A method for determining the enantiomer content of chiral crystals and its application

Abstract

The present application relates to a kind of method for determining the relative content of chiral crystal enantiomer and application, comprising the following steps: identifying and marking the coordinate position of all crystals on the grid;In imaging mode, successively collect the low-power image of all crystal samples in the rotation process, calculate the drift path of crystal in the rotation process;In electron diffraction mode, rotate all crystal samples in turn again, and according to the drift path of crystal, move electron beam spot, while collecting electron diffraction pattern, obtain crystal orientation, diffraction integral intensity information;According to crystal orientation information, simulate the three-dimensional electron diffraction data of two enantiomers in crystal sample under the same crystal orientation but different thickness and obtain its diffraction integral intensity;Compare experimental diffraction integral intensity with simulated diffraction integral intensity, determine the absolute structure of single crystal according to matching degree;Statistical analysis obtains the relative content of two enantiomers in crystal sample, solves the problem of enantiomer quantification in chiral powder crystal.

Description

Technical Field

[0001] This invention belongs to the field of compound molecular structure characterization technology, and in particular relates to a method and application for determining the enantiomer content of chiral crystals. Background Technology

[0002] Chirality is very common in drugs, proteins, and materials, and it is closely related to the properties of substances, especially chiral drugs, where enantiomers often exhibit completely different pharmacological activities. Single-crystal X-ray diffraction is a common method for characterizing the absolute structure of chiral crystals, but it is limited by the instrument and is generally only applicable to crystals larger than ten micrometers. Electron diffraction is an effective means of characterizing nanocrystals. Lukas et al. reported determining the absolute structure of chiral organic crystals through electron diffraction kinetic refinement, and Wang et al. proposed a method for determining the absolute structure of chiral organic crystals using selected area electron diffraction. In addition, crystal structure analysis using the cocrystallization method combined with cryo-electron microscopy three-dimensional electron diffraction has also been used to determine the absolute structure of organic molecules, but this method requires the target molecule to form a suitable cocrystallization with a molecule of known structure. However, all current methods can only analyze a small number of crystals (generally less than 30) to obtain qualitative analysis results, and cannot provide quantitative analysis results through the collection and analysis of large amounts of data. Therefore, it is necessary to provide a method for determining the enantiomer content of chiral crystals. Summary of the Invention

[0003] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a method and application for determining the enantiomer content of chiral crystals, thereby solving the problem that in the prior art, when the crystal size is small, it is impossible to use single-crystal X-ray diffraction to analyze the structure and to give quantitative analytical results.

[0004] To achieve the above and other related objectives, this 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, including the following steps:

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

[0006] S2. The mobile carrier network is used to capture a series of transmission electron microscope images, and the coordinate positions of all crystals are identified and marked.

[0007] S3. In imaging mode, acquire low-magnification images of all crystal samples during the rotation process in step S2, and calculate the drift path of the crystal during the rotation process.

[0008] S4. In electron diffraction mode, rotate all crystal samples in sequence and move the electron beam spot according to the drift path of the crystal in step S3. At the same time, collect the electron diffraction pattern and obtain information on crystal orientation and diffraction integral intensity.

[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 under the same crystal orientation but different thicknesses and obtain their diffraction integral intensity.

[0010] S6. Compare the diffraction integral intensity obtained in step S4 with the diffraction integral intensity obtained in step S5, and determine the absolute structure of a single crystal based on the matching degree.

[0011] S7. Through statistical analysis, the relative contents of the two enantiomers in the chiral powder crystal sample were obtained.

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

[0013] Preferably, the identification and marking method in step S2 is to automatically identify the crystals on the carrier and record their positions.

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

[0015] Preferably, in step S4, crystal morphology and size information are 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 different thicknesses mentioned in step S5 is as follows:

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

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

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

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

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

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

[0024] The present invention provides a method for determining the enantiomer content of chiral crystals. This method utilizes three-dimensional electron diffraction technology and a dual-rotation method, namely, rotating the crystal first in imaging mode and then rotating the crystal in 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 chiral analysis of chiral nanocrystals, and determines the content of left- and right-handed chiral enantiomers in the sample to be tested.

[0025] The method for determining the enantiomer content of chiral crystals in this invention, compared with previously reported electron diffraction-based methods, achieves high-throughput chiral structure analysis for the first time. It can not only perform qualitative analysis but also quantitative analysis by combining morphological images. Furthermore, the method for determining the enantiomer content of chiral crystals in this invention utilizes high-throughput three-dimensional electron diffraction data to analyze the content of two enantiomers in chiral crystals, currently reaching the level of hundreds of crystals. Especially for samples with small crystal sizes that cannot be analyzed using single-crystal X-ray diffraction, electron diffraction has advantages in characterizing nanoscale crystals compared to using single-crystal X-ray diffraction to determine crystal chirality. Attached Figure Description

[0026] Figure 1 This is a flowchart of the method of the present invention.

[0027] Figure 2 This is a schematic diagram illustrating the characterization steps in an embodiment of the present invention; Figure 2 a represents the crystal at the marked location on the carrier grid; Figure 2 b is a magnified transmission electron microscope image of the crystal; Figure 2 c represents the images and diffraction data collected by the two-step rotation method; Figure 2 d represents the collected high-throughput three-dimensional electron diffraction data.

[0028] Figure 3 This is a schematic diagram of the characterization results of Example 1; Figure 3 a represents the crystal size measurement results of 68 crystals; Figure 3 b is a histogram of crystal size distribution for crystals with different chiralities; Figure 3 c represents the content distribution of enantiomers.

[0029] Figure 4 This is a schematic diagram of the characterization results of Example 2; Figure 4a is a flowchart of the synthesis of chiral tellurium crystals; Figure 4 b represents the characterization results of the synthesized tellurium crystals when pure D-type penicillamine was used as the inducing agent; Figure 4 c represents the characterization results of the synthesized tellurium crystals when a mixture of L-type and D-type penicillamines was used as an inducer; Figure 4 d represents the characterization results of the synthesized tellurium crystals when pure L-type penicillamine was used as the inducer.

[0030] Figure 5 This is a schematic diagram of the characterization results for Example 3; Figure 5 a represents the (+)-cinkenin structure; Figure 5 b represents the (-)-cinkenin structure; Figure 5 c represents the chiral structure determination result of cinkingin. Detailed Implementation

[0031] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0032] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to an integer, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are included. For example, a specified range from “1 to 10” should be considered to include any and all subranges between the minimum value 1 and the maximum value 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, etc.

[0033] Furthermore, it should be understood that the one or more method steps mentioned in this invention do not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps, unless otherwise stated; moreover, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or limiting the scope of the invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered as within the scope of the invention.

[0034] The first aspect of this 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, including the following steps:

[0035] S1 disperses chiral powder crystals on a transmission electron microscope (TEM) grid and places them in the TEM.

[0036] The S2 mobile carrier network captured a series of transmission electron microscope images, identifying and marking the coordinate positions of all crystals;

[0037] S3. In imaging mode, acquire low-magnification images of all crystal samples during the rotation process in step S2, and calculate the drift path of the crystal during the rotation process.

[0038] S4. In electron diffraction mode, rotate all crystal samples in sequence and move the electron beam spot according to the drift path of the crystal in step S3. At the same time, collect the electron diffraction pattern and obtain information on crystal orientation and diffraction integral intensity.

[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 under the same crystal orientation but different thicknesses and obtain their diffraction integral intensity.

[0040] S6. Compare the diffraction integral intensity obtained in step S4 with the diffraction integral intensity obtained in step S5, and determine the absolute structure of a single crystal based on the matching degree.

[0041] S7. Through statistical analysis, the relative contents of the two enantiomers in the chiral powder crystal sample were obtained.

[0042] In this invention, the enantiomers refer to the two possible structures existing in the chiral crystal, which are mirror images of each other and cannot be superimposed by translation or rotation alone.

[0043] In this invention, absolute structure refers to the orientation specification of a non-centrosymmetric crystal structure under point reversal (i.e., parity, denoted as P) operations. Centrosymmetric crystal structures remain unchanged under the P transformation; therefore, no absolute crystal structure can be specified. For chiral crystal structures, due to their non-centrosymmetry, absolute structure can distinguish and specify enantiomers of the crystal structure. However, for achiral and non-centrosymmetric crystal structures, there are no enantiomers; these crystal structures can be superimposed on symmetric structures through translation and pure rotation. In this case, symmetry transformation is always equivalent to pure rotation.

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

[0045] In this invention, step S2, which involves capturing a series of transmission electron microscope (TEM) images, is performed at a low magnification. In a preferred embodiment of this invention, the low magnification is 4000x.

[0046] In this invention, the identification and marking method described in step S2 involves automatically identifying crystals on the carrier network and recording their positions. By importing the captured low-magnification images into the program, a threshold segmentation algorithm is used to identify crystals within the field of view and extract their positions (x and y coordinates within the field of view).

[0047] In this invention, the rotation angle in steps S3 and S4 is -50° to +50°. For example, it can be -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 acquiring low-magnification images of all crystal samples during rotation in step S2, the crystal marked in step S2 needs to be moved to the center of the field of view, and a suitable condenser aperture and magnification (e.g., 4000x) should be selected.

[0048] In this invention, step S4 also involves acquiring information such as crystal morphology and size rotation matrix. This data can be used to analyze the crystal structure.

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

[0050] In this invention, the thickness range of different thicknesses mentioned in step S5 is as follows: For example, or Etc. In a preferred embodiment of the present invention, the incremental step size of the thickness in the simulation is...

[0051] Chirality implies a broken symmetry in the atomic arrangement within a crystal. In electron diffraction, influenced by kinetic effects, two diffraction points symmetrical about the transmission spot (Frendel pair, hkl and -hkl) will exhibit one strong diffraction point and one relatively weak diffraction point in intensity. However, in selected area electron diffraction patterns of achiral crystals, a pair of diffraction points symmetrical about the transmission spot (Frendel pair) always satisfy Frendel's rule; the Frendel pair has equal intensity, i.e., I0 = I0. hkl =I -h-k-l For chiral crystals, considering the kinetic effects of electron diffraction, Frendel's rule is broken, resulting in unequal intensities of Frendel pairs, i.e., IF. hkl ≠I -h-k-lThe described dynamic simulation of electron diffraction intensity can simulate the strength relationship of diffraction points. The dynamic simulation process of this invention is based on the Bloch-wave method. The simulated three-dimensional electron diffraction method involves inputting the crystal structure model and rotation matrix of the crystal to be tested, and using the Bloch-wave method to simulate three-dimensional electron diffraction data at different thicknesses to extract the diffraction integral intensity. For example, if the rotation matrix of the crystal is determined in step S4 above, such as the initial crystal orientation being [u1, v1, w1], and an initial thickness is selected, the electron diffraction pattern is first simulated in this direction. Then, the crystal is rotated around a specific axis by different angles (e.g., from -50° to 50°, with an increment of 0.02°), and the electron diffraction pattern at the corresponding angle is calculated to obtain the simulated diffraction integral intensity. By changing the sample thickness, the above steps are repeated to obtain the simulated diffraction integral intensity at different thicknesses.

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

[0053] The matching degree determination method in step S6 of this invention is as follows: compare the experimental three-dimensional electron diffraction intensity with the simulated three-dimensional electron diffraction intensity at different thicknesses, and calculate the R1 value using the following formula. The structure 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 integral intensity); F c It is obtained from the simulation of diffraction integral intensity (F) c (Obtained by taking the square root of the simulated diffraction integral intensity); scale refers to F o With F c The proportion value.

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

[0056] A second aspect of this invention provides an application of the method for determining the enantiomer content of chiral crystals as described above in the characterization of nanocrystals, such as its application in determining the absolute structure of inorganic or organic nanocrystals.

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

[0058] Optionally, the computer-readable storage medium may include, but is not limited to, floppy disks, optical disks, CD-ROMs (Read-Only Optical Disk Memory), magneto-optical disks, ROMs (Read-Only Memory), RAMs (Random Access Memory), EPROMs (Erasable Programmable Read-Only Memory), EEPROMs (Electrically Erasable Programmable Read-Only Memory), magnetic cards or optical cards, 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 used in a computer device.

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

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

[0061] Optionally, the processor in the terminal will perform the following... Figure 1 Steps S2 to S7, as shown, load one or more instructions corresponding to the processes of an application into memory, and the processor runs the application stored in the first memory, thereby achieving the following: Figure 1 The 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. For example, 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 may be a general-purpose processor, including 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.

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

[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 P3121 or P3221 (the two are enantiomers), select racemic tellurium samples, and characterize them as follows: Figure 2 As shown:

[0067] 1) Take low-magnification (4000x) transmission electron microscope images of tellurium crystals. Figure 2 b) and mark the position of the crystal as shown in the figure. Figure 2 a);

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

[0069] 3) Using the crystal orientation information and crystal structure model obtained in step 2) above, at different sample thicknesses (thickness range of...), Step size is Simulate three-dimensional electron diffraction data and obtain the simulated diffraction integral intensity using XDS software;

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

[0071] 5) Based on the results of a large number of crystal statistics ( Figure 2 d) The relative contents of the two enantiomers were obtained ( Figure 3 Because this sample is racemic, the contents of the two enantiomers are each equal.

[0072] Example 2

[0073] This embodiment provides a non-racemic form of tellurium, synthesized using a method reported in the literature, employing chiral penicillin molecule-induced synthesis of a single chiral excess of tellurium sample. The characterization steps are the same as in Example 1. The measurement results are as follows: Figure 4 As shown, using pure D-type penicillamine as an inducer, the synthesized tellurium crystals had space groups P3121 and P3221 accounting for 87.8% and 12.2%, respectively. Figure 4 b); Using pure L-type penicillamine as an inducer, the synthesized tellurium crystals had space groups P3121 and P3221 accounting for 10.1% and 89.9%, respectively. Figure 4 d); while using a mixture of D and L (1:1) penicillamine as an inducer, the synthesized tellurium crystals had space groups P3121 and P3221 each accounting for approximately 50% ( Figure 4 c). The results of this test confirmed the circular dichroism spectrum.

[0074] Example 3

[0075] This embodiment provides cinnamylene powder crystals. Cinnamylene crystals belong to the monoclinic crystal system with space group P21. 25 crystals were tested as a sample. Figure 5 As shown, the result is a single chiral (+)-cinkenin, consistent with the expected result.

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

[0077] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can 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 those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method of determining the enantiomeric content of a chiral crystal, characterized in that, The quantitative analysis of the chiral structure of the chiral powder crystal sample by three-dimensional electron diffraction includes the following steps: S1, dispersing the chiral powder crystal sample on a transmission electron microscope grid, and placing it in a transmission electron microscope; S2, moving the grid to take a series of transmission electron microscope images, identifying and marking the coordinate positions of all the crystals; S3, in the imaging mode, sequentially collecting low-magnification images of all the crystal samples during the rotation process, and calculating the drift path of the crystals during the rotation process; S4, in the electron diffraction mode, sequentially rotating all the crystal samples again, and moving the electron beam spot according to the drift path of the crystals in step S3, while collecting electron diffraction patterns to obtain crystal orientation and diffraction integral intensity information; S5, according to the crystal orientation information obtained in step S4, simulating the three-dimensional electron diffraction data of two enantiomers in the same crystal orientation but different thicknesses in the crystal sample, and obtaining the diffraction integral intensity thereof; S6, comparing the diffraction integral intensity obtained in step S4 with the diffraction integral intensity simulated in step S5, and determining the absolute structure of a single crystal according to the matching degree; S7, by statistical analysis, obtaining the relative content of the two enantiomers in the chiral powder crystal sample.

2. The method of determining the enantiomeric content of a chiral crystal according to claim 1, wherein, In step S2, the series of transmission electron microscope images are taken at a low magnification of the electron microscope; And / or, the method of identification and marking in step S2 is automatic identification of the crystals on the grid and recording of the positions.

3. The method of determining the enantiomeric content of a chiral crystal according to claim 2, wherein, In step S2, the series of transmission electron microscope images are taken at a magnification of 4000 times of the electron microscope.

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

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

6. The method of determining the enantiomeric content of a chiral crystal of claim 1, wherein, In step S5, the simulation adopts Bloch wave method to simulate three-dimensional electron diffraction; And / or, in step S5, the thickness range of the different thicknesses is 50-3000 Å.

7. The method of determining the enantiomeric content of a chiral crystal according to claim 1, wherein, In steps S4 and S5, the tool for obtaining the diffraction integral intensity is XRD analysis software.

8. Use of the method for determining the content of enantiomers of a chiral crystal according to any one of claims 1-7 in the characterization of nanocrystals.

9. A computer-readable storage medium, characterized in that, The computer program stores data analysis, processing and control steps of steps S2-S7 in the method according to any one of claims 1-7 when the computer program is running.

10. A terminal comprising a memory and a processor, said memory comprising a computer program, characterized in that, The computer program is executed by a processor to perform data analysis, processing and control steps of steps S2-S7 in the method according to any one of claims 1-7.

11. A computer program product comprising a computer program, characterized in that, The computer program is executed by a processor to perform data analysis, processing and control steps of steps S2-S7 in the method according to any one of claims 1-7.

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