Method for determining content of enantiomer of chiral crystal and application

Abstract

The invention relates to a method for determining the content of enantiomers of chiral crystals and application. The method comprises the following steps: identifying and marking coordinate positions of all crystals on a recording net; sequentially collecting low-power images of all the crystal samples in the rotation process in the imaging mode, and calculating the drift path of the crystal in the rotation process; sequentially rotating all the crystal samples in an electron diffraction mode, moving an electron beam light spot according to a drift path of the crystal, collecting an electron diffraction pattern, and obtaining crystal orientation and diffraction integral intensity information; simulating three-dimensional electron diffraction data of two enantiomers in the crystal sample under the same crystal orientation but different thicknesses according to the crystal orientation information, and obtaining diffraction integral intensity of the two enantiomers; comparing the experimental diffraction integral intensity with the simulated diffraction integral intensity, and determining the absolute structure of the single crystal according to the matching degree; the relative content of the two enantiomers in the crystal sample is obtained through statistical analysis, and the problem of quantification of the enantiomers in the chiral powder crystal is solved.

Description

Technical Field

[0001] The present invention belongs to the technical field of compound molecular structure characterization, and particularly relates to a method for determining the enantiomer content of a chiral crystal and its application. Background Art

[0002] Chirality is very common in drugs, proteins, and materials, and is closely related to the properties of substances. This is particularly true for chiral drugs, where enantiomers often exhibit distinct pharmacological activities. Single-crystal X-ray analysis is a common method for characterizing the absolute structure of chiral crystals, but due to instrumental limitations, it is generally only suitable for crystals larger than ten microns. Electron diffraction is an effective tool for characterizing nanocrystals. Lukas et al. reported determining the absolute structure of chiral organic crystals using electron diffraction dynamics refinement, and Wang et al. proposed a method for determining the absolute structure of chiral organic crystals using selected-area electron diffraction. Furthermore, the cocrystal method combined with cryo-electron microscopy three-dimensional electron diffraction crystal structure analysis has also been used to determine the absolute structure of organic molecules. However, this method requires the target molecule to form a suitable cocrystal with a molecule of known structure. However, all current methods can only analyze a small number of crystals (typically less than 30) and obtain qualitative results, rather than providing quantitative results through the collection and analysis of large amounts of data. Therefore, a method for determining the enantiomeric content of chiral crystals is highly desirable. Summary of the Invention

[0003] In view of the shortcomings of the prior art described above, the object of the present invention is to provide a method and application for determining the enantiomer content of chiral crystals, so as to solve the problem in the prior art that when the crystal size is small, single crystal X-ray diffraction cannot be used to analyze the structure and provide quantitative analysis results.

[0004] To achieve the above and other related objectives, the present invention provides a method for determining the enantiomer content of chiral crystals, which utilizes three-dimensional electron diffraction to quantitatively analyze the chiral structure of powder crystals, comprising the following steps:

[0005] S1. Disperse chiral powder crystals on a transmission electron microscope grid and place it in a transmission electron microscope;

[0006] S2. Move the grid to take a series of transmission electron microscope images, identify and mark the coordinate positions of all crystals;

[0007] S3, in imaging mode, sequentially collecting low-magnification images of all crystal samples during the rotation process in step S2, and calculating the drift path of the crystal during the rotation process;

[0008] S4. Rotate all crystal samples in sequence again in electron diffraction mode, and move the electron beam spot according to the drift path of the crystal in step S3, while collecting electron diffraction patterns to obtain crystal orientation and diffraction integrated intensity information;

[0009] S5. Based on the crystal orientation information obtained in step S4, simulate the three-dimensional electron diffraction data of two enantiomers in the crystal sample at the same crystal orientation but different thicknesses and obtain the diffraction integrated intensity thereof;

[0010] S6, comparing the diffraction integrated intensity obtained in step S4 with the diffraction integrated intensity simulated in step S5, and determining the absolute structure of the single crystal based on the matching degree;

[0011] S7. Obtain the relative contents of the two enantiomers in the chiral powder crystal sample through statistical analysis.

[0012] Preferably, in step S2, the capturing of a series of transmission electron microscope images is performed under a low magnification of the electron microscope. More preferably, the low magnification is 4000 times.

[0013] Preferably, the identification and marking method in step S2 is to automatically identify the crystal on the grid and record the position.

[0014] Preferably, in steps S3 and S4, the rotation angle is -50° to +50°.

[0015] Preferably, in step S4, crystal morphology and size information is also obtained.

[0016] Preferably, the simulation in step S5 uses the Bloch wave method to simulate three-dimensional electron diffraction.

[0017] Preferably, the thickness range of the different thicknesses in step S5 is

[0018] Preferably, the tool for acquiring the diffraction integrated intensity in steps S4 and S5 is: XRD analysis software.

[0019] The present invention also provides an application of the above-mentioned method for determining the enantiomer content of chiral crystals in the characterization of nanocrystals.

[0020] The present invention also provides a computer-readable storage medium storing a computer program, which, when executed, implements the data analysis, processing, and control steps of steps S2 to S7 in the above-described method.

[0021] The present invention also provides a terminal comprising a memory and a processor, wherein the memory comprises a computer program, and when the computer program is executed by the processor, the data analysis, processing and control steps of steps S2 to S7 in the above method are executed.

[0022] The present invention also provides a computer program product, comprising a computer program, which, when executed by a processor, implements the data analysis, processing and control steps of steps S2 to S7 in the above method.

[0023] As described above, the method and application of determining the enantiomer content of chiral crystals of the present invention have the following beneficial effects:

[0024] The method for determining the enantiomer content of a chiral crystal of the present invention utilizes three-dimensional electron diffraction technology and a two-wheel rotation method, that is, first rotating the crystal in an imaging mode and then rotating the crystal in a diffraction mode to track the crystal drift trajectory, thereby confirming the content of the two enantiomers in the chiral crystal. The scheme is simple and effective, solves the problem of quantitative chirality analysis of chiral nanocrystals, and determines the content of the left and right chiral enantiomers of the sample to be tested.

[0025] Compared with previously reported electron diffraction-based methods, the method of the present invention for determining the enantiomer content of chiral crystals achieves high-throughput chiral structure analysis for the first time, enabling not only qualitative analysis but also quantitative analysis in combination with morphological images. Furthermore, the method of the present invention for determining the enantiomer content of chiral crystals utilizes high-throughput three-dimensional electron diffraction data to analyze the content of the two enantiomers in chiral crystals, currently measuring up to hundreds of particles. This is particularly true for samples with small crystal sizes that cannot be analyzed using single-crystal X-ray diffraction. Compared with determining crystal chirality using single-crystal X-ray diffraction, electron diffraction has advantages in characterizing nanosized crystals. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Flowchart of the method of the present invention.

[0027] Figure 2 A specific schematic diagram of the characterization steps of an embodiment of the present invention; Figure 2 a is the crystal at the mark on the grid; Figure 2 b is the transmission electron microscope image of the crystal after magnification; Figure 2 c is the image and diffraction data collected by the two-step rotation method; Figure 2 d is the collected high-throughput three-dimensional electron diffraction data.

[0028] Figure 3 Schematic diagram of the characterization results of Example 1; Figure 3 a is the crystal size measurement result of 68 crystals; Figure 3 b is the crystal size distribution histogram of crystals with different chirality; Figure 3 c is the content distribution of enantiomers.

[0029] Figure 4 Schematic diagram of the characterization results of Example 2; Figure 4a is the synthesis flow chart of chiral tellurium crystals; Figure 4 b is the characterization results of the synthesized tellurium crystals when pure D-type penicillamine was used as the inducer; Figure 4 c is the characterization results of the synthesized tellurium crystals when a mixture of L-type and D-type penicillamine was used as an inducer; Figure 4 d is the characterization results of the synthesized tellurium crystals when pure L-penicillamine was used as the inducer.

[0030] Figure 5 Schematic diagram of the characterization results of Example 3; Figure 5 a is the structure of (+)-cinconazole; Figure 5 b is the structure of (-)-cinconazole; Figure 5 c is the chiral structure determination result of cinchonine. DETAILED DESCRIPTION

[0031] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0032] When a numerical range is disclosed herein, the above range is deemed to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein. For example, a specified range from "1 to 10" should be deemed to include any and all subranges between a minimum of 1 and a maximum of 10. Exemplary subranges of the range 1 to 10 include, but are not limited to, 1 to 6.1, 3.5 to 7.8, 5.5 to 10, and the like.

[0033] In addition, it should be understood that one or more method steps mentioned in the present invention do not exclude the existence of other method steps before or after the combination step or the insertion of other method steps between these explicitly mentioned steps, unless otherwise specified; and, unless otherwise specified, the numbering of each method step is merely a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or to define the scope of implementation of the present invention. Changes or adjustments in their relative relationships, without substantially changing the technical content, should also be regarded as the scope of implementation of the present invention.

[0034] The first aspect of the present invention provides a method for determining the enantiomer content of chiral crystals, which quantitatively analyzes the chiral structure of powder crystals using three-dimensional electron diffraction, comprising the following steps:

[0035] S1 disperses chiral powder crystals on a transmission electron microscope grid and places it in a transmission electron microscope;

[0036] The S2 mobile grid takes a series of transmission electron microscope images to identify and mark the coordinate positions of all crystals;

[0037] S3, in imaging mode, sequentially collecting low-magnification images of all crystal samples during the rotation process in step S2, and calculating the drift path of the crystal during the rotation process;

[0038] S4. Rotate all crystal samples in sequence again in electron diffraction mode, and move the electron beam spot according to the drift path of the crystal in step S3, while collecting electron diffraction patterns to obtain crystal orientation and diffraction integrated intensity information;

[0039] S5. Based on the crystal orientation information obtained in step S4, simulate the three-dimensional electron diffraction data of two enantiomers in the crystal sample at the same crystal orientation but different thicknesses and obtain the diffraction integrated intensity thereof;

[0040] S6, comparing the diffraction integrated intensity obtained in step S4 with the diffraction integrated intensity simulated in step S5, and determining the absolute structure of the single crystal based on the matching degree;

[0041] S7. Obtain the relative contents of the two enantiomers in the chiral powder crystal sample through statistical analysis.

[0042] In the present invention, enantiomers refer to two possible structures that exist in chiral crystals, which are mirror images of each other and cannot be superimposed on each other only by translation or rotation.

[0043] In the present invention, absolute structure refers to the orientation specification of a non-centrosymmetric crystal structure under the operation of point inversion (i.e., parity, denoted as P). The centrosymmetric crystal structure remains unchanged under the P transformation, so there is no absolute crystal structure to specify. For chiral crystal structures, due to their non-centrosymmetry, the absolute structure can distinguish and specify the enantiomers of the crystal structure. For achiral and non-centrosymmetric crystal structures, there are no enantiomers, and these crystal structures can be superimposed with the symmetric structure through translation and pure rotation. In this case, symmetry transformation is always equivalent to pure rotation.

[0044] In the present invention, three-dimensional electron diffraction refers to a data set obtained by collecting electron diffraction patterns along different directions of the same crystal, which can be used to determine the unit cell, space group and crystal structure of the crystal.

[0045] In the present invention, in step S2, the taking of a series of transmission electron microscope images is performed under a low magnification of the electron microscope. In a preferred embodiment of the present invention, the low magnification is 4000 times.

[0046] In the present invention, the identification and marking method described in step S2 is to automatically identify the crystals on the grid and record their positions by importing the captured low-magnification image into the program, using a threshold segmentation algorithm to identify the crystals within the field of view, and extracting the crystal positions (x and y coordinates within the field of view).

[0047] In the present invention, in steps S3 and S4, the rotation angle is -50° to +50°. For example, it is -50° to -40°, -40° to -30°, -30° to -20°, -20° to -10°, -10° to 0°, 0° to 10°, 10° to 20°, 20° to 30°, 30° to 40°, or 40° to 50°. In step S3, before collecting low-magnification images of all crystal samples during rotation in step S2, it is necessary to move the crystal marked in step S2 to the center of the field of view and select an appropriate condenser aperture and magnification (e.g., 4000x).

[0048] In the present invention, in step S4, information such as crystal morphology, size rotation matrix, etc. can be obtained. The crystal structure can be analyzed through these data.

[0049] In the present invention, the simulation in step S5 uses the Bloch wave method to simulate three-dimensional electron diffraction.

[0050] In the present invention, the thickness range of the different thicknesses in step S5 is For example, or In a preferred embodiment of the present invention, the incremental step size of the thickness in the simulation is

[0051] Chirality means that the symmetry of the atomic arrangement inside the crystal is broken. In electron diffraction, due to the influence of dynamic effects, two diffraction points symmetrical about the center of the transmission spot (Freundl pair, hkl and -hkl) will show one strong diffraction point and one relatively weak diffraction point in terms of intensity. In the selected area electron diffraction pattern of non-chiral crystals, a pair of diffraction points symmetrical about the center of the transmission spot (Freundl pair) always satisfies Freundl's rule, and the intensity of the Freundl pair is equal, that is, I hkl =I -h-k-l For chiral crystals, the dynamic effect of electron diffraction is considered, and the Freundel rule is broken, resulting in unequal Freundel pair intensities, i.e., I hkl ≠I -h-k-l. The dynamic simulation of electron diffraction intensity can simulate the strength relationship of diffraction points. The dynamic simulation process of the present invention is based on the Bloch-wave method. The simulated three-dimensional electron diffraction method is as follows: the crystal structure model and rotation matrix of the crystal to be measured are input, and the Bloch-wave method is used to simulate the three-dimensional electron diffraction data at different thicknesses, and the diffraction integral intensity is extracted. For example, in the above step S4, the rotation matrix of the crystal is determined. If the initial crystal orientation is [u1, v1, w1], an initial thickness is selected, and the electron diffraction pattern is first simulated in this direction. Then, it is rotated around a specific axis at different angles (for example, from -50° to 50°, with an incremental step of 0.02°), and the electron diffraction pattern at the corresponding angle is calculated to obtain the simulated diffraction integral intensity. Change the thickness of the sample and repeat the above steps to obtain the simulated diffraction integral intensity at different thicknesses.

[0052] In the present invention, the tool for acquiring the diffraction integrated intensity in steps S4 and S5 is XRD analysis software. In steps S4 and S5, the three-dimensional electron diffraction integrated intensity refers to the intensity distribution of the diffraction point in actual experiments, which should be close to a Gaussian distribution. Extracting the integrated intensity involves considering the range of the diffraction point, fitting the peak shape, and calculating the integrated area. This function can be implemented using the electron diffraction pattern processing software XDS.

[0053] The matching degree determination method in step S6 of the present invention is as follows: comparing the experimental three-dimensional electron diffraction intensity with the simulated three-dimensional electron diffraction intensity at different thicknesses, and calculating the R1 value using the following formula. The one with the smallest R1 value is the correct chiral structure:

[0054] R1=∑ hkl ||F o |-scale*|F c || / ∑ hkl |F o |, where F o It is calculated based on the experimental diffraction integral intensity (F o Obtained by taking the square root of the experimental diffraction integrated intensity); F c is calculated from the simulated diffraction integral intensity (F c (obtained by taking the square root of the simulated diffraction integrated intensity); scale refers to F o With F c The ratio value.

[0055] In the present invention, step S7 obtains the relative contents of different enantiomers in the sample through statistical analysis of a large amount of data.

[0056] The second aspect of the present invention provides an application of the above-mentioned method for determining the enantiomer content of chiral crystals in nanocrystal characterization, such as application in determining the absolute structure of inorganic nanocrystals or organic nanocrystals.

[0057] A third aspect of the present invention provides a computer-readable storage medium storing a computer program, which, when executed, implements the data analysis, processing, and control steps of steps S2 to S7 in the above-described method.

[0058] Alternatively, the computer-readable storage medium may include, but is not limited to, a floppy disk, an optical disk, a CD-ROM (Compact Disc Read Only Memory), a magneto-optical disk, a ROM (Read Only Memory), a RAM (Random Access Memory), an EPROM (Erasable Programmable Read Only Memory), an EEPROM (Electrically Erasable Programmable Read Only Memory), a magnetic or optical card, a flash memory, or other types of media / machine-readable media suitable for storing machine-executable instructions. The computer-readable storage medium may be a product not connected to a computer device, or a component connected to a computer device for use.

[0059] A fourth aspect of the present invention provides a terminal comprising a memory and a processor, wherein the memory comprises a computer program, and when the computer program is executed by the processor, the data analysis, processing and control steps of steps S2 to S7 in the above method are executed.

[0060] Optionally, the number of the memories may be one or more, and the number of the processors may be one or more.

[0061] Optionally, the processor in the terminal will Figure 1 In steps S2 to S7, one or more instructions corresponding to the process of the application are loaded into the memory, and the processor runs the application stored in the first memory, thereby achieving the following. Figure 1 Various functions of steps S2 to S7 in the method.

[0062] Optionally, the memory may include, but is not limited to, high-speed random access memory and non-volatile memory, such as one or more disk storage devices, flash memory devices, or other non-volatile solid-state storage devices; the processor may include, but is not limited to, a central processing unit (CPU), a network processor (NP), etc.; it may also be a digital signal processor (DSP), an application-specific integrated circuit (ASIC), a field-programmable gate array (FPGA), or other programmable logic devices, discrete gate or transistor logic devices, or discrete hardware components.

[0063] Optionally, the processor can be a general-purpose processor, including a central processing unit (CPU), a network processor (NP), etc.; it can also be a digital signal processor (DSP), an application-specific integrated circuit (ASIC), a field-programmable gate array (FPGA) or other programmable logic devices, discrete gate or transistor logic devices, discrete hardware components.

[0064] A fifth aspect of the present invention provides a computer program product, comprising a computer program, which, when executed by a processor, implements the data analysis, processing and control steps of steps S2 to S7 in the above method.

[0065] Example 1

[0066] The tellurium crystal provided in this embodiment belongs to the trigonal crystal system, and its unit cell is: α=90°、β=90°、γ=120°,space group is P3121 or P3221 (the two are enantiomers of each other), a racemic sample of tellurium is selected, and the characterization steps are as follows, such as Figure 2 As shown:

[0067] 1) Take a low-magnification (4000x) transmission electron microscope image of a tellurium crystal ( Figure 2 b) and mark the position of the crystal ( Figure 2 a);

[0068] 2) Collect the image and electron diffraction data of the crystal in step 1) by the two-step rotation method (rotation angle: -50° to +50°) Figure 2 c), the image data is used to estimate the sample volume, and the electron diffraction data are used to extract the unit cell, crystal orientation (rotation matrix), and diffraction integrated intensity using XDS software;

[0069] 3) Using the crystal orientation information and crystal structure model obtained in step 2), the thickness of the samples was varied (thickness range: The step size is ) simulate three-dimensional electron diffraction data and obtain the simulated diffraction integrated intensity using XDS software;

[0070] 4) comparing the diffraction integral intensity of the experimental data in step 2) with the diffraction integral intensity of the simulated data in step 3) to determine the absolute structure of the crystal;

[0071] 5) According to the results of a large number of crystal statistics ( Figure 2 d) Determine the relative content of the two enantiomers ( Figure 3 Since this sample is racemic, the content of the two enantiomers is half each.

[0072] Example 2

[0073] The non-racemic tellurium provided in this example was synthesized using a method reported in the literature, using chiral penicillamine molecules to induce the synthesis of a single chiral excess tellurium sample. The characterization steps were the same as in Example 1. The measurement results are shown in Figure 1. Figure 4 As shown, using pure D-penicillamine as an inducer, the synthesized tellurium crystals P3121 and P3221 space groups accounted for 87.8% and 12.2% respectively ( Figure 4 b); Using pure L-penicillamine as an inducer, the synthesized tellurium crystals P3121 and P3221 space groups accounted for 10.1% and 89.9% respectively ( Figure 4 d); When D and L (1:1) were mixed with penicillamine as an inducer, the synthesized tellurium crystals P3121 and P3221 space groups each accounted for about 50% ( Figure 4 c) The results of this method were confirmed by circular dichroism spectroscopy.

[0074] Example 3

[0075] The present embodiment provides the powder crystal of cinchoninic acid, which belongs to the monoclinic system and has a space group of P21. 25 crystals were tested in the sample. Figure 5 As shown, the result was single chiral (+)-cinconazole, which was consistent with the expected result.

[0076] Therefore, the present invention effectively overcomes various shortcomings of the prior art and has high industrial utilization value.

[0077] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A method for determining the enantiomer content of a chiral crystal, characterized in that: The quantitative analysis of the chiral structure of a chiral powder crystal sample using three-dimensional electron diffraction includes the following steps: S1. Disperse the chiral powder crystal sample on a transmission electron microscope grid and place it in a transmission electron microscope; S2. Move the grid to take a series of transmission electron microscope images, identify and mark the coordinate positions of all crystals; S3, in imaging mode, sequentially collecting low-magnification images of all crystal samples during the rotation process in step S2, and calculating the drift path of the crystal during the rotation process; S4. Rotate all crystal samples in sequence again in electron diffraction mode, and move the electron beam spot according to the drift path of the crystal in step S3, while collecting electron diffraction patterns to obtain crystal orientation and diffraction integrated intensity information; S5. Based on the crystal orientation information obtained in step S4, simulate the three-dimensional electron diffraction data of two enantiomers in the crystal sample at the same crystal orientation but different thicknesses and obtain the diffraction integrated intensity thereof; S6, comparing the diffraction integrated intensity obtained in step S4 with the diffraction integrated intensity simulated in step S5, and determining the absolute structure of the single crystal based on the matching degree; S7. Obtain the relative contents of the two enantiomers in the chiral powder crystal sample through statistical analysis.

2. The method for determining the enantiomer content of a chiral crystal according to claim 1, wherein In step S2, the taking of a series of transmission electron microscope images is performed under a low magnification of the electron microscope; preferably, the low magnification is 4000 times; And / or, the identification and marking method in step S2 is to automatically identify the crystal on the grid and record the position.

3. The method for determining the enantiomer content of a chiral crystal according to claim 1, wherein: In steps S3 and S4, the rotation angle is -50° to +50°.

4. The method for determining the enantiomer content of a chiral crystal according to claim 1, wherein: In step S4, crystal morphology and size information is also obtained.

5. The method for determining the enantiomer content of a chiral crystal according to claim 1, wherein: The simulation in step S5 uses the Bloch wave method to simulate three-dimensional electron diffraction; And / or, the thickness range of the different thicknesses in step S5 is 6. The method for determining the enantiomer content of a chiral crystal according to claim 1, wherein: The tool for acquiring the diffraction integrated intensity in steps S4 and S5 is: XRD analysis software.

7. Use of the method for determining the enantiomer content of chiral crystals according to any one of claims 1 to 6 in the characterization of nanocrystals.

8. A computer-readable storage medium, characterized in that A computer program is stored, and when the computer program is run, the data analysis, processing and control steps of steps S2 to S7 in the method according to any one of claims 1 to 6 are implemented.

9. A terminal comprising a memory and a processor, wherein the memory comprises a computer program, characterized in that: When the computer program is executed by a processor, the data analysis, processing and control steps of steps S2 to S7 in the method according to any one of claims 1 to 6 are executed.

10. A computer program product comprising a computer program, characterized in that When the computer program is executed by a processor, the data analysis, processing and control steps of steps S2 to S7 in the method according to any one of claims 1 to 6 are implemented.

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