NMR detection method for PAMAM and guest small molecule interaction mode
阅读说明：本技术 一种pamam与客体小分子相互作用方式的nmr检测方法 (NMR detection method for PAMAM and guest small molecule interaction mode ) 是由 乔岩 马敏珺 王英雄 赵奇 郭朝晖 张书怀 郭晓樱 于 2019-10-30 设计创作，主要内容包括：本发明涉及一种PAMAM与客体小分子相互作用方式的NMR检测方法,所述的NMR检测方法是基于一维氢谱、DOSY谱、NOESY谱、CSSFs-TOCSY谱和pure shift谱进行PAMAM与客体小分子相互作用方式的检测,至少选择PAMAM与客体小分子的一维氢谱、DOSY谱和NOESY谱,当所述PAMAM与客体小分子的一维氢谱中出现谱峰重叠难以进行指认与归属时,再进一步选择CSSFs-TOCSY谱和pure shift谱中的一种或两种进行分离重叠谱峰,通过对比PAMAM与客体小分子相互作用前后谱图的变化,即可得知PAMAM与客体小分子的相互作用方式。本发明的检测方法具有所需测试样品量小、测试精确度高、分辨率高,可实现对样品的无损检测等优点。本发明的检测方法针对研究PAMAM与客体小分子相互作用方式中存在的谱峰重叠归属不明确现象提供有效可靠的解决方法。(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.)
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.
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
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.
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