CN110806421A - NMR detection method for PAMAM and guest small molecule interaction mode - Google Patents
NMR detection method for PAMAM and guest small molecule interaction mode Download PDFInfo
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Abstract
The invention relates to an NMR detection method of PAMAM and guest small molecule interaction mode, the NMR detection method is based on a one-dimensional hydrogen spectrum, a DOSY spectrum, a NOESY spectrum, a CSSFs-TOCSY spectrum and a pure shift spectrum to detect the PAMAM and guest small molecule interaction mode, at least the one-dimensional hydrogen spectrum, the DOSY spectrum and the NOESY spectrum of the PAMAM and the guest small molecule are selected, when the overlapping of the peaks in the one-dimensional hydrogen spectrum of the PAMAM and the guest small molecule is difficult to identify and belong to, one or two of the CSSFs-TOCSY spectrum and the pure shift spectrum are further selected to separate the overlapping peaks, and the interaction mode of the PAMAM and the guest small molecule can be known by comparing the changes of the spectra before and after the interaction of the PAMAM and the guest small molecule. The detection method has the advantages of small amount of samples to be tested, high test accuracy, high resolution, capability of realizing nondestructive testing of the samples and the like. The detection method provided by the invention provides an effective and reliable solution for researching the ambiguous phenomenon of spectral peak overlapping attribution existing in the interaction mode of PAMAM and guest small molecules.
Description
Technical Field
The invention relates to the technical field of nuclear magnetic resonance detection, in particular to an NMR detection method of PAMAM and guest small molecule interaction mode.
Background
The PAMAM is a novel three-dimensional and highly ordered polymer compound, and has the characteristics of relatively controllable molecular weight, abundant surface functional groups and nonpolar internal hydrophobic cavities. When PAMAM interacts with the guest small molecule, the PAMAM not only can be used as a carrier of the guest small molecule, but also can improve the chemical or biological activity of the guest small molecule. At present, the interaction mode of PAMAM and guest small molecules has been widely applied to many fields, such as reducing the surface tension and critical micelle concentration of a surfactant by utilizing the interaction of PAMAM and the surfactant; the activity of the catalyst is improved by utilizing the interaction of the PAMAM and the catalyst; the interaction between PAMAM and slow-release drug molecules is utilized to increase the water solubility of insoluble drugs. However, there is no systematic NMR detection method to determine the mode of interaction of PAMAM with guest small molecules.
The method for determining the interaction mode of the PAMAM and the guest small molecules can adopt a calorimetric titration method and an ultraviolet spectrophotometry method, but the methods have large sample demand and low accuracy. At present, the method for detecting the interaction mode of the PAMAM and the guest small molecule can also use the combination of one-dimensional hydrogen spectrometry and NOESY spectra, but spectral peaks are easy to overlap, so that the signal attribution is unclear, the accuracy is not high, and the method has limitation on the research on the interaction mode of the PAMAM and the guest small molecule.
Disclosure of Invention
The invention provides an NMR detection method of PAMAM and guest micromolecule interaction mode, aiming at the defects of large required sample amount, low test accuracy and the like of the existing calorimetric titration method and ultraviolet spectrophotometry method for determining PAMAM and guest micromolecule interaction and the existing method only using one-dimensional hydrogen spectrometry and NOESY.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
an NMR detection method of a PAMAM and guest small molecule interaction mode is characterized in that the NMR detection method is used for detecting the PAMAM and guest small molecule interaction mode based on a one-dimensional hydrogen spectrum, a DOSY spectrum, a NOESY spectrum, a CSSFs-TOCSY spectrum (chemical selective filter excitation spectrum) and a pure chemical shift spectrum, at least the one-dimensional hydrogen spectrum, the DOSY spectrum and the NOESY spectrum of the PAMAM and the guest small molecule are selected, when the overlapping of peaks in the one-dimensional hydrogen spectrum of the PAMAM and the guest small molecule is difficult to identify and attribute, one or two of the CSSFs-TOCSY spectrum and the pure shift spectrum are further selected for separating the overlapping peaks, and the interaction mode of the PAMAM and the guest small molecule can be obtained by comparing the changes of the spectra before and after the interaction of the PAMAM and the guest small molecule. The invention is based on one-dimensional hydrogen spectrum, DOSY spectrum, NOESY spectrum, CSSFs-TOCSY spectrum and pure shift spectrum to carry out the detection method of the interaction between PAMAM and the object small molecule, in the detection process, the required sample amount is small, the sample is not damaged, the sample pretreatment is not needed, the interaction mode of the PAMAM and the object small molecule can be obtained by comparing the change of the spectrogram before and after the interaction between the PAMAM and the object small molecule, the spectrogram has accurate attribution, the analysis result is accurate, the broad spectrum is provided, and the invention is suitable for the determination of the interaction between the PAMAM and various object small molecules. CSSFs-TOCSY spectrum can selectively excite the side peak of the overlapped spectrum peak, excite the proton signal directly or indirectly coupled with the overlapped spectrum peak, and extract the pure spectrum of a certain component. The Pure shift spectrum can eliminate the J coupling effect (namely fusing multiple splits caused by coupling between adjacent protons into a single peak), and the resolution is obviously improved. The overlapping phenomenon of signals of the one-dimensional hydrogen spectrum, the DOSY spectrum and the NOESY spectrum can seriously interfere the accuracy of an analysis interaction result, and one or two of the CSSFs-TOCSY spectrum and the pure shift spectrum are selected to separate overlapped spectrum peaks, thereby being beneficial to the accuracy of the analysis result.
Still further, the one-dimensional hydrogen spectra use zg30 pulse sequence spectra based on 30 degree excitation; the DOSY spectrum uses a pulse sequence based on BPPLED orThe diffusion sequencing spectrum of the stimulated echo STE pulse sequence changes the molecule position due to self-diffusion movement, so that the magnetization intensity is incompletely refocused to further cause signal attenuation, the pulse can greatly reduce the eddy current effect in a shielding probe, the phase of an obtained spectrogram is not distorted, and the obtained data is more accurate; the NOESY spectra use a sequence of NOESygppppp pulses that use a square wave standard waveform that results in a spatial magnitude less than
(angstrom) H signal, even different H separate multiple bond, and then detect the small molecule of the object and PAMAM space configuration; the CSSFs-TOCSY spectrum uses a selcsfdizs.2 pulse sequence, chemical shift selective filtering is added on the basis of a one-dimensional TOCSY method, proton signals directly or indirectly coupled with the CSSFs-TOCSY spectrum are excited, and a spectrum is purified so as to facilitate identification and attribution of a spectrum peak; the pureshift spectrum comprises a spectrum obtained by adopting any one of ZS, PSYCHE and TSE-PSYCHE. By adopting any one of the ZS method, the PSYCHE method and the TSE-PSYCHE method, multiple splits caused by coupling between adjacent protons are fused into a single peak, the resolution ratio is obviously improved, overlapping spectral peaks are separated, the identification and attribution of the spectral peaks are facilitated, and the detection accuracy is greatly improved.
Still further, the ZS, PSYCHE and TSE-PSYCHE methods use PSYCHE. mf, pushpr1dzs and TSE-PSYCHE pulses, respectively.
Still further, the detecting instrument used in the NMR detecting method is a liquid nuclear magnetic resonance spectrometer with a gradient field, and the specific detecting steps are as follows:
(1) preparing a test sample and placing the test sample in a sample tube of a liquid nuclear magnetic resonance spectrometer;
(2) adjusting the temperature and airflow rate of the liquid nuclear magnetic resonance spectrometer without rotating the sample tube; the sample is kept for a certain number of minutes under the set temperature and airflow;
(3) sequentially testing the one-dimensional hydrogen spectrum, the DOSY spectrum and the NOESY spectrum of the sample, and processing the obtained data by using Bruk Topspin 3.1 or Dynamics center2.2.4 software;
(4) when no spectrum peak overlapping occurs in the one-dimensional hydrogen spectrum of the test sample, analyzing the interaction mode of the PAMAM and the guest small molecules according to the data obtained in the step (3);
(5) when the one-dimensional hydrogen spectra of the test samples show spectral peak overlap, one or two of CSSFs-TOCSY spectra and pure shift spectra of the test samples are further selected, the data obtained are processed by using Bruk Topspin 3.1 or Dynamics center2.2.4 software, and the interaction mode of the PAMAM and the guest small molecules is analyzed according to the data obtained in the step (3).
The sample is constant for 15-30 minutes under the set airflow and temperature, and then the one-dimensional hydrogen spectrum, the DOSY spectrum and the NOESY spectrum of the sample are tested, so that the convection effect of the sample solution during the test is avoided, and the accuracy of the test result is influenced. The data obtained by using software of Bruker Topspin 3.1 or Dynamics center2.2.4 is processed to make the spectrogram clear and facilitate the judgment of the interaction mode of PAMAM and the guest small molecule.
Still further, the gradient field of the liquid nuclear magnetic resonance spectrometer is above 400 MHz. The gradient field with the frequency of 400MHz can ensure that the field intensity is more than 9.5T so as to ensure the test sensitivity and the accuracy of detecting the interaction of the PAMAM and the object small molecules.
Still further, the specific steps for preparing the test sample in the step (1) are as follows: dissolving PAMAM and the guest micromolecules in the same solvent, magnetically stirring for 24 hours, adding 350-500 mu l of solution into the internal standard substance, placing the solution into a nuclear magnetic sample tube, uniformly mixing, and placing the sample tube into a liquid nuclear magnetic resonance spectrometer for detection. The internal standard substance, the PAMAM and the guest micromolecule are placed in the same solvent, so that experimental errors can be reduced, the influence of the solution environment on the system can be eliminated, and the accuracy of the test interaction can be improved.
Still further, the internal standard substance is one or more of tetramethoxysilane, 3- (trimethylsilyl) -1-propanesulfonic acid sodium salt or 1, 4-dioxane; the solvent is one or more of heavy water, deuterated DMSO, deuterated chloroform or deuterated methanol. The selected internal standard does not interact with the PAMAM and guest small molecules, and the spectral peak signals do not overlap with the PAMAM and guest small molecules.
Still further, in the step (2), the temperature of the liquid nuclear magnetic resonance spectrometer is adjusted to 298K to 333K, the gas flow rate is adjusted to 400 to 500lph, and the constant time is 15 to 30 minutes.
Still further, the parameters of the DOSY spectrum tested in the step (3) are as follows: the value interval of the adopted gradient field intensity GPZ6 is 2 to 98 percent; the diffusion time delta is 100-300 ms; the gradient field pulse width value delta/2 is 1000-3000 mu s; obtaining 2% -10% residual signal of the sample at the maximum gradient field intensity; the number of scans NS is a multiple of 8; the number of empty sweeps DS is a multiple of 4; the sampling frequency TD F1 of the used two-dimensional spectrogram is 8-128 times, and the F2 dimension is 16-128 k.
Still further, the parameters for testing NOESY spectra in step (3) are: the mixing time D8 is 0.1-2 s; the number of pulse scans NS used is a multiple of 2; the number of null sweeps DS is a multiple of 16.
Compared with the prior art, the invention has the following beneficial effects:
(1) the detection method of the invention has the advantages of small sample amount, high test accuracy and high resolution, and can realize nondestructive test of the sample;
(2) the detection method provided by the invention provides an accurate and systematic NMR detection method about the interaction mode of the PAMAM and the guest small molecules;
(3) the detection method provided by the invention provides an effective and reliable solution for researching the ambiguous phenomenon of spectral peak overlapping attribution existing in the interaction mode of PAMAM and guest small molecules;
(4) the detection method disclosed by the invention has universality for the research of the interaction mode of the PAMAM and the guest small molecules.
Drawings
FIG. 1 is a graph of the one-dimensional hydrogen spectrum of the interaction of PAMAM with vitamin B7 in example 1;
FIG. 2 is a NOESY spectrum of the interaction of PAMAM with vitamin B7 in example 1;
FIG. 3 is a graph comparing the CSSFs-TOCSY spectra and the one-dimensional hydrogen spectra of the interaction between PAMAM and vitamin B7 in example 1;
FIG. 4 is a plot of pure shift versus one-dimensional hydrogen spectra of the interaction of PAMAM with vitamin B7 in example 1;
FIG. 5 is a comparison of CSSFs-TOCSY, pure shift and one-dimensional hydrogen spectra of PAMAM interaction with vitamin B7 in example 1;
FIG. 6 is a one-dimensional hydrogen spectrum of the interaction of PAMAM with 5-fluorouracil (5-FU) in example 2;
FIG. 7 is a NOESY spectrum of the interaction of PAMAM with 5-fluorouracil (5-FU) in example 2;
FIG. 8 is a one-dimensional hydrogen spectrum of the interaction of PAMAM with tryptophan in example 3;
FIG. 9 is the NOESY spectrum of the interaction of PAMAM with tryptophan in example 3.
Detailed Description
The technical solution of the present invention is further described below with reference to the accompanying drawings and specific embodiments. It should be understood by those skilled in the art that the specific embodiments are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Example 1
In the NMR detection method of the interaction mode of the PAMAM and the guest small molecules, vitamin B7 is selected as the guest small molecules, the NMR detection method is based on a one-dimensional hydrogen spectrum, a DOSY spectrum, a NOESY spectrum, a CSSFs-TOCSY spectrum and a pure shift spectrum to detect the interaction mode of the PAMAM and the vitamin B7, at least the one-dimensional hydrogen spectrum, the DOSY spectrum and the NOESY spectrum of the PAMAM and the vitamin B7 are selected, when the overlapping of peaks appears in the one-dimensional hydrogen spectrum of the PAMAM and the vitamin B7 and is difficult to identify and belong, one or two of the CSSFs-TOCSY spectrum and the pure shift spectrum are further selected to separate the overlapping peaks, and the interaction mode of the PAMAM and the vitamin B7 can be known by comparing the changes before and after the interaction of the PAMAM and the vitamin B7.
The one-dimensional hydrogen spectra use zg30 pulse sequence spectra based on 30 degree excitation; the DOSY spectrum uses a diffusion-ordered spectrum based on a BPPLED pulse sequence or a stimulated echo STE pulse sequence; the NOESY spectra use a Noesygpphpp pulse sequence standard for nuclear magnetic resonance spectroscopy; the CSSFs-TOCSY spectrum uses a selcsfdizs.2 pulse sequence, the spectrum is added with chemical shift selective filtering on the basis of a one-dimensional TOCSY method, proton signals directly or indirectly coupled with the spectrum are excited, and a spectrum is purified to facilitate the identification and attribution of spectral peaks; the pure shift spectrum comprises a spectrum obtained by adopting any one of ZS, PSYCHE and TSE-PSYCHE; the ZS, PSYCHE and TSE-PSYCHE methods use PSYCHE. mf, pushpr1dzs and TSE-PSYCHE pulses, respectively.
The detection instrument used in the NMR detection method is a liquid nuclear magnetic resonance spectrometer with a 400MHz gradient field, and the detection method comprises the following specific steps:
(1) preparation of test samples: dissolving 0.8mg of the 5 th generation PAMAM and 0.41mg of vitamin B7 in 500 microliters of deuterated methanol, magnetically stirring for 24 hours, adding 400 microliters of the solution into 5 microliters of internal standard 1, 4-dioxane (10 microliters of 1, 4-dioxane is diluted by 20 times with heavy water) and placing the mixture into a nuclear magnetic sample tube, uniformly mixing, and placing the sample tube into a sample tube of a liquid nuclear magnetic resonance spectrometer for detection.
(2) Adjusting the temperature of a liquid nuclear magnetic resonance spectrometer to 333K, the airflow rate to 500lph, and the sample tube does not rotate; the sample was held constant at the set temperature and airflow for 15 minutes.
(3) Sequentially testing a one-dimensional hydrogen spectrum, a DOSY spectrum and a NOESY spectrum of a sample, wherein the DOSY spectrum is characterized in that the value of gradient field strength GPZ6 is set to be 2%, the diffusion time delta is 180ms, the gradient field pulse width value delta/2 is 1000 mus, a hydrogen spectrum is adopted, and the adopted gradient field strength GPZ6 is 95%; the diffusion time delta is 300 ms; the gradient field pulse width value delta/2 is 2000 mu s; acquiring a second hydrogen spectrum, and matching the two hydrogen spectra to enable the sample to obtain a residual signal of 3% at the maximum gradient field intensity; the number of scans NS is 16; the number of empty sweeps DS is 8; the used two-dimensional spectrogram sampling times TD F1 is 32 times, and F2 is 32 k. Wherein the parameters of the NOESY spectrum are: the range of mixing time D8 values was 2 s; the number of pulse scans NS used was 16; the number of empty sweeps DS is 16. The interaction of PAMAM with vitamin B7 shows the one-dimensional hydrogen spectrum and NOESY spectrum as shown in fig. 1 and 2, respectively.
(4) Analyzing a hydrogen spectrum by using Bruk Topspin 3.1 software, finding that G5-PAMAM and vitamin B7 have an overlapping signal, further detecting a CSSFs-TOCSY spectrum of a test sample by using a CSSFs-TOCSY method, setting the central frequency O1 to be 962.13Hz, scanning times NS to be 16 times, and empty scanning times to be four times; the pure shift spectrum of the test sample can also be detected by using a pure shift method, wherein the scanning times NS are 16 times, and the empty scanning times are 2 times; the test sample can likewise be examined by combining the CSSFs-TOCSY spectra with the pureshift spectra. The CSSFs-TOCSY spectra and the plot for comparing the single-dimensional hydrogen spectra, the plot for comparing the pure shift spectra and the single-dimensional hydrogen spectra, and the CSSFs-TOCSY spectra, the pure shift spectra and the plot for comparing the single-dimensional hydrogen spectra of the interaction of PAMAM and vitamin B7 are shown in FIGS. 3-5.
(5) Processing the residual data by using Bruk Topspin 3.1 software or Dynamics center2.2.4 software, comparing the chemical shift and peak shape change of proton peak in spectrogram before and after interaction of PAMAM and vitamin B7, finding that only two methylene groups connected with terminal amino in one-dimensional hydrogen spectrum move to low field direction, carboxyl of vitamin B7 moves to high field, and no cross signal related to PAMAM and vitamin B7 exists in NOESY spectrum, calculating by software to obtain that PAMAM after interaction is slower than diffusion before interaction, indicating that the interaction of PAMAM and small molecule of object is surface combination, comparing (1) CSSFs-TOCSY spectrum with original one-dimensional hydrogen spectrum, and missing a group of peaks at 2.34 ppm; (2) the pure shift spectrum showed two peaks at 2.39ppm and 2.34 ppm; (3) the CSSFs-TOCSY spectrum is combined with the pure shift spectrum, the peak which disappears in the CSSFs-TOCSY spectrum is the peak at 2.34ppm separated from the pure shift spectrum, and the results of the two are verified mutually and more accurate. The three modes can show that the chemical environments of the inner layer and the outer layer of the PAMAM are different after the interaction, and further verify that the interaction mode of the PAMAM and the vitamin B7 guest micromolecules is surface bonding. The diffusion coefficient versus time before and after interaction of PAMAM with vitamin B7 is shown in table 1.
TABLE 1 comparison of diffusion coefficients before and after interaction of PAMAM with vitamin B7
Example 2
In the NMR detection method of the interaction mode of PAMAM and guest small molecules, 5-fluorouracil (5-FU) is selected as guest small molecules, the NMR detection method is based on a one-dimensional hydrogen spectrum, a DOSY spectrum, a NOESY spectrum, a CSSFs-TOCSY spectrum and a pure shift spectrum to detect the interaction mode of PAMAM and 5-fluorouracil (5-FU), the one-dimensional hydrogen spectrum, the DOSY spectrum and the NOESY spectrum of PAMAM and 5-fluorouracil (5-FU) are selected firstly, one or two of the CSSFs-TOCSY spectrum and the pure shift spectrum are selected according to requirements to identify and attribute the spectrum peak, and the interaction mode of PAMAM and 5-fluorouracil (5-FU) can be known by comparing the change of the spectrums before and after the interaction of PAMAM and 5-fluorouracil (5-FU).
Wherein, the pulse sequences of the one-dimensional hydrogen spectrum, the DOSY spectrum, the NOESY spectrum, the CSSFs-TOCSY spectrum and the pure shift spectrum are respectively selected and used in the same way as the example 1.
The detection instrument used in the NMR detection method is a liquid nuclear magnetic resonance spectrometer with a 600MHz gradient field, and the detection steps are as follows:
(1) preparation of test samples: 0.8mg of the 3 rd generation PAMAM and 0.90mg of 5-fluorouracil (5-FU) are dissolved in 500 microliters of heavy water, after magnetic stirring is carried out for 24 hours, 500 microliters of the solution is added into 5 microliters of the internal standard substance 1, 4-dioxane (10 microliters of 1, 4-dioxane is diluted by 20 times with the heavy water) and placed in a nuclear magnetic sample tube, the mixture is uniformly mixed, and the sample tube is placed in a liquid nuclear magnetic resonance spectrometer for testing.
(2) Adjusting the temperature of the liquid nuclear magnetic resonance spectrometer to 303K, the airflow to 450lph, and the sample tube does not rotate; the sample was held constant at the set temperature and airflow for 25 minutes.
(3) Sequentially testing a one-dimensional hydrogen spectrum, a DOSY spectrum and a NOESY spectrum of a sample, wherein the DOSY spectrum is obtained by firstly setting the value of gradient field strength GPZ6 to be 2%, the diffusion time delta to be 100ms, the gradient field pulse width value delta/2 to be 1000 mu s, and adopting a hydrogen spectrum with the gradient field strength GPZ6 of 98%; the diffusion time delta is 100 ms; the gradient field pulse width value delta/2 is 3000 mu s; acquiring a second hydrogen spectrum, and matching the two hydrogen spectra to enable the sample to obtain 5% of residual signals at the maximum gradient field intensity; the number of scans NS is 32; the number of empty sweeps DS is 4; the used two-dimensional spectrogram sampling times TD F1 is 8 times, and F2 is 16 k. Wherein the parameters of the NOESY spectrum are: the range of mixing time D8 values was 1 s; the number NS of pulse scans used is 32; the number of empty sweeps DS is 32. The one-dimensional hydrogen spectrum and NOESY spectrum of interaction of PAMAM and fluorouracil (5-FU) are respectively shown in FIGS. 6 and 7.
(4) The hydrogen spectrum was analyzed using the Brooks Topspin 3.1 software and it was found that G5-PAMAM has no overlapping signal with 5-fluorouracil (5-FU), and therefore the CSSFs-TOCSY spectrum and pure shift spectrum need not be tested.
(5) The data obtained by using Bruk Topspin 3.1 software or Dynamics center2.2.4 software for processing is compared, after PAMAM is added, 5-FU in a one-dimensional spectrogram before and after interaction of PAMAM and 5-FU moves to a high field, only two methylene groups connected with terminal amino groups of PAMAM move to a low field direction, a cross peak of 5-FU and PAMAM obviously appears in a NOESY spectrum, and the PAMAM after interaction obtained by software calculation is slower than the diffusion before the interaction, which indicates that 5-FU and PAMAM not only have surface interaction, but also enter an inner cavity of the PAMAM. PAMAM and 5-fluorouracil (5-FU) interaction diffusion coefficient pairs are shown in Table 2.
TABLE 2 comparison of the diffusion coefficient of interaction between PAMAM and 5-fluorouracil (5-FU)
Example 3
An NMR detection method of a PAMAM and guest small molecule interaction mode selects tryptophan as a guest small molecule in the embodiment, the NMR detection method is based on a one-dimensional hydrogen spectrum, a DOSY spectrum, a NOESY spectrum, a CSSFs-TOCSY spectrum and a pure shift spectrum to detect the PAMAM and tryptophan interaction mode, the one-dimensional hydrogen spectrum, the DOSY spectrum and the NOESY spectrum of the PAMAM and the tryptophan are selected firstly, one or two of the CSSFs-TOCSY spectrum and the pure shift spectrum are selected according to requirements to identify and attribute a spectrum peak, and the interaction mode of the PAMAM and the tryptophan can be known by comparing the change of the spectrums before and after the interaction of the PAMAM and the tryptophan.
Wherein, the pulse sequences of the one-dimensional hydrogen spectrum, the DOSY spectrum, the NOESY spectrum, the CSSFs-TOCSY spectrum and the pure shift spectrum are respectively selected and used in the same way as the example 1.
The detection instrument used in the NMR detection method is a liquid nuclear magnetic resonance spectrometer with a 500MHz gradient field, and the detection method comprises the following specific steps:
(1) preparation of test samples: dissolving 0.8mg of the 4 th generation PAMAM and 1mg of tryptophan in 500 microliters of heavy water, magnetically stirring for 24 hours, adding 350 microliters of the solution into 20 microliters of Tetramethoxysilane (TMS) and placing the mixture in a nuclear magnetic sample tube, uniformly mixing, and placing the sample tube in a liquid nuclear magnetic resonance spectrometer to be detected;
(2) adjusting the temperature of a liquid nuclear magnetic resonance spectrometer to 298K, the airflow to 400lph, and the sample tube does not rotate; the sample is kept constant for 30 minutes at the set temperature and airflow;
(3) sequentially testing a one-dimensional hydrogen spectrum, a DOSY spectrum and a NOESY spectrum of a sample, wherein the DOSY spectrum is obtained by firstly setting the value of gradient field strength GPZ6 to be 2%, the diffusion time delta to be 150ms, the gradient field pulse width value (delta/2) to be 1000 mu s, and adopting a hydrogen spectrum with the gradient field strength GPZ6 of 98%; the diffusion time delta is 150 ms; the gradient field pulse width value (delta/2) is 2500 mu s; acquiring a second hydrogen spectrum, and matching the two hydrogen spectra to enable the sample to obtain 6% of residual signals at the maximum gradient field intensity; the number of scans NS is 24; the number of empty sweeps DS is 8; the two-dimensional spectrogram is sampled by 128 times in the dimension TD 1 and 128k in the dimension F2. Wherein the parameters of the NOESY spectrum are: the range of mixing time D8 values was 0.1 s; the number NS of pulse scans used was 64; the number of empty sweeps DS is 16. The interaction of PAMAM and tryptophan shows the one-dimensional hydrogen spectrum and NOESY spectrum as shown in FIGS. 8 and 9, respectively.
(4) The hydrogen spectrum was analyzed using the Brooks Topspin 3.1 software and it was found that G4-PAMAM had no overlapping signal with tryptophan and therefore no CSSFs-TOCSY and pure shift spectra were required to be tested.
(5) The data obtained by using Bruk Topspin 3.1 software or Dynamics center2.2.4 software for processing is compared, the tryptophan in the one-dimensional spectrograms before and after the interaction between the PAMAM and the tryptophan is added and then moves to a high field, the PAMAM only moves to a low field direction through two methylene groups connected with terminal amino groups, the cross peak of the tryptophan and the PAMAM does not appear in a NOESY spectrum, and the PAMAM after the interaction is calculated by the software to be slower than the diffusion before the interaction, which indicates that the tryptophan and the PAMAM only have surface interaction and do not enter the inner cavity of the PAMAM. The diffusion coefficient versus the diffusion coefficient before and after interaction of PAMAM with tryptophan is shown in table 3.
TABLE 3 comparison of diffusion coefficients before and after interaction of PAMAM with tryptophan
The internal standard substance used for preparing the test sample in the above embodiment can also be 3- (trimethylsilyl) -1-propanesulfonic acid sodium, or a mixture of tetramethoxysilane, 3- (trimethylsilyl) -1-propanesulfonic acid sodium or 1, 4-dioxane; the solvent can also be deuterated DMSO, deuterated chloroform, or mixture of deuterium oxide, deuterated DMSO, deuterated chloroform or deuterated methanol.
The above embodiments are merely illustrative, and not restrictive, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions also belong to the scope of the present invention, and the protection scope of the present invention should be defined by the claims.
Claims (10)
1. An NMR detection method of PAMAM and object small molecule interaction mode is characterized in that the NMR detection method is based on a one-dimensional hydrogen spectrum, a DOSY spectrum, a NOESY spectrum, a CSSFs-TOCSY spectrum and a pure shift spectrum to detect the PAMAM and object small molecule interaction mode, at least the one-dimensional hydrogen spectrum, the DOSY spectrum and the NOESY spectrum of the PAMAM and the object small molecule are selected, when the overlapping of the peaks in the one-dimensional hydrogen spectrum of the PAMAM and the object small molecule is difficult to identify and belong, one or two of the CSSFs-TOCSY spectrum and the pure shift spectrum are further selected to separate the overlapping peaks, and the interaction mode of the PAMAM and the object small molecule can be known by comparing the changes of the spectra before and after the interaction of the PAMAM and the object small molecule.
2. The method for NMR detection of PAMAM mode of interaction with guest small molecules as claimed in claim 1, wherein said one-dimensional hydrogen spectra uses zg30 pulse sequence spectra based on 30 degree excitation; the DOSY spectrum uses a diffusion-ordered spectrum based on a BPPLED pulse sequence or a stimulated echo STE pulse sequence; the NOESY spectra use a noesygpphpp pulse sequence; the CSSFs-TOCSY spectrum uses a selcsfdizs.2 pulse sequence, the spectrum is added with chemical shift selective filtering on the basis of a one-dimensional TOCSY method, proton signals directly or indirectly coupled with the spectrum are excited, and a spectrum is purified to facilitate the identification and attribution of spectral peaks; the pure shift spectrum comprises a spectrum obtained by any one of ZS, PSYCHE and TSE-PSYCHE.
3. The method of claim 2, wherein the ZS, PSYCHE, and TSE-PSYCHE methods use PSYCHE. mf, pushpr1dzs, and TSE-PSYCHE pulses, respectively.
4. The NMR detection method of PAMAM and guest small molecule interaction mode, according to any one of claims 1-3, wherein the detection instrument used in the NMR detection method is a liquid nuclear magnetic resonance spectrometer with gradient field, and the detection steps are as follows:
(1) preparing a test sample and placing the test sample in a sample tube of a liquid nuclear magnetic resonance spectrometer;
(2) adjusting the temperature and airflow rate of the liquid nuclear magnetic resonance spectrometer without rotating the sample tube; the sample is kept for a certain number of minutes under the set temperature and airflow;
(3) sequentially testing the one-dimensional hydrogen spectrum, the DOSY spectrum and the NOESY spectrum of the sample, and processing the obtained data by using Bruk Topspin 3.1 or dynamic marker 2.2.4 software;
(4) when no spectrum peak overlapping occurs in the one-dimensional hydrogen spectrum of the test sample, analyzing the interaction mode of the PAMAM and the guest small molecules according to the data obtained in the step (3);
(5) when the one-dimensional hydrogen spectra of the test samples show spectral peak overlap, one or two of CSSFs-TOCSY spectra and pure shift spectra of the test samples are further selected, the data obtained are processed by using Bruk Topspin 3.1 or Dynamics center2.2.4 software, and the interaction mode of the PAMAM and the guest small molecules is analyzed according to the data obtained in the step (3).
5. The method of claim 4, wherein the gradient field of the liquid NMR spectrometer is 400MHz or higher.
6. The method for NMR detection of PAMAM mode of interaction with guest small molecules as claimed in claim 4, wherein the specific steps for preparing the test sample in step (1) are as follows: dissolving PAMAM and the guest micromolecules in the same solvent, magnetically stirring for 24 hours, adding 350-500 mu l of solution into the internal standard substance, placing the solution into a nuclear magnetic sample tube, uniformly mixing, and placing the sample tube into a liquid nuclear magnetic resonance spectrometer for detection.
7. The method for NMR detection of PAMAM mode of interaction with guest small molecules as claimed in claim 4, wherein the internal standard is one or more of tetramethoxysilane, sodium 3- (trimethylsilyl) -1-propanesulfonate or 1, 4-dioxane; the solvent is one or more of heavy water, deuterated DMSO, deuterated chloroform or deuterated methanol.
8. The method for detecting the interaction mode of PAMAM and guest small molecules through NMR as claimed in claim 4, wherein the temperature of the liquid NMR in step (2) is adjusted to 298K to 333K, the gas flow rate is 400 to 500lph, and the constant time is 15 to 30 minutes.
9. The method for NMR detection of PAMAM mode of interaction with guest small molecules as claimed in claim 4, wherein the parameters of DOSY spectra tested in step (3) are: the value interval of the adopted gradient field intensity GPZ6 is 2 to 98 percent; the diffusion time delta is 100-300 ms; the gradient field pulse width value delta/2 is 1000-3000 mu s; obtaining 2% -10% residual signal of the sample at the maximum gradient field intensity; the number of scans NS is a positive integer multiple of 8; the number of null sweeps DS is a positive integer multiple of 4; the sampling frequency TD F1 of the used two-dimensional spectrogram is 8-128 times, and the F2 dimension is 16-128 k.
10. The method for NMR detection of PAMAM mode of interaction with guest small molecules as claimed in claim 4, wherein the parameters for NOESY spectrum measurement in step (3) are: the mixing time D8 is 0.1-2 s; the number of pulse scans NS used is a positive integer multiple of 2; the number of null sweeps DS is a positive integer multiple of 16.
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