Nucleoside phosphoramidite crystal and crystallization method thereof

Abstract

The invention relates to the technical field of nucleic acid raw materials, in particular to a nucleoside phosphoramidite crystal and a crystallization method thereof, and the nucleoside phosphoramidite crystal is high in purity, rod-like in crystal habit and good in stability; the nucleoside phosphoramidite crystal is prepared by adopting a solvent-cooling crystallization method, and the crystallization process is simple, mild in condition and easy to operate; the method has the advantages of low energy consumption, short time consumption, many solvent combinations and high selectivity, and is beneficial to large-scale production.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid raw materials, in particular to nucleoside phosphoramidite crystals and a crystallization method thereof. Background Art

[0002] Small nucleic acid drugs, such as oligonucleotide drugs, are usually composed of a dozen to dozens of nucleotides in series. Among the many methods for synthesizing oligonucleotides, the most mainstream method is to synthesize them through solid-phase phosphoramidite chemistry. Phosphoramidite monomers provide the necessary chemical activity in the synthesis process and are the basic raw materials for synthesizing small nucleic acid drugs.

[0003] The synthesis process of phosphoramidite monomers is complex and involves multiple chemical processes, and its purity and quality directly affect the quality and efficacy of small nucleic acid drugs. Therefore, it is of great significance to prepare high-purity phosphoramidite monomers. Among the many purification and separation methods, the crystallization method has the advantages of high product purity, low purification cost, simple operation and easier scale-up, making it one of the preferred methods for purifying phosphoramidite monomers.

[0004] As one of the monomers for synthesizing small nucleic acid drugs by the phosphoramidite method, nucleoside phosphoramidite monomers are usually amorphous and easily contain specific impurities, which affects their synthesis efficiency. In addition, the amorphous state is highly hygroscopic and unstable. Patent WO2010021897A1 reports a method for preparing uracil nucleoside phosphoramidite crystals by crystallizing at -42 °C for 18 h. This is the first attempt to obtain crystals from powders, but the experimental process has a low temperature, a long time, high energy consumption, and a single type of solvent.

[0005] Therefore, it is very necessary to provide a nucleoside phosphoramidite crystal with stable crystal form, simple preparation process and low energy consumption. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the related art. To this end, the first object of the present invention is to provide a nucleoside phosphoramidite crystal; the second object of the present invention is to provide a crystallization method.

[0007] In order to achieve the first purpose, the technical solution adopted by the present invention is: Nucleoside phosphoramidite crystal, the structural formula is as follows: ; Wherein, X is any group selected from CH3O-, -F and OTBS, and R is any base selected from A, G, C and U; Among them, OTBS is dimethyl tert-butylsilyl ether, A is adenine, G is guanine, C is cytosine, and U is uracil.

[0008] Further, its structural formula is shown below: ; The X-ray diffraction pattern includes the following multiple peaks of diffraction peaks at 2θ diffraction angles: Preferably, the X-ray diffraction pattern has the following diffraction peaks at 2θ diffraction angles: Preferably, the X-ray diffraction pattern has the following diffraction peaks at 2θ diffraction angles: Preferably, the X-ray diffraction pattern has the following diffraction peaks at 2θ diffraction angles: In order to achieve the second purpose, the technical solution adopted by the present invention is: A crystallization method for preparing the nucleoside phosphoramidite crystals described in any one of the above, comprising the following steps: S100, dissolving nucleoside phosphoramidite powder in a good solvent to prepare a nucleoside phosphoramidite solution with a concentration of 0.05 to 1.0 g / mL; the concentration of the crystalline substance directly affects the saturation state of the solution. When the concentration of the nucleoside phosphoramidite solution is less than 0.05 g / mL, it is difficult to form a supersaturated solution due to the low concentration of the solution, which also reduces the chance of solute molecules diffusing to the crystal surface, thereby slowing down the crystal growth rate and increasing the time cost and technical difficulty of subsequent crystallization operations; when the concentration of the nucleoside phosphoramidite solution is greater than 1.0 g / mL, the high concentration of the solution will accelerate the molecular motion, resulting in an increase in defects in the crystal growth process, affecting the crystal quality. In addition, high concentrations are prone to explosive nucleation, affecting the product particle size; Wherein, the good solvent is selected from at least one of 1,2-dichloroethane, 1,4-dioxane, ethyl acetate, tetrahydrofuran, dichloromethane, acetonitrile and methyl tert-butyl ether, preferably dichloromethane or 1,2-dichloroethane; S200, rotation speed 100-300 rpm, stirring, adding a poor solvent to the nucleoside phosphoramidite solution for dissolution and crystallization, cooling to 0-5°C at a rate of 5-10°C / h while adding the poor solvent, and continuing to stir until the nucleoside phosphoramidite solution forms a uniform nucleoside phosphoramidite suspension; when the stirring rate is less than 100 rpm, the mass transfer rate of the solute molecules is reduced due to the low rate, affecting the crystallization rate and causing irregular crystal growth and serious agglomeration; when the stirring rate is greater than 300 rpm, the stirring rate is too high and does not increase the crystallization rate, but instead causes crystal breakage, affecting the size and distribution of the crystals; The poor solvent is at least one selected from n-heptane, n-hexane, cyclohexane and n-octane, preferably n-heptane or n-hexane.

[0009] S300, filtering and drying the nucleoside phosphoramidite suspension to obtain nucleoside phosphoramidite crystals.

[0010] Furthermore, in step S100, the temperature during the dissolution process is not higher than 50° C. If the dissolution temperature is higher than 50° C., the raw materials are prone to deterioration and decomposition, resulting in failure to obtain the desired crystal product.

[0011] Furthermore, in step S200, the dropping rate of the poor solvent is 0.01 to 2.0 ml / min; when the dropping rate is less than 0.01 ml / min, the rate is too slow and the poor solvent will evaporate during the dropping process, thereby reducing the yield of the crystal product or even failing to precipitate the crystal product; when the dropping rate is greater than 2.0 ml / min, the rate is too fast and the concentration fluctuation is too large, resulting in crystal nucleation explosion and large-scale agglomeration, which affects the dispersion and crystal morphology of the product.

[0012] Furthermore, the amount of poor solvent added is 2 to 6 times the amount of good solvent added; outside this limited range, too little poor solvent will make the solubility of the raw material too high, making it difficult to form supersaturation and easy to crystallize, while too much poor solvent will make the supersaturation of the system too high, resulting in nucleation being inhibited, and the solid cannot precipitate, thus forming oil or gel, and the excessive use of solvent not only does not significantly improve the product quality, but also greatly increases the production cost.

[0013] Further, in step 300, the drying temperature is not higher than 50°C, and the drying time is 5 to 12 hours; Outside this limited range, too high a temperature will cause the product to deteriorate and decompose, while too low a temperature will increase the drying time and increase production energy consumption.

[0014] The above one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects: The nucleoside phosphoramidite crystals provided by the present invention have high purity, rod-shaped crystal habit and good stability. The nucleoside phosphoramidite crystals are prepared by a solvent-cooling crystallization method. The crystallization process is simple, the conditions are mild and the operation is easy. The crystallization process has low energy consumption, short time consumption, multiple solvent combinations and high selectivity, which is conducive to large-scale production.

[0015] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the powder X-ray diffraction spectrum of the uridine-type nucleoside phosphoramidite single crystal provided in Example 1 of the present invention.

[0017] Figure 2 A structural perspective view of a uracil-type nucleoside phosphoramidite single crystal provided in Example 1 of the present invention.

[0018] Figure 3 The morphology of the uridine nucleoside phosphoramidite single crystal provided in Example 2 of the present invention.

[0019] Figure 4 Thermogravimetric analysis diagram of the uridine nucleoside phosphoramidite single crystal provided in Example 2 of the present invention.

[0020] Figure 5 The differential scanning calorimetry analysis diagram of the uridine-type nucleoside phosphoramidite single crystal provided in Example 2 of the present invention. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The following embodiments are used to illustrate the present invention, but cannot be used to limit the scope of the present invention.

[0022] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0023] In the following examples, powder X-ray diffraction was performed under the following conditions: Instrument used: X-ray powder diffractometer (MiniFlex600, Rigaku, Japan); Target: Cu; X-ray tube current: 15 mA; X-ray tube voltage: 40 kV; Scanning range: 2θ = 2~35º.

[0024] Example 1 A uracil-type nucleoside phosphoramidite crystal, the structural formula of which is shown below: ; Its X-ray diffraction pattern is as follows Figure 1 As shown, the diffraction peaks at the following 2θ diffraction angles are included: Generally speaking, the diffraction angle (2θ) in powder X-ray diffraction sometimes includes an error range of less than 5%. Therefore, in addition to crystals with completely consistent peak diffraction angles in powder X-ray diffraction, crystals with consistent peak diffraction angles with an error of less than 5% are also included in uracil-type nucleoside phosphoramidite crystals, that is, diffraction peaks with 2θ diffraction angles in the following range in the X-ray diffraction spectrum all belong to the uracil-type nucleoside phosphoramidite crystals provided by the present invention: The crystal parameters of the uracil nucleoside phosphoramidite crystal are shown in Table 1: Among them, a, b, and c represent the unit cell lengths of the crystal in three mutually perpendicular directions, α, β, and γ refer to the angles between the sides of the unit cell, V refers to the unit cell volume, and Z refers to the number of molecules in the unit cell.

[0025] The atomic parameters of the uridine nucleoside phosphoramidite crystal are shown in Table 2: The fractional atomic coordinates are ×10 4 , the equivalent isotropic displacement parameter is Å 2 ×10 3 .

[0026] The interatomic bond lengths of the uracil nucleoside phosphoramidite crystals are shown in Table 3: The interatomic bond angles of the uracil nucleoside phosphoramidite crystal are shown in Table 4: The structural perspective diagram of the uracil nucleoside phosphoramidite crystal calculated based on the crystal coordinates, such as Figure 2 shown.

[0027] Example 2 The uracil nucleoside phosphoramidite monomer powder (250 mg) was added to dichloromethane (5 mL), and the mixture was stirred at 45°C and 200 rpm until the solid raw material was completely dissolved. Then, n-heptane (10 mL) was added at a drop rate of 0.33 mL / min, and the temperature was lowered at a rate of 5°C / h. After the temperature was lowered to 5°C, the mixture was stirred at a constant temperature for 5 h to obtain a suspension, which was filtered, and the filter cake was washed with n-heptane and dried at 45°C for 5 h to obtain uracil nucleoside phosphoramidite crystals. The X-ray powder diffraction pattern of the crystal is consistent with the diffraction pattern of the uridine nucleoside phosphoramidite crystal provided in Example 1, including the following diffraction peaks at 2θ diffraction angles: The morphology of the crystal is Figure 3 As shown in the thermogravimetric analysis Figure 4 The calorimetric analysis results are shown in Figure 5 shown.

[0028] Example 3 The uracil nucleoside phosphoramidite monomer powder (500 mg) was added to dichloromethane (10 mL), and the mixture was stirred at 30 °C and 300 rpm until the solid raw material was completely dissolved. Then, n-heptane (30 mL) was added at a drop rate of 0.17 mL / min, and the temperature was lowered at a rate of 5 °C / h. After the temperature was lowered to 5 °C, the mixture was stirred at a constant temperature for 5 h to obtain a suspension, which was filtered, and the filter cake was washed with n-heptane and dried at 40 °C for 8 h to obtain uracil nucleoside phosphoramidite crystals. The X-ray powder diffraction pattern of the crystal is consistent with the diffraction pattern of the uridine nucleoside phosphoramidite crystal provided in Example 1, including the following diffraction peaks at 2θ diffraction angles: Example 4 The uracil nucleoside phosphoramidite monomer powder (250 mg) was added to 1,2-dichloroethane (5 mL), and the mixture was stirred at 30 °C and 200 rpm until the solid raw material was completely dissolved. Then, n-heptane (15 mL) was added at a drop rate of 0.33 mL / min, and the temperature was lowered at a rate of 5 °C / h. After the temperature was lowered to 5 °C, the mixture was stirred at a constant temperature for 5 h to obtain a suspension, which was filtered, and the filter cake was washed with n-heptane and dried at 40 °C for 8 h to obtain uracil nucleoside phosphoramidite crystals. The X-ray powder diffraction pattern of the crystal is consistent with the diffraction pattern of the uridine nucleoside phosphoramidite crystal provided in Example 1, including the following diffraction peaks at 2θ diffraction angles: Example 5 The uracil nucleoside phosphoramidite monomer powder (250 mg) was added to 1,2-dichloroethane (5 mL), and the mixture was stirred at 30°C and 200 rpm until the solid raw material was completely dissolved. Then, n-hexane (15 mL) was added at a drop rate of 0.33 mL / min, and the temperature was lowered at a rate of 5°C / h. After the temperature was lowered to 5°C, the mixture was stirred at a constant temperature for 5 h to obtain a suspension, which was filtered, and the filter cake was washed with n-heptane and dried at 35°C for 12 h to obtain uracil nucleoside phosphoramidite crystals. The X-ray powder diffraction pattern of the crystal is consistent with the diffraction pattern of the uridine nucleoside phosphoramidite crystal provided in Example 1, including the following diffraction peaks at 2θ diffraction angles: Example 6 The uracil nucleoside phosphoramidite monomer powder (50 mg) was added to ethyl acetate (1 mL), and the mixture was stirred at 35°C and 200 rpm until the solid raw material was completely dissolved. Then, n-hexane (6 mL) was added at a drop rate of 2.0 mL / min, and the temperature was lowered at a rate of 5°C / h. After the temperature was lowered to 5°C, the mixture was stirred at a constant temperature for 5 h to obtain a suspension, which was filtered, and the filter cake was washed with n-hexane and dried at 45°C for 5 h to obtain uracil nucleoside phosphoramidite crystals. The X-ray powder diffraction pattern of the crystal is consistent with the diffraction pattern of the uridine nucleoside phosphoramidite crystal provided in Example 1, including the following diffraction peaks at 2θ diffraction angles: Example 7 The uracil nucleoside phosphoramidite monomer powder (250 mg) was added to dichloromethane (5 ml), and the mixture was stirred at 10 °C and 100 rpm until the solid raw material was completely dissolved. Then, n-heptane (10 mL) was added at a drop rate of 0.01 mL / min, and the temperature was lowered at a rate of 5 °C / h. After the temperature was lowered to 5 °C, the mixture was stirred at a constant temperature for 5 h to obtain a suspension, which was filtered, and the filter cake was washed with n-hexane and dried at 35 °C for 12 h to obtain uracil nucleoside phosphoramidite crystals. The X-ray powder diffraction pattern of the crystal is consistent with the diffraction pattern of the uridine nucleoside phosphoramidite crystal provided in Example 1, including the following diffraction peaks at 2θ diffraction angles: Example 8 The uracil nucleoside phosphoramidite monomer powder (1 g) was added to dichloromethane (1 ml), and the mixture was stirred at 30°C and 300 rpm until the solid raw material was completely dissolved. Then, n-heptane (3 mL) was added at a drop rate of 1.0 mL / min, and the temperature was lowered at a rate of 5°C / h. After the temperature was lowered to 5°C, the mixture was stirred at a constant temperature for 5 h to obtain a suspension, which was filtered, and the filter cake was washed with n-hexane and dried at 40°C for 8 h to obtain uracil nucleoside phosphoramidite crystals. The X-ray powder diffraction pattern of the crystal is consistent with the diffraction pattern of the uridine nucleoside phosphoramidite crystal provided in Example 1, including the following diffraction peaks at 2θ diffraction angles: Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Nucleoside phosphoramidite crystal, characterized in that: The structural formula is as follows: ； Wherein, X is any group selected from CH3O-, -F and OTBS, and R is any base selected from A, G, C and U; Among them, OTBS is dimethyl tert-butylsilyl ether, A is adenine, G is guanine, C is cytosine, and U is uracil.

2. The nucleoside phosphoramidite crystal according to claim 1, characterized in that: Its structural formula is as follows: ； The X-ray diffraction pattern includes the following multiple peaks of diffraction peaks at 2θ diffraction angles:

3. The nucleoside phosphoramidite crystal according to claim 2, characterized in that: The X-ray diffraction pattern has the following diffraction peaks at 2θ diffraction angles:

4. The nucleoside phosphoramidite crystal according to claim 2, characterized in that: The X-ray diffraction pattern has the following diffraction peaks at 2θ diffraction angles:

5. The nucleoside phosphoramidite crystal according to claim 2, characterized in that: The X-ray diffraction pattern has the following diffraction peaks at 2θ diffraction angles:

6. A crystallization method, characterized in that: The method for preparing the nucleoside phosphoramidite crystal according to any one of claims 1 to 5 comprises the following steps: S100, dissolving nucleoside phosphoramidite powder in a good solvent to prepare a nucleoside phosphoramidite solution with a concentration of 0.05 to 1.0 g / mL; Wherein, the good solvent is selected from at least one of 1,2-dichloroethane, 1,4-dioxane, ethyl acetate, tetrahydrofuran, dichloromethane, acetonitrile and methyl tert-butyl ether; S200, rotating speed 100-300 rpm, stirring, adding a poor solvent to the nucleoside phosphoramidite solution to dissolve and crystallize, while adding the poor solvent, cooling to 0-5°C at a rate of 5-10°C / h, and continuing to stir until the nucleoside phosphoramidite solution forms a uniform nucleoside phosphoramidite suspension; Wherein, the poor solvent is selected from at least one of n-heptane, n-hexane, cyclohexane and n-octane; S300, filtering and drying the nucleoside phosphoramidite suspension to obtain nucleoside phosphoramidite crystals.

7. The crystallization method according to claim 6, characterized in that In step S100, the temperature during the dissolution process is not higher than 50°C.

8. The crystallization method according to claim 6, characterized in that In step S200, the dropping rate of the poor solvent is 0.01 to 2.0 ml / min.

9. The crystallization method according to claim 6, characterized in that The amount of poor solvent added is 2 to 6 times the amount of good solvent added.

10. The crystallization method according to claim 6, characterized in that In step 300, the drying temperature is not higher than 50°C, and the drying time is 5 to 12 hours.

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