM of different generations in a solution, and drawing a standard curve of the correlation relationship between the relative diffusion coefficients of the PAMAM of different generations in the solution and the molecular weight of the PAMAM, wherein the standard curve specifically comprises the following steps: the diffusion rates of different generations of PAMAM and internal standard substance 3- (trimethylsilyl) -1-propanesulfonic acid sodium salt in the solution are measured, the relative diffusion coefficients of the PAMAM in the solution relative to the diffusion rate of the added 3- (trimethylsilyl) -1-propanesulfonic acid sodium salt are calculated by fitting (table 9), the logarithm of the relative diffusion coefficients of the different generations of PAMAM in the solution is used as an abscissa, the logarithm of the molecular weights of the different generations of PAMAM is used as an ordinate (table 10), and a standard curve of the correlation relationship between the relative diffusion coefficients of the different generations of PAMAM in the heavy water and the molecular weights of the PAMAM is obtained by fitting (figure 3).
Step three: the determination of the relative diffusion coefficient of the PAMAM after the adsorption of the phenylbutazone molecules in the deuterated methanol specifically comprises the following steps: the diffusion rates of the PAMAM and the 3- (trimethylsilyl) -1-propanesulfonic acid sodium salt (DSS) in the solution after the adsorption of the phenylbutazone molecules were measured, and fitting was performed to obtain the self-diffusion coefficients of the PAMAM and the 3- (trimethylsilyl) -1-propanesulfonic acid sodium salt respectively, and then the relative diffusion coefficient of the PAMAM in the solution after the adsorption of the phenylbutazone molecules was obtained by dividing the self-diffusion coefficient of the PAMAM by the self-diffusion coefficient of the 3- (trimethylsilyl) -1-propanesulfonic acid sodium salt (Table 11).
Step four: according to the standard curve of the correlation between the relative diffusion coefficient of different generations of PAMAM in deuterated methanol and the molecular weight thereof, the molecular weight of the PAMAM in the solution after adsorption of phenylbutazone molecules is calculated, so that the adsorption amount of the PAMAM on the phenylbutazone molecules is calculated (table 12), and the method specifically comprises the following steps: substituting the relative diffusion coefficient of PAMAM after adsorbing the phenylbutazone molecules into a standard curve of correlation between the relative diffusion coefficient of PAMAM in different generations in the solution and the molecular weight of the PAMAM to calculate the molecular weight of the PAMAM, subtracting the molecular weight of the PAMAM from the molecular weight, and dividing the molecular weight by the molecular weight of the phenylbutazone molecules to obtain the adsorption quantity of the PAMAM to the phenylbutazone molecules.
Table 9 shows the relative diffusion coefficient values of PAMAM in heavy water and the related values.
Table 10 shows the logarithmic values of the relative diffusion coefficients of PAMAM of different generations and the relative values of the logarithms of the corresponding molecular weights.
FIG. 3 is a standard curve of the correlation between the relative diffusion coefficient of PAMAM of different generations in deuterated methanol and its molecular weight.
Table 11 shows the relative diffusion coefficient values and the related values of the PAMAM in the present invention after adsorbing the phenylbutazone molecules with different molar ratios.
Table 12 shows the calculated values of the amount of adsorption of PAMAM on phenylbutazone in accordance with the present invention.
TABLE 9
Watch 10
TABLE 11
TABLE 12
Example 4
In this embodiment, an NMR measurement method for PAMAM adsorbing mycophenolic acid molecular weight is based on a bi-directional gradient longitudinal eddy delay, that is, a BPPLED pulse sequence nuclear magnetic resonance diffusion sequencing spectrum, and includes the steps of measuring relative diffusion coefficients of PAMAM of different generations in a solution, drawing a standard curve of a correlation between the relative diffusion coefficients of PAMAM of different generations in the solution and the molecular weight of PAMAM of different generations, measuring the relative diffusion coefficients of PAMAM adsorbing mycophenolic acid molecules in the solution, and calculating the molecular weight of PAMAM adsorbing mycophenolic acid molecules in the solution according to the standard curve of the correlation between the relative diffusion coefficients of PAMAM of different generations in the solution and the molecular weight of PAMAM, so as to calculate the mycophenolic acid molecular weight adsorbed by PAMAM, specifically including the following steps:
the method comprises the following steps: the nuclear magnetic resonance diffusion sequencing spectrum test specifically comprises the following steps:
1) adjusting the temperature of the nuclear magnetic resonance spectrometer to 298K, the airflow to 300lph, and the sample tube does not rotate; the nuclear magnetic resonance spectrometer is a 600MHz liquid nuclear magnetic resonance spectrometer with a gradient field.
2) The sample is constant for 20 minutes under the set temperature and airflow; the preparation process of the sample comprises the following specific steps: dissolving PAMAM samples (mycophenolic acid: G5PAMAM is 5:1,10:1,20:1,30:1) which are respectively 4mg of different generations of PAMAM of G0, G2, G3, G3.5 and G5 and adsorb four mycophenolic acid molecules in a molar ratio, 5 mu L of an internal standard substance 1, 4-dioxane (10 mu L of the 1, 4-dioxane is diluted by 20 times by a mixed solution of heavy water and deuterated methanol) in a mixed solution of heavy water and deuterated methanol to prepare 400 mu L of solution, placing the sample tube in a nuclear magnetic resonance spectrometer for testing, wherein the internal standard substance and the solvent used and the internal standard substance and the solvent used in drawing a standard curve are both mixed solutions of 1, 4-dioxane, deuterated methanol and heavy water.
3) Measuring a one-dimensional hydrogen nuclear magnetic spectrum of the sample;
4) and calling out a diffusion sorting spectrum pulse, optimizing parameters, wherein in the process of acquiring a two-dimensional map, the value of gradient field strength GPZ6 is set to be 2%, and the diffusion time delta (G0, G2, G3, G3.5, G5, mycophenolic acid: g5PAMAM ═ 5:1,10:1,20:1,30:1 systems) for 40ms, 80ms, 100ms, 100ms, 200ms, 200ms, 200ms, 300ms, respectively; gradient application time/2 is 1000 mus, and a first one-dimensional hydrogen spectrum is collected; then, the first pulse sequence is copied, the value of the gradient field strength GPZ6 is set to be 98%, the gradient application time/2 is changed to be (the set values of G0, G2, G3, G3.5, G5, mycophenolic acid: G5PAMAM ═ 5:1,10:1,20:1,30:1 are respectively 600 mus, 750 mus, 1300 mus, 1700 mus, 2200 mus, 2000 mus, 1230 mus, 1360 mus and 1800 mus), and a second hydrogen spectrum is collected under the condition; then copying a second hydrogen spectrum pulse sequence, changing the one-dimensional spectrum into a two-dimensional spectrum, wherein the used pulse scanning times are 16, namely NS is 8 x 2; the number of empty sweeps used was 8, i.e. DS 4 x 2; the used two-dimensional map sampling times TD F1 are 16 times, and the used sampling point number TD F2 is 16 k; so as to obtain a one-dimensional hydrogen spectrum with the matching proportion range of 2 percent under the maximum gradient field and the minimum gradient field; collecting a two-dimensional map;
5) the resulting data were processed by the bruker Topspin 3.1 software to obtain the self-diffusion coefficient values of the samples.
Step two: the method comprises the following steps of measuring the relative diffusion coefficients of PAMAM of different generations in a solution, and drawing a standard curve of the correlation relationship between the relative diffusion coefficients of the PAMAM of different generations in the solution and the molecular weight of the PAMAM, wherein the standard curve specifically comprises the following steps: the diffusion rates of different generations of PAMAM and internal standard substance 1, 4-dioxane in G0, G2, G3, G3.5 and G5 in the solution are measured, the relative diffusion coefficients of the PAMAM in the solution relative to the diffusion rate of the added 1, 4-dioxane are calculated by fitting (table 13), then the logarithm of the relative diffusion coefficients of different generations of PAMAM in the solution is used as the abscissa, the logarithm of the molecular weights of different generations of PAMAM is used as the ordinate (table 14), and a standard curve of the correlation between the relative diffusion coefficients of different generations of PAMAM in the mixed solution of heavy water and deuterium methanol and the molecular weights thereof is obtained by fitting (figure 4).
Step three: the method for measuring the relative diffusion coefficient of the PAMAM after the mycophenolic acid molecule is adsorbed in the solution specifically comprises the following steps: the diffusion rates of PAMAM and 1, 4-dioxane in the solution after adsorption of the mycophenolic acid molecules were measured, fitting was performed to obtain the self-diffusion coefficients of PAMAM and 1, 4-dioxane in the solution, respectively, and the relative diffusion coefficients of PAMAM and 1, 4-dioxane in the solution after adsorption of the mycophenolic acid molecules were obtained by dividing the self-diffusion coefficients of PAMAM by the self-diffusion coefficients of 1, 4-dioxane (table 15).
Step four: according to the standard curve of the correlation between the relative diffusion coefficient of different generations of PAMAM in the mixed solution of heavy water and deuterated methanol and the molecular weight thereof, the molecular weight of the PAMAM in the solution after mycophenolic acid molecule adsorption is calculated, so that the adsorption amount of the PAMAM to mycophenolic acid molecules is calculated (table 16), and the specific steps are as follows: substituting the relative diffusion coefficient of the PAMAM after the mycophenolic acid molecule is adsorbed into a standard curve of a correlation relation between the relative diffusion coefficient of the PAMAM in different generations in the solution and the molecular weight of the PAMAM, calculating the molecular weight of the PAMAM, subtracting the molecular weight of the PAMAM from the molecular weight, and dividing the molecular weight by the molecular weight of the mycophenolic acid molecule to obtain the adsorption quantity of the PAMAM to the mycophenolic acid molecule.
Table 13 shows the relative diffusivity values of PAMAM in the mixed solution of heavy water and deuterated methanol and the related values thereof in accordance with the present invention.
Table 14 shows the logarithmic values of the relative diffusion coefficients of PAMAM of different generations and the relative values of the logarithms of the corresponding molecular weights.
FIG. 4 is a standard curve of the correlation between the relative diffusion coefficient of PAMAM of different generations in a mixed solution of heavy water and deuterated methanol and the molecular weight thereof.
Table 15 shows the relative diffusivity values of PAMAM adsorbing mycophenolic acid molecules in different molar ratios and the related values thereof.
Table 16 shows the calculated values of the adsorption amounts of mycophenolic acid by PAMAM in the present invention.
Watch 13
TABLE 14
Watch 15
TABLE 16
In order to show that the invention has the advantages of direct observation of test results and high accuracy, the test analysis results of the adsorption of 5-fluorouracil to G3PAMAM and G5PAMAM are compared with the test analysis results of the adsorption of 5-fluorouracil to G3PAMAM and G5PAMAM by methods such as ultraviolet spectrophotometry, equilibrium dialysis, calorimetric titration and the like (table 17), and the comparison of the data shows that the results of the invention are more accurate and precise to specific values.
Table 17 shows the comparison of the adsorption quantity of PAMAM adsorption object small molecules measured by the DOSY method in the present invention with other methods
TABLE 17
Claims (9)
1. An NMR determination method of PAMAM for the adsorption quantity of a guest small molecule is characterized in that: the NMR determination method is based on a nuclear magnetic resonance diffusion sequencing spectrum, the relative diffusion coefficients of different generations of PAMAM in a solution are determined, a standard curve of a correlation relation between the relative diffusion coefficients of different generations of PAMAM in the solution and the molecular weight of the PAMAM is drawn, then the relative diffusion coefficients of PAMAM adsorbing the guest micromolecules in the solution are determined, and the molecular weight of PAMAM adsorbing the guest micromolecules in the solution is calculated according to the standard curve of the correlation relation between the relative diffusion coefficients of different generations of PAMAM in the solution and the molecular weight of the PAMAM, so that the adsorption amount of the PAMAM to the guest micromolecules is calculated; the NMR measurement method is based on nuclear magnetic resonance diffusion sequencing of a two-way gradient longitudinal eddy current delay, BPPLED pulse sequence or stimulated echo STE pulse sequence.
2. The NMR method for measuring the adsorption amount of a guest small molecule by PAMAM according to claim 1, wherein: the testing steps of the nuclear magnetic resonance diffusion sequencing spectrum are as follows: 1) adjusting the temperature of the nuclear magnetic resonance spectrometer to be 273K-323K, the airflow to be 200-500 lph, and the sample tube does not rotate; 2) the sample is kept constant for 10-30 minutes at a set temperature and under a set airflow; 3) measuring a one-dimensional hydrogen nuclear magnetic spectrum of the sample; 4) calling out diffusion sequencing spectrum pulses, optimizing parameters and collecting a two-dimensional spectrum; 5) the resulting data were processed by the brook Topspin 3.1 or Dynamics Center 2.2.4 software to obtain the self-diffusion coefficient values of the samples.
3. The NMR method for measuring the adsorption amount of a guest small molecule by PAMAM according to claim 2, wherein: the nuclear magnetic resonance spectrometer in the step 1) is a 400-600 MHz liquid nuclear magnetic resonance spectrometer with a gradient field.
4. The NMR method for measuring the adsorption amount of a guest small molecule by PAMAM according to claim 3, wherein: in the step 4), in the process of calling out diffusion sequencing spectrum pulses, optimizing parameters and acquiring a two-dimensional map, the value range of gradient field intensity GPZ6 adopted by each nuclear magnetic resonance diffusion sequencing spectrum is between 2% and 98%; the used diffusion time delta is 40-300 ms; the pulse width/2 of the used pulse field gradient is 600-2500 mu s; the number of pulse scans used is a multiple of 8, i.e., NS-8 × n; the number of empty sweeps used is a multiple of 4, i.e., DS 4 × n; the dimension of the used two-dimensional map sampling time TD F1 is 8-128 times, and the dimension of the used sampling point number TD F2 is 16-128 k; so as to obtain the one-dimensional hydrogen spectrum with the matching proportion range of 2 to 10 percent under the maximum gradient field and the minimum gradient field.
5. The NMR method for measuring the adsorption amount of a guest small molecule by PAMAM according to claim 4, wherein: the method comprises the following specific operation steps of measuring the relative diffusion coefficients of different generations of PAMAM in a solution, and drawing a standard curve of the correlation relationship between the relative diffusion coefficients of different generations of PAMAM in the solution and the molecular weight of the PAMAM, wherein the specific operation steps comprise: measuring diffusion rates of different generations of PAMAM and an internal standard substance in the solution, calculating the relative diffusion coefficients of the PAMAM in the solution relative to the diffusion rate of the added internal standard substance by fitting, taking the logarithm of the relative diffusion coefficients of the different generations of PAMAM in the solution as the abscissa, taking the logarithm of the molecular weights of the different generations of PAMAM as the ordinate, and fitting to obtain a standard curve of the correlation relationship between the relative diffusion coefficients of the different generations of PAMAM in the solution and the molecular weights of the PAMAM.
6. The NMR method for measuring the adsorption amount of a guest small molecule by PAMAM according to claim 5, wherein: the method for measuring the relative diffusion coefficient of PAMAM after the adsorption of guest micromolecules in the solution comprises the following specific operation steps: and measuring the diffusion rates of the PAMAM after the adsorption of the guest micromolecules and the internal standard substance in the solution, fitting to respectively obtain the self-diffusion coefficients of the PAMAM and the internal standard substance in the solution, and dividing the self-diffusion coefficient of the internal standard substance by the self-diffusion coefficient of the PAMAM to obtain the relative diffusion coefficient of the PAMAM after the adsorption of the guest micromolecules in the solution.
7. The NMR method for measuring the adsorption amount of a guest small molecule by PAMAM according to claim 6, wherein: the method comprises the following steps of calculating the molecular weight of PAMAM in a solution after adsorbing guest small molecules according to a standard curve of a correlation relation between the relative diffusion coefficient of the PAMAM in the solution and the molecular weight of the PAMAM, wherein the relative diffusion coefficient of the PAMAM in different generations and the molecular weight of the PAMAM are different in generations, so that the adsorption amount of the PAMAM to the guest small molecules is calculated, and the specific operation steps are as follows: substituting the relative diffusion coefficient of PAMAM after adsorbing the guest micromolecule into a standard curve of correlation between the relative diffusion coefficient of PAMAM in different generations in the solution and the molecular weight of the PAMAM to calculate the molecular weight of the PAMAM, subtracting the molecular weight of the PAMAM from the molecular weight, and dividing the molecular weight of the PAMAM by the molecular weight of the guest micromolecule to obtain the adsorption quantity of the PAMAM to the guest micromolecule.
8. The method of claim 7, wherein the method comprises the steps of: the preparation process of the sample in the step 2) comprises the following specific steps: dissolving PAMAM with different generations, PAMAM samples after adsorption of guest micromolecules and an internal standard substance into the same solvent, preparing 350-450 mu L of solution into a nuclear magnetic resonance sample tube, and placing the sample tube into a nuclear magnetic resonance spectrometer for detection, wherein the internal standard substance and the solvent are the same as the internal standard substance and the solvent selected when a standard curve is drawn.
9. The method of claim 8, wherein the method comprises the following steps: the internal standard substance is one or more of 3- (trimethylsilyl) -1-propanesulfonic acid sodium, tetramethylsilane or 1, 4-dioxane; the solvent is one or more of heavy water, deuterated methanol or deuterated chloroform.
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