Method for culturing organic small-molecule single crystals

Abstract

The application discloses a kind of organic small molecule single crystal culture methods, belong to single crystal culture technical field.The application is mainly based on the method principle of gas phase diffusion cooperates Mosquito automatic sample application robot and carries out medicine organic small molecule single crystal culture.First, sample is dissolved in good solvent and placed on sample plate, and sample solution is added to sample application hole on crystal plate using automatic sample application robot, while adding poor solvent in the pool liquid hole of crystal plate, and the required single crystal can be prepared after sealing the crystallization plate and culturing at a specific temperature.The application is simple to operate, requires less sample, is basically automated, is easy to observe, and has high single crystal culture efficiency.Compared with traditional single crystal culture method, only milligram level sample is needed to design a large number of crystallization conditions for experiment in a short time, and high-throughput screening preparation of small molecule crystal is realized.

Description

Technical Field

[0001] This invention relates to a method for cultivating single crystals of small organic molecules, belonging to the field of single crystal cultivation technology. Background Technology

[0002] Vapor diffusion is a common method for cultivating small molecule single crystals. Typically, two miscible solvents are selected, with a significant difference in solubility between the compound and the solvent: a good solvent A and a poor solvent B. Generally, good solvent A is a non-volatile solvent, while poor solvent B is a volatile solvent. The compound is dissolved in good solvent A and placed in the same sealed space as poor solvent B. Poor solvent B evaporates into good solvent A, causing the solubility of the compound to continuously decrease, thus precipitating crystals. By adjusting the concentration of the compound and changing the type of solvent, the crystallization conditions can be optimized to cultivate single crystals of sufficient mass and size.

[0003] Currently, the cultivation of single crystals using the vapor-phase diffusion method is typically carried out in vials. A volatile, poorly formulated solvent is added to the larger vial, while a better solvent solution containing the compound is added to the smaller vial. After sealing, the vials are allowed to diffuse statically, and the precipitation of crystals is observed periodically. Vapor-phase diffusion cultivation of single crystals usually requires a large sample volume and is quite complex. Designing experiments with multiple different crystallization conditions often takes a significant amount of time, and there is no guarantee that single crystals of sufficient size will be obtained.

[0004] The Mosquito automated sample dispensing robot is an instrument that provides rapid and convenient technical support for the complex experimental process of protein crystallization screening. Using the Mosquito instrument, high-throughput screening of protein crystals can be achieved using the sitting drop, pendant drop, and additive methods. The Mosquito instrument works by using a high-precision stepper motor to drive a 0.4mm inner diameter dispensing needle, employing a volumetric displacement method for sample dispensing. It can dispense samples without adjusting pipetting parameters for different liquid types. Controlled by high-throughput screening software, the Mosquito instrument automatically dispenses a fixed volume of protein solution sample sequentially into each well on the crystallization plate. By sealing the entire crystallization plate, multiple closed crystallization chambers are formed for crystallization. In experiments, by controlling the type and concentration of solvent and the volume of liquid dispensed per cycle, high-throughput screening of protein crystals under various conditions can be achieved quickly and efficiently.

[0005] While experiments using the Mosquito instrument can achieve large-scale screening and preparation of protein crystals with only trace amounts of sample, for small organic molecules, existing literature only reports cases of using the Mosquito instrument to cultivate single crystals via oil droplet evaporation (DOI: 10.1016 / j.chempr.2020.04.009). Evaporation is also a common method for cultivating single crystals of small molecules. Typically, sample solutions of different concentrations are prepared in vials, and as the solvent evaporates, the sample solution reaches a supersaturated state, thereby precipitating crystals. The desired single crystal size can be obtained by controlling the solvent evaporation rate through operations such as oil droplets and by changing the ambient temperature.

[0006] In small molecule single crystal cultivation experiments, evaporation alone is often insufficient to obtain single crystals of the desired size. Therefore, using a Mosquito instrument for gas-phase diffusion cultivation of small molecule single crystals is only a possible technique. Whether sufficient small molecule single crystals of the required size can be obtained depends on adjusting the compound concentration, selecting the appropriate solvent, and optimizing the crystallization conditions.

[0007] Cyclic adenosine monophosphate (molecular formula: C 10 H 12 Cyclic adenosine monophosphate (cAMP) is an important substance involved in intracellular metabolism and various biological functions. Its effects are very broad; injection of large doses can enhance myocardial contractility, increase cardiac output, and has effects such as relaxing smooth muscle, dilating coronary arteries, improving liver function, and alleviating myocardial hypoxia. However, cAMP has poor solubility in organic solvents and is only slightly soluble in water, making single-crystal cultivation using traditional methods difficult.

[0008] Listatin (molecular formula: C 29 H 24 Litahistamine (Cl2N2O7S) is a novel small-molecule integrin antagonist with wide applications in real-world applications. It primarily inhibits T-cell-mediated inflammation by blocking the binding of two important cell surface proteins, thereby alleviating the overall inflammatory response. Litahistamine has generally low solubility in common solvents and tends to form an oil during crystallization. In conventional single-crystal culture experiments, even with extensive experimental design, it is difficult to obtain crystals of sufficient size. Summary of the Invention

[0009] The purpose of this invention is to address the shortcomings of existing small molecule single crystal cultivation methods, such as requiring a large amount of sample, complex operation, and low efficiency in obtaining single crystals, by providing a high-throughput crystal cultivation method based on an automated spotting robot. This method is simple to operate, highly efficient, and requires less sample.

[0010] The technical solution adopted to achieve the purpose of this invention is: a method for cultivating single crystals of organic small molecules, which includes the following steps:

[0011] 1) Dissolve the small organic molecule compound in a good solvent to obtain a solution of the small organic molecule compound;

[0012] 2) Use an automated spotting robot to sequentially dispense the organic small molecule compound solution into the spotting wells on the crystal plate, while adding unsuitable solvents into the pooling wells of the crystal plate.

[0013] 3) Seal the crystallization plate to form multiple closed crystallization chambers for crystallization until organic small molecule single crystals of sufficient size are obtained.

[0014] Preferably, the single crystal culture of the present invention is carried out on a 96-well crystal plate, which has 8 rows and 12 columns, and each well position includes 1 pooling liquid well and 3 spot wells.

[0015] More preferably, the upper sample plate matching the 96-well crystal plate has 8 grooves, which can hold up to 8 different concentrations or different solvents of organic small molecule compound solutions; a poor solvent is added to the pool liquid wells, and the 96-well crystal plate has 12 columns, which can hold up to 12 poor solvents; the good solvent is a non-volatile solvent; the poor solvent is a volatile solvent and is miscible with the good solvent.

[0016] As a preferred embodiment, the present invention is used for single-crystal culture of cyclic adenosine monophosphate (cAMP), wherein the molecular formula of cAMP is C1. 10 H 12 N5O6P.

[0017] Preferably, the cyclic adenosine monocrystalline solution of the present invention is obtained by the following steps:

[0018] 1) Water was chosen as a good solvent, and aqueous solutions of cyclic adenosine monophosphate (cAMP) at different concentrations were prepared on the sample plate. The concentrations of the aqueous solutions corresponding to the eight grooves on the sample plate were 10 mg / mL, 10 mg / mL, 9 mg / mL, 9 mg / mL, 8 mg / mL, 8 mg / mL, 7 mg / mL, and 7 mg / mL, respectively.

[0019] 2) Using an automated sampling robot, the cyclic adenosine monophosphate aqueous solution was sequentially dispensed into the sampling wells on the crystal plate. The solution volumes corresponding to the three sampling well positions were 100 nL, 150 nL, and 200 nL, respectively.

[0020] 3) Use a multichannel pipette to add unsuitable solvents into the wells of the crystal plate. Five solvents were selected as unsuitable solvents: methanol, ethanol, tetrahydrofuran, acetone, and isopropanol.

[0021] 4) After sealing the crystallization plate, incubate at a constant temperature and observe the crystal condition using a stereomicroscope.

[0022] Preferably, the crystal structure of the cyclic adenosine monophosphate single crystal of the present invention is monoclinic, with space group P21 and cell parameters as follows: α=90°, β=98.7250(10)°, γ=90°,

[0023] In another preferred embodiment, the present invention is used for single-crystal cultivation of ristatin, wherein the molecular formula of ristatin is C1. 29 H 24 Cl2N2O78.

[0024] Preferably, the ristatin single crystal of the present invention is obtained by the following steps:

[0025] 1) Using acetone as a good solvent, different concentrations of listatin solutions were prepared on the sample plate. The aqueous solution concentrations corresponding to the 8 grooves on the sample plate were 5 mg / mL, 5 mg / mL, 4 mg / mL, 4 mg / mL, 3 mg / mL, 3 mg / mL, 2 mg / mL, and 2 mg / mL, respectively.

[0026] 2) An automated dispensing robot sequentially dispenses the sample solution into the dispensing wells on the crystal plate. The solution volumes at the three dispensing locations are 100 nL, 150 nL, and 200 nL, respectively.

[0027] 3) Use a multichannel pipette to add antisolvents to the wells of the crystal plate. Four solvents were selected as antisolvents: ethyl acetate, acetonitrile, methyl tert-butyl ether, and n-heptane.

[0028] 4) After sealing the crystallization plate, incubate at a constant temperature and observe the crystal condition using a stereomicroscope.

[0029] Preferably, the crystal structure of the ristat single crystal of the present invention is monoclinic, with space group P21 and cell parameters as follows: α=90°, β=104.6520(10)°, γ=90°,

[0030] The technical advantages of this invention are as follows:

[0031] 1) This invention is based on the principle of gas-phase diffusion for single crystal cultivation. Small molecule single crystals are cultivated using gas-phase diffusion with the help of a Mosquito instrument. By adjusting the compound concentration and selecting the solvent, and optimizing the crystallization conditions, small molecule single crystals of cyclic adenosine monophosphate and ristatin of sufficient size are obtained.

[0032] 2) This invention requires a small sample volume and is largely automated. It can replace traditional single-crystal cultivation methods, requiring only milligram-level samples to design numerous crystallization conditions for experiments in a short time, thus achieving high-throughput screening and preparation of small molecule crystals. Attached Figure Description

[0033] Figure 1 This is a 96-well crystal plate used in the experiments of this invention.

[0034] Figure 2 This is a morphology diagram of the crystals cultured in Example 1 of the present invention.

[0035] Figure 3 This is a morphology diagram of the crystals cultured in Example 2 of the present invention.

[0036] Figure 4 This is a diagram of the asymmetric unit of cyclic adenosine monophosphate (cAMP).

[0037] Figure 5 This is a morphology diagram of the crystals cultured in Example 3 of the present invention.

[0038] Figure 6 For the asymmetric unit diagram of Littors. Detailed Implementation

[0039] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The embodiments and descriptions of the present invention are only for explaining the present invention and are not intended to limit the present invention.

[0040] This invention is based on the principle of gas-phase diffusion and combines the high throughput and rapid sample loading capabilities of the Mosquito instrument for single-crystal cultivation of small organic molecules. First, the solubility of the small organic molecule compound is roughly determined. A certain mass of sample is weighed, and the selected solvent is gradually added. The dissolution is observed and the solubility is calculated using vortexing or ultrasound. Based on the solubility results, suitable good and bad solvents are selected.

[0041] Then, the sample is dissolved in a good solvent and placed on a sample plate. Under program control, an automated sampling robot sequentially dispenses the sample solution from the sample plate into each well of the crystal plate. Simultaneously, a multi-channel pipette adds a poor solvent to the wells of the crystal plate. After sealing the entire crystal plate, it is incubated at a specific temperature to obtain the desired single crystal.

[0042] like Figure 1In this invention, the single crystal culture experiment is conducted on a 96-well crystal plate. The 96-well crystal plate has 8 rows and 12 columns, with each well containing one pooling well and three sample wells. The matching sample plate has 8 grooves, accommodating up to 8 different concentrations or solvent types of sample solutions. The 96-well crystal plate has 12 columns, and a multi-channel pipette is used to add antisolvents to the pooling wells, allowing for up to 12 different antisolvents. Through program editing, three different volumes of sample solution can be added to the three sample wells. The Mosquito instrument can perform high-throughput screening of up to 288 conditions simultaneously.

[0043] In this invention, the good solvent selected when preparing the sample solution is generally a non-volatile solvent; the anti-solvent added to the pool liquid hole is generally a volatile poor solvent, and is ensured to be miscible with the good solvent used to dissolve the sample.

[0044] In this invention, sealing the entire crystallization plate is to create multiple sealed crystallization chambers in the pores of the crystallization plate, thereby facilitating gas-phase diffusion experiments. The crystallization plate is then incubated at a specific temperature, determined according to the properties of the compound (18°C can be selected), and observed periodically using a stereomicroscope to obtain the desired single crystals.

[0045] Example 1

[0046] Selection of compound and single crystal culture method:

[0047] Choose cyclic adenosine monophosphate (molecular formula: C 10 H 12 Single crystal culture experiments were conducted using N5O6P.

[0048] Cyclic adenosine monophosphate (cAMP) was cultured in single crystals using a Mosquito instrument via a volatilization method.

[0049] Compound selection and solubility determination:

[0050] Single-crystal culture of cyclic adenosine monophosphate (cAMP) was performed using an oil droplet evaporation method. First, the solubility of cAMP samples in different solvents was roughly determined using 11 solvents. Based on the solubility data, solvents were selected for experimental design. The solubility data of cAMP samples are shown in Table 1.

[0051] Table 1. Results of rough determination of cyclic adenosine monophosphate solubility

[0052]

[0053]

[0054] Single crystal cultivation:

[0055] Based on the preliminary solubility results in Table 1, water was selected as the best solvent. Aqueous solutions of cyclic adenosine monophosphate (cAMP) at different concentrations were prepared on the sample plate. The concentrations of the solutions corresponding to the eight grooves on the sample plate were 10 mg / mL, 10 mg / mL, 9 mg / mL, 9 mg / mL, 8 mg / mL, 8 mg / mL, 7 mg / mL, and 7 mg / mL, respectively. An automated dispensing robot sequentially dispensed the sample solutions into the wells on the crystal plate. The solution volumes corresponding to the three locations in each well were 100 nL, 150 nL, and 200 nL, respectively. Subsequently, the automated dispensing robot also sequentially dispensed dimethyl silicone oil into the wells on the crystal plate. The oil volumes corresponding to each row of wells were 50 nL, 100 nL, 150 nL, 200 nL, and 300 nL, respectively. The crystal plate was placed at a constant temperature of 18℃ for 24 hours for evaporation, and the crystals were observed using a stereomicroscope.

[0056] Cyclic adenosine monophosphate (cAMP) single crystals were cultured on 96-well crystal plates using a Mosquito instrument (model: Mosquito LCP). A total of 120 experimental groups were conducted at once, divided into 60 different crystallization conditions. Crystal growth was observed using a stereomicroscope, and the results are shown in Table 2. Under different conditions, single crystals of sufficient size were not obtained. Some experimental phenomena are recorded and labeled as follows: A2-2, A3-4, B2-4, C2-6, D1-3, E4-2, F2-3, and G4-4.

[0057] The results from the examples show that the desired single crystals could not be successfully grown using the Mosquito instrument for evaporation crystallization.

[0058] Table 2 Results of Cyclic Adenosine Volatilization Experiment

[0059]

[0060]

[0061] Example 2

[0062] Selection of compound and single crystal culture method:

[0063] Choose cyclic adenosine monophosphate (molecular formula: C 10 H 12 Single crystal culture experiments were conducted using N5O6P.

[0064] The method based on gas-phase diffusion was used to culture cyclic adenosine monophosphate (cAMP) in single crystals using a Mosquito instrument.

[0065] Single crystal cultivation:

[0066] Based on the preliminary solubility results in Example 1, water was selected as the good solvent. Different concentrations of cyclic adenosine monophosphate (cAMP) aqueous solutions were prepared on the sample plate. The concentrations of the aqueous solutions corresponding to the eight wells on the sample plate were 10 mg / mL, 10 mg / mL, 9 mg / mL, 9 mg / mL, 8 mg / mL, 8 mg / mL, 7 mg / mL, and 7 mg / mL, respectively. An automated dispensing robot was used to sequentially dispense the sample solutions into the wells on the crystal plate. The solution volumes corresponding to the three locations in the wells were 100 nL, 150 nL, and 200 nL, respectively. Simultaneously, a multi-channel pipette was used to add unsuitable solvents to the wells of the crystal plate. Five solvents were selected as unsuitable solvents: methanol, ethanol, tetrahydrofuran, acetone, and isopropanol. After sealing the crystal plate, it was incubated at 18°C ​​for 24 hours, and the crystals were observed using a stereomicroscope.

[0067] Cyclic adenosine monophosphate (cAMP) single crystals were cultured on 96-well crystal plates using a Mosquito instrument (model: Mosquito LCP). A total of 120 experimental groups were conducted at once, divided into 60 different crystallization conditions. Crystal growth was observed using a stereomicroscope. The results are shown in Table 3. When the concentration of cAMP aqueous solution was 10 mg / mL, good crystals were obtained in methanol, ethanol, tetrahydrofuran, and isopropanol solvent systems, respectively labeled as A2-3, A5-3, B1-4, and B3-2. Under other crystallization conditions, no solid was produced or only powder was obtained.

[0068] Table 3 Results of the gas-phase diffusion experiment of cyclic adenosine monophosphate.

[0069]

[0070] Characterization of single crystals:

[0071] The single crystal was characterized using an OLYMPUS SZX16 stereo microscope, and the crystal morphology is as follows: Figure 3 As shown.

[0072] The crystal structure of the cyclic adenosine monophosphate single crystals obtained in the examples was determined:

[0073] The structural data of the single crystals prepared in this example were obtained using a Bruker D8 VENTURE dual-microspot single-crystal X-ray diffractometer with an enhanced Cu light source. The test temperature was 193K.

[0074] Single-crystal diffraction data of cyclic adenosine monophosphate indicate that the crystal structure is monoclinic, space group P21, and the cell parameters are: α=90°, β=98.7250(10)°, γ=90°, Crystal asymmetric unit cell diagram as follows Figure 4 As shown, the crystal data is shown in Table 4.

[0075] Table 4. Data on Cyclic Adenosine Crystals

[0076]

[0077] Example 3

[0078] Compound selection and solubility determination:

[0079] Select listatin (molecular formula: C) 29 H 24 Single crystal culture experiments were conducted using Cl2N2O7S.

[0080] This invention uses a gas-phase diffusion method to cultivate ristatin into single crystals.

[0081] First, the solubility of listatin samples in different solvents was roughly determined using 11 solvents. Based on the solubility data, solvents were selected for experimental design. The solubility data of listatin samples are shown in Table 5.

[0082] Table 5. Results of rough determination of ristatin solubility

[0083] 1 methanol 8.0～10.0 2 ethanol 3.0～5.0 3 acetone 2.0～5.0 4 Ethyl acetate ～1.0 5 Tetrahydrofuran 50.0～100.0 6 Acetonitrile 1.0～2.0 7 Methyl tert-butyl ether ＜1.0 8 water ＜1.0 9 n-Heptane ＜1.0

[0084] Selection of compound and single crystal culture methods:

[0085] Select listatin (molecular formula: C) 29 H 24 Single crystal culture experiments were conducted using Cl2N2O78.

[0086] This invention utilizes a gas-phase diffusion method to cultivate ristatin single crystals using a Mosquito instrument.

[0087] Single crystal cultivation:

[0088] Based on the preliminary solubility results, acetone was selected as the good solvent. Different concentrations of ristatin solutions were prepared on the sample plate. The aqueous solution concentrations corresponding to the eight wells on the sample plate were 5 mg / mL, 5 mg / mL, 4 mg / mL, 4 mg / mL, 3 mg / mL, 3 mg / mL, 2 mg / mL, and 2 mg / mL, respectively. An automated dispensing robot sequentially dispensed the sample solutions into the wells on the crystal plate. The solution volumes corresponding to the three well positions were 100 nL, 150 nL, and 200 nL, respectively. Simultaneously, a multi-channel pipette was used to add unsuitable solvents to the wells of the crystal plate. Four solvents were selected as unsuitable solvents: ethyl acetate, acetonitrile, methyl tert-butyl ether, and n-heptane. After sealing the crystal plate, it was incubated at 18℃ for 24 hours, and the crystals were observed using a stereomicroscope.

[0089] Rittalact single crystals were cultured on 96-well crystal plates using a Mosquito instrument (model: Mosquito LCP). 96 experimental groups were conducted simultaneously, divided into 48 different crystallization conditions. Crystal growth was observed using a stereomicroscope. The results are shown in Table 6. When the rittalact aqueous solution concentration was 3 mg / mL, better crystals were obtained in the n-heptane solvent system, labeled as E4-4 and F4-3, respectively. Under other crystallization conditions, no solid was produced or only powder was obtained.

[0090] Table 6 Results of the gas phase diffusion experiment of rituximab

[0091]

[0092] Characterization of single crystals:

[0093] The single crystal was characterized using an OLYMPUS SZX16 stereo microscope, and the crystal morphology is as follows: Figure 5 As shown.

[0094] The crystal structure of the prepared ristatin single crystal was determined:

[0095] The structural data of the single crystals prepared in this example were obtained using a Bruker D8 VENTURE dual-microspot single-crystal X-ray diffractometer with an enhanced Cu light source. The test temperature was 193K.

[0096] The single-crystal diffraction data from Rittalt indicate that the crystal structure is monoclinic, the space group is P21, and the cell parameters are: α=90°, β=104.6520(10)°, γ=90°, Crystal asymmetric unit cell diagram as follows Figure 6 As shown, the crystal data is shown in Table 7.

[0097] Table 7. Rittal crystal data

[0098]

[0099]

[0100] This invention's method is primarily used for small molecule single crystal cultivation, enabling it to accomplish single crystal cultivation tasks that are impossible with the oil droplet evaporation method. Furthermore, the method used in this invention is based on the principle of gas-phase diffusion. Therefore, this invention is compared with traditional gas-phase diffusion methods, and the results are shown in Table 8:

[0101] Table 8 Comparison between traditional gas phase diffusion methods and the method used in this invention

[0102]

[0103] As can be seen from the table above, compared with the traditional vapor-phase diffusion method for single crystal cultivation, this invention requires less sample and is largely automated. This invention can replace traditional single crystal cultivation methods, requiring only milligrams of rare samples to design large-scale crystallization conditions for experiments in a short time, achieving high-throughput screening and preparation of small molecule crystals.

Claims

1. A method for cultivating single crystals of small organic molecules, characterized in that: Includes the following steps: 1) Dissolve the small organic molecule compound in a good solvent to obtain a solution of the small organic molecule compound; 2) Use an automated spotting robot to sequentially dispense the organic small molecule compound solution into the spotting wells on the crystal plate, while adding unsuitable solvents into the pooling wells of the crystal plate at the same time; 3) Seal the crystal plate to form multiple closed crystallization chambers for crystallization until organic small molecule single crystals of sufficient size are obtained; Single crystal culture was carried out on a 96-well crystal plate, which has 8 rows and 12 columns. Each well contains 1 pooling liquid well and 3 spot wells. The organic small molecule compound is cyclic adenosine monophosphate (cAMP) or ristatin, and the molecular formula of cAMP is C10. 10 H 12 N5O6P, the molecular formula of ristat is C 29 H 24 Cl2N2O7S.

2. The method for cultivating single crystals of small organic molecules according to claim 1, characterized in that: The upper sample plate, which matches the 96-well crystal plate, has 8 grooves, which can hold up to 8 different concentrations or different solvents of organic small molecule compound solutions; undesirable solvents are added to the pool liquid wells, and the 96-well crystal plate has 12 columns, which can hold up to 12 undesirable solvents; the good solvent is a non-volatile solvent; the undesirable solvent is a volatile solvent and is miscible with the good solvent.

3. The method for cultivating single crystals of small organic molecules according to claim 1, characterized in that: When the organic small molecule compound is cyclic adenosine monophosphate, the organic small molecule single crystal is obtained by the following steps: 1) Water was chosen as a good solvent, and aqueous solutions of cyclic adenosine monophosphate (cAMP) at different concentrations were prepared on the sample plate. The concentrations of the aqueous solutions corresponding to the eight grooves on the sample plate were 10 mg / mL, 10 mg / mL, 9 mg / mL, 9 mg / mL, 8 mg / mL, 8 mg / mL, 7 mg / mL, and 7 mg / mL, respectively. 2) Using an automated spotting robot, the cyclic adenosine monophosphate aqueous solution was sequentially dispensed into the spotting wells on the crystal plate. The solution volumes corresponding to the three spots were 100 nL, 150 nL, and 200 nL, respectively. 3) Use a multichannel pipette to add unsuitable solvents into the wells of the crystal plate. Five solvents were selected as unsuitable solvents: methanol, ethanol, tetrahydrofuran, acetone, and isopropanol. 4) After sealing the crystallization plate, incubate at a constant temperature and observe the crystal condition using a stereomicroscope.

4. The method for cultivating single crystals of small organic molecules according to claim 3, characterized in that: The crystal structure of the cyclic adenosine monophosphate single crystal is monoclinic, space group P21, and the cell parameters are: a=7.7287(2) Å, b=10.5825(2) Å, c=17.6266(4) Å, α=90°, β=98.7250(10)°, γ=90°, V=1424.98(6) Å. 3 .

5. The method for cultivating single crystals of small organic molecules according to claim 1, characterized in that: When the organic small molecule compound is ristat, the organic small molecule single crystal is obtained by the following steps: 1) Using acetone as a good solvent, different concentrations of listatin solutions were prepared on the sample plate. The solution concentrations corresponding to the 8 grooves on the sample plate were 5 mg / mL, 5 mg / mL, 4 mg / mL, 4 mg / mL, 3 mg / mL, 3 mg / mL, 2 mg / mL, and 2 mg / mL, respectively. 2) The sample solution is sequentially dispensed into the spotting wells on the crystal plate using an automated spotting robot. The solution volumes corresponding to the three positions of the spotting wells are 100 nL, 150 nL, and 200 nL, respectively. 3) Use a multichannel pipette to add unsuitable solvents into the wells of the crystal plate. Four solvents were selected as unsuitable solvents: ethyl acetate, acetonitrile, methyl tert-butyl ether, and n-heptane. 4) After sealing the crystal plate, incubate at a constant temperature and observe the crystal condition using a stereomicroscope.

6. The method for cultivating single crystals of organic small molecules according to claim 5, characterized in that: The rituxima single crystal has a monoclinic crystal system with space group P21 and cell parameters of: a = 15.4997(5) Å, b = 5.2271(2) Å, c = 19.2919(6) Å, α = 90°, β = 104.6520(10)°, γ = 90°, V = 1512.17(9) Å. 3 .

Images