A high-resolution D-glucose structure analysis method based on nuclear magnetic resonance spectroscopy

Abstract

The application relates to a high-resolution D-glucose structure analysis method based on nuclear magnetic resonance spectrum, which comprises the following steps: placing 2%-5% D-glucose deuterium water solution in a magnetic resonance spectrometer, applying a 90-degree hard pulse to acquire 1 H spectrum; determining the chemical shift information of the proton pair and the 90-degree pulse time width through 1 H spectrum, performing multi-frequency point pulse selective filtering to obtain the signal in the chemical shift region of the proton pair, separating and phase correcting the multi-quantum signals through a multi-quantum filtering module and a CLIP-COSY module, and obtaining two one-dimensional nuclear magnetic resonance phase-sensitive correlation spectra; and analyzing the phase-coded signals through Hadamard coding to reveal the molecular structure of D-glucose. The application provides an efficient and lossless nuclear magnetic resonance detection method for analyzing the molecular structure of D-glucose through selective filtering and phase coding.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of nuclear magnetic resonance spectroscopy detection, and particularly relates to a high-resolution D-glucose structure analysis method based on nuclear magnetic resonance spectroscopy. BACKGROUND

[0002] Glucose is the most common and important colorless monosaccharide in living organisms, with the molecular formula C6H 12 O6, which is the main source of energy for life activities. The human body converts carbohydrates in food into glucose through digestion and absorption, and then transports it to various tissues and organs through the blood to provide energy for cells. Glucose is also an intermediate product of many biochemical reactions, participating in the metabolism of fats and proteins. The structural analysis of glucose is crucial for understanding its function and metabolic pathways, and understanding its molecular structure helps explain its function in cell metabolism, energy production, etc.

[0003] One-dimensional selective proton homonuclear correlation spectroscopy (one-dimensional COSY) in nuclear magnetic resonance spectroscopy technology is a key technology for displaying direct coupling relationships between atomic nuclei and analyzing molecular structures. However, the current one-dimensional nuclear magnetic resonance correlation spectroscopy technology still cannot completely overcome the problems of crowded spectra and signal overlap caused by J coupling splitting, or cannot achieve phase-sensitive display, because the selective pulse is difficult to accurately select the required signal, and there is a lack of phase encoding strategy to achieve phase-sensitive correlation spectrum display. SUMMARY

[0004] To solve the above problems, the present application proposes a high-resolution D-glucose structure analysis method based on nuclear magnetic resonance spectroscopy, which selectively observes the direct coupling network of the direct coupling proton pair of D-glucose. Without losing signal-to-noise ratio, the method uses filtering technology in combination with phase encoding to obtain the phase-sensitive correlation spectrum of the direct coupling network of the coupling proton pair of glucose, and realizes the molecular structure analysis of glucose.

[0005] The specific scheme is as follows:

[0006] A high-resolution D-glucose structure analysis method based on nuclear magnetic resonance spectroscopy, comprising:

[0007] S1, placing a pre-configured deuterium water solution of D-glucose in a magnetic resonance spectrometer;

[0008] S2, applying a 90° hard pulse to the deuterium water solution of D-glucose in the magnetic resonance spectrometer to obtain the 1 H spectrum of the D-glucose molecule;

[0009] S3, selectively observing the direct coupling network of the direct coupling proton pair of the D-glucose molecule by using the 1The H spectrum determines the chemical shift information of the proton pair having a direct coupling relationship in the D-glucose molecule, and determines the 90° pulse time width required for exciting the proton pair having a direct coupling relationship;

[0010] S4, based on the 90° pulse time width and the chemical shift information, performing first selective filtering by a first multi-frequency point pulse to obtain a signal in the chemical shift region of the proton pair having a direct coupling relationship;

[0011] S5, based on the signal in the chemical shift region of the proton pair, performing filtering operation by a multi-quantum filtering module to obtain a multi-quantum signal evolved by the proton pair having a direct coupling relationship, performing second selective filtering on the multi-quantum signal evolved by the proton pair having a direct coupling relationship by a second multi-frequency point pulse to obtain a multi-quantum signal after phase information correction;

[0012] S6, by performing chemical shift encoding on the first 180° hard pulse in the CLIP-COSY module to separate the multi-quantum signal after phase information correction, obtaining multi-quantum signal peaks of D-glucose having a direct coupling relationship in different phases, and performing J coupling evolution extension on the multi-quantum signal peaks to obtain one-dimensional nuclear magnetic resonance phase-sensitive correlation spectrum;

[0013] S7, changing the evolution time, and performing S4-S6 again to obtain two one-dimensional nuclear magnetic resonance phase-sensitive correlation spectra in different phases, and performing phase encoding on the two one-dimensional nuclear magnetic resonance phase-sensitive correlation spectra to obtain one-dimensional nuclear magnetic resonance phase-sensitive correlation spectra of two signals in the multi-quantum signal peaks of D-glucose having a direct coupling relationship respectively;

[0014] S8, by the one-dimensional nuclear magnetic resonance phase-sensitive correlation spectra of two signals in the multi-quantum signal peaks of D-glucose respectively, obtaining direct coupling information of two signals in the proton pair having a direct coupling relationship of D-glucose, and selecting different coupling proton pairs for excitation by the direct coupling information, thereby tracking all coupling proton pairs and revealing the entire molecular structure of D-glucose.

[0015] Further, in S1, the mass concentration of the deuterium water solution of D-glucose is 2%-5%.

[0016] Further, in S4, the first selective filtering by the first multi-frequency point pulse to obtain the signal in the chemical shift region of the proton pair having a direct coupling relationship specifically includes:

[0017] The first gradient field and the second gradient field are added on the left and right sides of the first multi-frequency point pulse, the coherent path selection is performed through the first gradient field and the second gradient field, and selective filtering is realized in cooperation with the first multi-frequency point pulse, so that the signal in the proton pair chemical shift region with the direct coupling relationship is obtained; the first gradient field and the second gradient field have equal strength and direction.

[0018] Further, in S5, the multi-quantum filtering module comprises a first group of 90° hard pulses, a second group of 90° hard pulses, a third gradient field and a fourth gradient field; the strength of the third gradient field after the first group of 90° hard pulses is proportional to the strength of the fourth gradient field after the second group of 90° hard pulses by -1:2.

[0019] Further, in S6, the CLIP-COSY module is mixed on both sides of a period of two single-scan z-filter elements, through which the in-phase to in-phase coherent transfer between directly coupled spins in the multi-quantum signal after phase information correction is ensured, and the anti-phase and zero-quantum contributions in the multi-quantum signal after phase information correction are eliminated.

[0020] Further, in S6, the signal model after chemical shift encoding is:

[0021]

[0022] Wherein, ω I and ω S represent chemical shifts; I and S represent spin operators; the raising operator I + and the lowering operator I - represent +1 and -1 quantum orders respectively; Δ1 and Δ2 represent two different evolution times.

[0023] Further, in S7, the specific implementation mode of phase encoding on the two one-dimensional nuclear magnetic resonance phase-sensitive correlation spectra is Hadamard encoding.

[0024] The present application adopts the above technical solution and has the beneficial effects:

[0025] (1) The present application obtains the direct coupling network information of the selected D-glucose hydrogen nucleus through the selective multi-quantum filtering module, the improved CLIP-COSY module, the chemical shift encoding and the phase encoding, and displays in a phase-sensitive mode, has higher sensitivity and resolution, strong anti-interference ability, good selectivity, short experimental time and the like, and will not cause damage to the experimental sample, and can facilitate subsequent other measurement purposes;

[0026] (2) The present application only selects the hydrogen nucleus for selective detection and obtains the direct coupling network information, and there is no other signal interference spectrum result;

[0027] (3) The present application does not need to separate the D-glucose to be tested before the test, and can be directly used for molecular structure detection in a mixed sample;

[0028] (4) The present application has strong robustness and no dependence on nuclear magnetic equipment, and can be realized on various types of nuclear magnetic resonance instruments, only by using 1 H spectrum information, the parameter data required for subsequent multi-frequency point pulse generation can be obtained;

[0029] (5) The present application has high signal-to-noise ratio and wide application range, and can accurately analyze the molecular structure of specific substances in a complex chemical and biological system, and can also be used to analyze any system with coupling in addition to D-glucose. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The present application is a high-resolution D-glucose structure analysis method based on nuclear magnetic resonance spectroscopy;

[0031] Figure 2 The present application is a high-resolution D-glucose structure analysis method based on nuclear magnetic resonance spectroscopy;

[0032] Figure 3 The present application is a high-resolution D-glucose structure analysis method based on nuclear magnetic resonance spectroscopy; DETAILED DESCRIPTION

[0033] The present application will be further described in detail below in combination with embodiments and drawings, but the embodiments of the present application are not limited thereto.

[0034] As shown in Figure 1 , the present application is a high-resolution D-glucose structure analysis method based on nuclear magnetic resonance spectroscopy, comprising:

[0035] S1, a deuterium water solution of D-glucose is pre-configured, and the pre-configured deuterium water solution of D-glucose is placed in a magnetic resonance spectrometer.

[0036] Specifically, the pre-configured deuterium water solution of D-glucose has a mass concentration of 2%-5%;

[0037] S2, a 90° hard pulse is applied to the pre-configured deuterium water solution of D-glucose, and a 1 H spectrum of the D-glucose molecule is obtained.

[0038] Specifically, as shown in Figure 3 (a), in the present embodiment, the 1 H spectrum of the D-glucose molecule comprises:

[0039] The doublet at 4.62 ppm represents the H signal on carbon 1 of β-D-glucose, the triplet at 3.22 ppm represents the H signal on carbon 2 of β-D-glucose, the quartet at 3.70 ppm represents the H signal on carbon 5 of β-D-glucose, and the doublet at 3.87 ppm represents the H signal on carbon 6 of β-D-glucose. The H signals on carbon 1 and carbon 2 of β-D-glucose, which are directly coupled with it, were selected as the target peaks, as were the H signals on carbon 5 and carbon 6 of β-D-glucose, which are directly coupled with it.

[0040] Specifically, in this embodiment, the instrument used for high-resolution structural analysis of glucose is a Varian 500MHz nuclear magnetic resonance spectrometer.

[0041] S3, through D-glucose molecules 1 H-spectroscopy determines the chemical shift information of directly coupled proton pairs in the D-glucose molecule and determines the 90° pulse time width required to excite directly coupled proton pairs.

[0042] Specifically, in this embodiment, the D-glucose molecule... 1 The H-spectrum acquisition process uses a single pulse consisting of a 90° radio frequency hard pulse and a signal sampling period. 1 After H-spectroscopy, the 90° pulse time width pw required to excite the target signal is measured to calibrate the calibrator in the solution. This aims to fix the chemical shift information and prevent inconsistencies in the chemical shift of the same substance in multiple experiments due to reasons such as unstable instrument magnetic field.

[0043] S4, based on the 90° pulse time width and chemical shift information, performs first selective filtering through the first multi-frequency pulse to obtain the signal in the chemical shift region of the proton pair with direct coupling relationship.

[0044] Specifically, the step of performing first selective filtering through a first multi-frequency pulse to obtain the signal within the chemical shift region of the proton pair with a direct coupling relationship includes:

[0045] A first gradient field and a second gradient field are added to the left and right sides of the first multi-frequency pulse. Coherent path selection is performed through the first gradient field and the second gradient field, and selective filtering is achieved in conjunction with the first multi-frequency pulse to obtain the signal in the chemical shift region of the proton pair with direct coupling relationship. The intensity and direction of the first gradient field and the second gradient field are equal.

[0046] The first multi-frequency pulse is used to excite the signal of proton pairs that have a direct coupling relationship within D-glucose;

[0047] By applying a deuterium water solution of glucose in the y direction, time τ, excitation frequency point is 180° first multi-frequency point pulse of two different chemical shifts of a pair of protons with direct coupling relationship, the specific parameters are the vector addition calculation of the vector formed by the parameters of the single frequency excitation pulse, with equal intensity and the first gradient field and the second gradient field in the z direction, the target signal corresponding to the signal of two different chemical shift regions is obtained by filtering from the full spectrum signal;

[0048] Specifically, as shown in the figure, Figure 2 First, a 90° hard pulse is used to excite the full spectrum signal, and the 180° multi-frequency pulse required for excitation is generated according to the chemical shift information of the target signal to be extracted and the time width pw of the measured 90° pulse; The 180° multi-frequency pulse is calculated by vector addition principle to generate a multi-frequency pulse that can excite two frequency points at the same time after inputting the chemical shift information of the glucose hydrogen nucleus to be excited, with reference to the time width pw of the 90° pulse; By 180° multi-frequency pulse with first gradient field and second gradient field, selective filtering is realized, and the signal in the two chemical shift regions of glucose hydrogen nucleus is filtered; wherein, the action time of 90° hard pulse is 12.25μs, the first multi-frequency pulse used is rsnob type, and the bandwidth is 40Hz;

[0049] After density operator derivation, take a pair of AX nuclei with weak coupling of spin-1 / 2 as an example, whose spin operators are I and S, and the coupling constant is JIS, and the chemical shifts are ω I and ω S In order to facilitate the description of quantum signal order, the raising and lowering operators I + and I - represent +1 and-1 quantum order respectively, in the magnetic resonance spectrometer, the initial density operator in thermal equilibrium state can be written as:

[0050] δ1=I z +S z

[0051] Where, I z and S z represent the angular momentum operator expression in z direction, I y and S y represent the angular momentum operator expression in y direction; The formula of single quantum coherence formed by the first 90° hard pulse is as follows:

[0052]

[0053] The first 180° double-color pulse with two gradient pulses is applied on two different chemical shift sites of the target coupled spin pair of I and S simultaneously, and the signals near the two chemical shift frequencies are reserved by the coherent path selection of the gradient pulses, and during the period of τ, the magnetization of I and S evolves into anti-phase single quantum coherence (SQC) terms 2I ± S z and 2I z S ± .

[0054] S5, based on the signals in the proton pair chemical shift region, a filtering operation is performed by a multi-quantum filtering module to obtain a multi-quantum signal evolved by the proton pairs with direct coupling relationship, and a second selective filtering is performed on the multi-quantum signal evolved by the proton pairs with direct coupling relationship by the second multi-frequency point pulse to obtain the multi-quantum signal after phase information correction.

[0055] Specifically, the multi-quantum filtering module comprises a first group of 90° hard pulses, a second group of 90° hard pulses, a third gradient field and a fourth gradient field; the intensity of the third gradient field after the first group of 90° hard pulses is proportional to the intensity of the fourth gradient field after the second group of 90° hard pulses by -1:2.

[0056] As Figure 2 shown, the multi-quantum signal evolved by the glucose hydrogen nucleus is filtered out by the multi-quantum filtering module through the first 180° multi-frequency point pulse (first multi-frequency point pulse) and its corresponding first gradient field and second gradient field, and the second 180° multi-frequency point pulse (second multi-frequency point pulse) and its corresponding third gradient field and fourth gradient field are used to realize the effect of re-coupling, adjust the signal phase information, filter out the selected signal, suppress the remaining background signal, and the signal expression returns to δ2=I + +I - +S + +S - ; wherein δ2 represents the signal evolution form after the second multi-frequency point pulse.

[0057] S6, the first 180° hard pulse in the CLIP-COSY module is subjected to chemical shift encoding to separate the multi-quantum signal after phase information correction, and obtain the multi-quantum signal peaks of D-glucose with direct coupling relationship in different phases, and the multi-quantum signal peaks are subjected to J coupling evolution extension to obtain one-dimensional nuclear magnetic resonance phase-sensitive correlation spectrum.

[0058] Specifically, the CLIP-COSY module is mixed with two single-scan z-filter elements on both sides, which ensures the in-phase to in-phase coherent transfer between directly coupled spins in the phase information corrected multi-quantum signal and eliminates the anti-phase and zero quantum contributions in the phase information corrected multi-quantum signal.

[0059] Two different evolution times Δ1 and Δ2 are set before and after the first 180° hard pulse of the perfect echo sequence; Δ is a constant evolution time; two different phase coupling network information spectra of proton pair signal peaks of D-glucose with direct coupling relationship are obtained through two experiments according to the two different evolution times.

[0060] Specifically, referring to Figure 2 In the embodiment, the improved perfect echo sequence is used in the CLIP-COSY module, and the two z-filters before and after the perfect echo sequence act on the sample to be tested. The first 180° hard pulse in the CLIP-COSY is subjected to chemical shift encoding, and the two signals after re-aggregation are separated. Two different evolution times Δ1 and Δ2 are set before and after the 180° hard pulse. The chemical shift encoding mainly evolves the chemical shift by setting different evolution times in the two experimental samplings, so that the signals extracted after the selective multi-quantum filter module obtain different phases. The evolution times Δ1 and Δ2 of the coupled signals β1 and β2 of β-D-glucose are 14.75 ms and 15.25 ms, respectively. The evolution times Δ1 and Δ2 of the coupled signals β5 and β6 of β-D-glucose are 13.16 ms and 16.84 ms, respectively. By setting different evolution times in the two experiments, the signals obtained by the selective multi-quantum filter module are in different phases, so that the subsequent transformation processing of the spectra of the two experiments can be performed to obtain one of the signals.

[0061] S7, changing the evolution time, and performing S4-S6 again to obtain two one-dimensional nuclear magnetic resonance phase-sensitive correlation spectra in different phases. The two one-dimensional nuclear magnetic resonance phase-sensitive correlation spectra are subjected to phase encoding to obtain one-dimensional nuclear magnetic resonance phase-sensitive correlation spectra of two signals in the multi-quantum signal peaks of D-glucose with direct coupling relationship.

[0062] Specifically, the specific implementation mode of the phase encoding of the two one-dimensional nuclear magnetic resonance phase-sensitive correlation spectra is Hadamard encoding.

[0063] In the CLIP-COSY module, the density operator in the simplified signal model after chemical shift encoding is:

[0064]

[0065] Through the signal expression, it can be known that the separate spectrum of two signals can be obtained by the Hadamard coding processing in F1 dimension after the two experimental spectra are juxtaposed.

[0066] S8, obtaining the direct coupling information of the two signals in the proton pair having the direct coupling relationship of D-glucose through the one-dimensional nuclear magnetic resonance phase-sensitive correlation spectrum of the two signals of the multi-quantum signal peak of D-glucose, and selecting different coupling proton pairs for excitation through the direct coupling information, so as to track all coupling proton pairs and reveal the entire molecular structure of D-glucose.

[0067] Specifically, the entire molecular structure of D-glucose is revealed, and specifically includes: repeatedly selecting different hydrogen nuclei for excitation through the direct coupling information exhibited by the obtained phase-sensitive correlation spectrum, obtaining the coupling network information of all coupling proton pairs, and revealing the entire molecular structure of D-glucose.

[0068] Through Figure 3 As can be seen from the experimental result graphs of (b)-(e), the embodiment of the present application can accurately extract the coupling network information of the specific atomic nucleus of D-glucose from the nuclear magnetic resonance spectrum graph split by J coupling and overlapping, and exhibit in the form of phase sensitivity. The arrows in the graphs represent the selected hydrogen nuclear signals, and the other marked signals are the coupling network information thereof.

[0069] Although the present application is specifically shown and introduced in combination with the preferred embodiments, it should be understood by those skilled in the art that various changes can be made in form and details without departing from the spirit and scope of the present application as defined in the appended claims, and all such changes are within the protection scope of the present application.

Claims

1. A high-resolution D-glucose structure analysis method based on nuclear magnetic resonance spectroscopy, characterized by, The method comprises the following steps: S1, placing a pre-configured deuterium water solution of D-glucose into a magnetic resonance spectrometer; S2, a 90° hard pulse is applied to a deuterium water solution of D-glucose in a magnetic resonance spectrometer to obtain a H spectrum of D-glucose molecules 1 H spectrum; S3, by way of a D-glucose molecule 1 H spectrum determines chemical shift information for pairs of protons in the D-glucose molecule that have direct coupling relationships, and determines the 90° pulse time width required to excite the pairs of protons that have direct coupling relationships; S4, performing first selective filtering through a first multi-frequency point pulse based on a 90° pulse time width and chemical shift information to obtain signals within a proton pair chemical shift region having a direct coupling relationship; S5, performing filtering operation through a multi-quantum filtering module based on the signals within the proton pair chemical shift region to obtain multi-quantum signals evolved by the proton pairs having the direct coupling relationship, and performing second selective filtering on the multi-quantum signals evolved by the proton pairs having the direct coupling relationship through a second multi-frequency point pulse to obtain multi-quantum signals after phase information correction; S6, separating the multi-quantum signals after phase information correction by performing chemical shift encoding on a first 180° hard pulse in a CLIP-COSY module to obtain multi-quantum signal peaks of D-glucose having direct coupling relationship at different phases, and extending the multi-quantum signal peaks through J coupling evolution to obtain one-dimensional nuclear magnetic resonance phase-sensitive correlation spectra; S7, changing an evolution time, and performing S4-S6 again to obtain two one-dimensional nuclear magnetic resonance phase-sensitive correlation spectra at different phases, and performing phase encoding on the two one-dimensional nuclear magnetic resonance phase-sensitive correlation spectra to obtain one-dimensional nuclear magnetic resonance phase-sensitive correlation spectra of two signals in the multi-quantum signal peaks of D-glucose having the direct coupling relationship respectively; S8, obtaining direct coupling information of two signals in the proton pairs of D-glucose having the direct coupling relationship through the one-dimensional nuclear magnetic resonance phase-sensitive correlation spectra of the two signals in the multi-quantum signal peaks of D-glucose, and selecting different coupled proton pairs for excitation through the direct coupling information to track all coupled proton pairs and reveal the entire molecular structure of D-glucose.

2. The high-resolution D-glucose structure solving method based on nuclear magnetic resonance spectroscopy according to claim 1, characterized in that, In S1, the pre-configured deuterium water solution of D-glucose has a mass concentration of 2%-5%.

3. The high-resolution D-glucose structure solving method based on nuclear magnetic resonance spectroscopy according to claim 1, characterized in that, In S4, the first selective filtering performed through the first multi-frequency point pulse to obtain the signals within the proton pair chemical shift region having the direct coupling relationship specifically comprises: first and second gradient fields are added to the left and right of the first multi-frequency point pulse, coherent path selection is performed through the first and second gradient fields, and selective filtering is realized through the first multi-frequency point pulse to obtain the signals within the proton pair chemical shift region having the direct coupling relationship; the first and second gradient fields have equal intensity and direction.

4. The method for high-resolution D-glucose structure analysis based on nuclear magnetic resonance spectroscopy according to claim 1, characterized by, In S5, the multi-quantum filtering module comprises a first group of 90° hard pulses, a second group of 90° hard pulses, a third gradient field and a fourth gradient field; the intensity of the third gradient field after the first group of 90° hard pulses is proportional to the intensity of the fourth gradient field after the second group of 90° hard pulses at a ratio of -1:

2.

5. The high-resolution D-glucose structure solving method based on nuclear magnetic resonance spectroscopy according to claim 1, characterized in that, In S6, the CLIP-COSY module has two single-scan z-filter elements on both sides of a mixed period, the two single-scan z-filter elements ensure in-phase to in-phase coherent transfer between directly coupled spins in the multi-quantum signals after phase information correction, and eliminate anti-phase and zero quantum contributions in the multi-quantum signals after phase information correction.

6. The high-resolution D-glucose structure solving method based on nuclear magnetic resonance spectroscopy according to claim 1, characterized in that, In S6, the signal model after chemical shift encoding is: where ω I and ω S denote the chemical shift; I and S denote spin operators; the raising operator I + and the lowering operator I - denote +1 and -1 quantum steps, respectively; Δ1 and Δ2 denote two different evolution times.

7. The high-resolution D-glucose structure solving method based on nuclear magnetic resonance spectroscopy according to claim 1, characterized in that, In S7, the specific implementation of phase encoding on the two one-dimensional NMR phase-sensitive correlation spectra is Hadamard encoding.

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