CN110376235B - Measurement method of spatial correlation spectrum of mannine based on nuclear magnetic resonance technology - Google Patents

Abstract

The invention relates to the field of chemical analysis, in particular to a cephalomannine space correlation spectrum (ROESY) testing method based on a nuclear magnetic resonance technology. The method measures the space correlation spectrum ROESY of the cephalomannine by taking hydrogen as an observation nucleus through the optimization design of a pulse sequence. Most of the spatial correlation spectra used by the current instruments are NOESY spectra, but the NOE signals of some medium molecules are almost zero, so that the spatial correlation condition cannot be represented by NOESY. Aiming at the characteristics of cephalomannine molecules, the method changes the idea of introducing ROESY spectra and optimizes related parameters on the basis. The invention can overcome the defect of weak NOE signal and provides an effective and simple means for representing the spatial structure for medium-sized molecules.

Description

Measurement method of spatial correlation spectrum of mannine based on nuclear magnetic resonance technology

technical field

The invention relates to the field of chemical analysis, in particular to a method for testing spatial correlation spectroscopy (ROESY) based on nuclear magnetic resonance technology.

Background technique

Mandarine is derived from Taxus chinensis and is an important pharmaceutical intermediate, material intermediate and fine chemical with anti-inflammatory and anti-tumor effects. At the same time, mannine can also be converted into an important anti-tumor drug, taxol. The chemical structure is different only in the amide side chain. It can be used as an important raw material for the chemical synthesis of taxol, and can be used as a new type of taxane. precursor.

The structural formula of mannine is as follows:

In recent years, two-dimensional nuclear magnetic resonance spectroscopy has been applied to the determination and analysis of the spatial conformation of drug molecules in liquids, and the spatial geometric information between atoms can be obtained. One of the most commonly used two-dimensional nuclear magnetic resonance spectroscopy is NOESY. However, NOESY is not applicable to all molecules, and the NOE signal is almost zero when the molecular motion correlation time Tc ≈ 1/ω ₀ (ω ₀ Larmor frequency) of medium size molecules. As a complement to NOESY technology, ROESY technology is a reliable method. ROESY is a new method for the determination of homonuclear NOE under spin-locked conditions, which is the Overhauser Enhancement Spectroscopy (Rotatingframe Overhauser Enhancement Spectroscopy, ROESY) in the rotating coordinate system. It is an ideal technology to solve the stereo structure of medium-sized compounds, but there are two problems with ROESY technology: one is how to suppress similar two-dimensional NMR signals so as not to affect the identification of ROESY signals; the other is how to eliminate off-resonance effects. Impact. These two problems are related to each other, because reducing the locking field strength can suppress the formants, but the increase of off-resonance effect will reduce the sensitivity of the ROESY spectrum, especially for the formants with relatively large off-resonance frequencies.

There is no report about the test method of mannine spatial correlation spectrum (ROESY), but there are some about the separation method of mannine and paclitaxel, mannine derivatives and their preparation methods and their drug combinations Reports on the preparation of 10-deacetyl baccadine by hydrolysis of mannine base and its use. The present invention provides a new modified pulse sequence for the test of mannine spatial correlation spectrum (ROESY).

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method that can be directly used for determining the spatial correlation spectrum (ROESY) of mannine in view of the deficiencies of the prior art.

The present invention adopts the following technical scheme: a method for analyzing mannine spatial correlation spectrum (ROESY), the method comprises the following steps:

1) Weigh the sample and put it into the test tube, add the deuterated solvent DMSO, dissolve fully ultrasonically, transfer it into the nuclear magnetic tube, and then put the test tube with the loaded sample into the detection magnet of the nuclear magnetic spectrometer;

2) Select the corresponding deuterated solvent in the instrument workstation to perform tuning, shimming and field locking;

3) Set the test parameters of ROESY: turn on the pulse sequence, and set the experimental conditions including spectral width, pre-pulse relaxation delay time, pulse width setting, mixing time setting, scan times, and acquisition mode;

Among them, the main content of the pulse sequence is: taking hydrogen as the observation nucleus, after an empty sweep time, a rectangular π/2 pulse (pw), and a relaxation delay time (d1), the magnetization vector is in thermal equilibrium; plus A rectangular π/2 pulse (pw); a mixing time (mixR) followed by a sampling period (at) for acquiring the final signal;

4) After completing the experimental parameter setting, directly perform data sampling;

5) After the data sampling is completed, post-processing the data to obtain the spatial correlation spectrum of ROESY.

Preferably, in step 3), the pre-pulse relaxation delay time (d1) is 0.5-3s.

Preferably, the pre-pulse relaxation delay time (d1) is 1.0s.

Preferably, the pulse time (pw) of a rectangular π/2 pulse is 9.4 μs.

Preferably, step 3) includes the step of setting the mixing time, the minimum value is set to 100ms, the maximum value is set to 500ms, preferably 400ms.

Preferably, in step 3), the sampling period (at) is 150.005ms.

Preferably, in step 3), for samples with different concentrations, the number of scans is set to 4-32 times, preferably 16 times.

The beneficial effect of the present invention is that: the present invention determines the spatial correlation spectrum (ROESY) by optimizing and improving the pulse sequence for mannine. The current methods for measuring spatially correlated structures mainly use NOESY spectra, but the NOE signals for medium-sized molecules are weak, so they are not suitable for spatially correlated detection of mannine. The invention can overcome the deficiency of the NOE effect-based detection method, optimize the relevant parameters, and provide a simple and effective means for the spatial correlation conformation determination of the mannine sample.

Description of drawings

Fig. 1 Pulse sequence of the ¹ H spectrum of mannine,

Among them, the time sequence of the pulse sequence is divided into three periods: preparatory period, evolution period and detection period. In the preparatory period, the relaxation delay time (d1) before a pulse passes; a rectangular π excitation pulse (p1) is added on the x-axis of the evolution period. ), add a rectangular π/2 pulse (pw) after the pulse interval time (d2); set the sampling time (at) in the detection period, and the receiver records the free decay signal.

The relaxation delay time (d1) before the pulse is 1.0s, the rectangular π pulse (p1) is 0.0 μs, the pulse interval time (d2) is 0.0 μs, the rectangular π/2 pulse (pw) is 4.7 μs, and the sampling after the pulse The time (at) was 1.703 s, and the number of scans (nt) was 8 times.

Fig. 2 ¹ H spectrum of mannine;

Fig. 3 Pulse sequence of ROESY spectrum of mannine;

Among them, the time sequence of the pulse sequence is divided into three periods: the preparatory period, the evolution period and the detection period. With hydrogen as the observation nucleus, in the preparatory period, an empty sweep time, a rectangular π/2 pulse (pw), and a relaxation delay time are passed. (d1), make the magnetization vector in thermal equilibrium; add a rectangular π/2 pulse (pw) on the x-axis of the evolution period; add a mixing time (mixR) after a pulse interval (d2); set it in the detection period Sampling period (at), the receiver records the free decay signal;

The first rectangular π/2 pulse (pw) is 9.4 μs, the relaxation delay time (d1) is 1.0 s, the rectangular π/2 pulse (pw) is 9.4 μs, the pulse interval (d2) is 0 μs, and the mixing time ( mixR) is 400ms, the pulse receiver gate control time rof2 is 30.796μs; the sampling time (at) after the pulse is 150.005ms, and the number of scans (nt) is 16 times.

Figure 4 ROESY spectrum of mannine.

Detailed ways

The method proposed in the invention can determine the ROESY spectrum of mannine, and has very important guiding significance for the structural analysis and research of the spatial conformation and interaction of mannine.

Example 1:

The method proposed in the present invention is used to determine the ROESY spectrum of mannine as an example, and this specific example is used to verify the feasibility of the present invention in the determination of the ROESY spectrum of mannine. The sample used in the experiment was mannine, and the experimental test was carried out under an Agilent 600MHz NMR spectrometer (Agilent, USA). The whole experiment process did not perform any sample pretreatment on the sample and did not change the instrument hardware. According to the operational flow of the proposed method of the present invention, the concrete steps are as follows:

Step 1, weigh the sample and put it into a test tube, add the deuterated solvent DMSO, dissolve it fully by ultrasonic, transfer it into a nuclear magnetic tube, and then put the test tube with the sample into the detection magnet of the nuclear magnetic spectrometer;

Step 2, select the corresponding deuterated solvent in the instrument workstation, and perform tuning, shimming and field locking;

Step 3, set the test parameters of ¹ H: turn on the pulse sequence, and set the experimental conditions including the spectral width range, the pre-pulse relaxation delay time, the setting of the pulse width, the number of scans, and the acquisition mode;

Among them, the main content of the pulse sequence is: taking hydrogen as the observation nucleus, after a pre-pulse relaxation delay time (d1), the magnetization vector is in a thermal equilibrium state; adding a rectangular π excitation pulse (p1), after a pulse interval A rectangular π/2 pulse (pw) is added after the time (d2); the last is followed by a sampling period (at), which is used to collect the final signal; in this embodiment, the experimental parameters are set as follows: the direct dimension spectral width sw is 9615Hz, the pre-pulse relaxation delay time d1 is 1.0s, the pulse time of the rectangular π/2 pulse is 4.7μs, the sampling time at of a single sampling period is 1.703s, the number of repeated scans nt is 8, and the entire sampling time is 22s, As shown in Figure 1.

Step 4: After completing the experimental parameter setting, directly perform data pre-sampling;

In step 5, after all the data pre-sampling is completed, data post-processing is performed to obtain the ¹ H spectrum of mannine, as shown in FIG. 2 .

Step 6: Based on the test results of ¹ H in step 3, set the test parameters of ROESY; turn on the pulse sequence, and set the experimental conditions including spectral width range, pre-pulse relaxation delay time, pulse width setting, mixing time setting, scanning times, collection mode;

Among them, the main content of the pulse sequence is: taking hydrogen as the observation nucleus, in the preparatory period, after an empty sweep time, a rectangular π/2 pulse (pw), and a relaxation delay time (d1), the magnetization vector is in thermal equilibrium. Add a rectangular π/2 pulse (pw) on the x-axis of the evolution period; after a pulse interval (d2), add a mixing time (mixR); set the sampling period (at) in the detection period, and the receiver records free decay signal;

In this embodiment, the experimental parameters are set as follows: the direct dimension spectral width sw is 6313 Hz, the pre-pulse relaxation delay time d1 is 1.0s, the pulse time of the rectangular π/2 pulse is 9.4μs, and the relaxation delay time (d1) is 1.0 s, the pulse interval time (d2) is 0s, the mixing time (mixR) is 400ms, the sampling time at of a single sampling period is 150ms, the number of repeated scans nt is 16 times, and the entire sampling time is 3h, as shown in Figure 3.

Step 7: After completing the experimental parameter setting, directly perform data pre-sampling;

Step 8, when the data pre-sampling is all completed, perform data post-processing to obtain the ROESY spectrum of mannine, as shown in FIG. 4 .

Example 2

As shown in Table 1, this embodiment optimizes the mixing time (mixR) on the basis of Example 1, and sets the mixing time (mixR) to be 100ms, 200ms, 300ms, 400ms, and 500ms respectively. It is found that when mixR is 400ms, The measured ROE correlation points have the largest number and are basically stable, so it is preferable to set mixR to 400ms.

Table 1. Signal correspondence table of ROESY spectra of samples at different mixing times (mixR)

Example 3

As shown in Table 2, in this embodiment, the detected direct dimension spectral width sw is 6313 Hz. On the basis of Embodiment 1, the sampling time at of the sampling period is adjusted, and is set from 100ms to 150ms and 200ms. It is found that the measured ROE The number of relevant points varies greatly, indicating that the optimization of the sampling time is more effective, so the preferred sampling time at is 150ms.

Table 2 The signal corresponding table of the ROESY spectrum of the samples at different sampling times (at)

serial number Sampling time (ms) Number of ROE related points 1 at=100 12 2 at=150 15 3 at=200 13

Example 4

As shown in Table 3, on the basis of Example 1, in this embodiment, the number of scans (nt) is optimized, and 8 times, 16 times and 32 times are set respectively, and it is found that the results of 32 times and 16 times of scanning have not changed. It is large and tends to be stable, indicating that the optimization of the number of scans is more effective and 16 scans is enough, so the number of scans nt is 16 times.

Table 3 Correspondence table of signals of ROESY spectra of samples at different scan times (nt)

serial number number of scans Number of ROE related points 1 nt=8 13 2 nt=16 15 3 nt=32 14

It can be seen from the above table that the method proposed in the present invention can determine the ROESY spectrum of mannine, which is helpful for analyzing the information of the spatial configuration of mannine. It can be seen that the ROESY spectrum of mannine can be measured by the method of the present invention, and when the direct dimension spectrum width sw is 6313 Hz, the mixing time (mixR) is 400 ms, and the sampling time at of a single sampling period is 150 ms, When the number of repeated scans nt is 16 times, and the whole sampling time is 3h, the effect is the best.

Claims (4)

1. a test method based on the spatial correlation spectrum of mannine based on nuclear magnetic resonance technology, is characterized in that, comprises the steps: (1) take the sample into test tube, add deuterated solvent DMSO, ultrasonically dissolve, transfer into the nuclear magnetic tube, then put the test tube loaded with the sample into the detection magnet of the nuclear magnetic spectrometer; (2) Select the corresponding deuterated solvent in the instrument workstation to perform tuning, shimming and field locking; (3) Set the test parameters of ROESY: turn on the pulse sequence, and set the experimental conditions including the spectral width range, the pre-pulse relaxation delay time, the pulse width, the mixing time, the number of scans and the acquisition mode, wherein the content of the pulse sequence is: Taking hydrogen as the observation nucleus, after an empty sweep time, a rectangular π/2 pulse pw, and a relaxation delay time d1, the magnetization vector is in thermal equilibrium; a rectangular π/2 pulse pw is added; after a mixing time mixR, finally It is followed by the sampling period at, which is used to collect the final signal; Described mixing time mixR is 100-500ms; The pw of a rectangular π/2 pulse is 9.4μs; The sampling period at is 100ms, 150ms or 200ms; The number of scans is 8, 16 or 32; The relaxation delay time d1 is 1.0s; (4) After completing the experimental parameter setting, directly perform data sampling; (5) After the data sampling is completed, post-processing of the data is performed to obtain the spatial correlation spectrum (ROESY) of mannine. 2. The method according to claim 1, wherein in step (3), the mixing time mixR is 400ms. 3. The method according to claim 1, wherein in step (3), the sampling period at is 150ms. 4 . The method according to claim 1 , wherein, in step (3), the number of scans is 16 times. 5 .

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