CN114751949A

CN114751949A - 5' -cytidine monophosphate monohydrate crystal and preparation method thereof

Info

Publication number: CN114751949A
Application number: CN202210586687.6A
Authority: CN
Inventors: 杨朋朋; 应汉杰; 徐梦洁; 李姝姝; 张磊; 李子涵; 赵谷林; 温庆仕; 吴菁岚; 庄伟�
Original assignee: Nanjing Tech University
Current assignee: Nanjing Tech University
Priority date: 2022-05-26
Filing date: 2022-05-26
Publication date: 2022-07-15

Abstract

The invention discloses a 5' -cytidine monophosphate monohydrate crystal with a molecular formula of C₉H₁₆N₃O₉P belongs to monoclinic system and has a space group of P₂₁The unit cell parameters a (a) =4.858, b (a) =8.3267, c (a) =17.0704, β =90.126 °, unit cell volume V =690.515 a³The minimum number of asymmetric units Z =2 in the unit cell. The invention also provides a preparation method of the crystal. The crystallization method is simple and easy to control, the obtained 5' -cytidylic acid monohydrate crystal has good fluidity, good stability, uniform particle size distribution and high yield, and the product can be stably stored for a long time.

Description

5' -cytidine monophosphate monohydrate crystal and preparation method thereof

Technical Field

The invention belongs to the field of refining of food additives, and particularly relates to a crystal structure of 5' -cytidine monophosphate monohydrate and a crystallization method thereof.

Background

5' -cytidine monophosphate, alternative names: cytidine 5 ' -monophosphate, cytosine (ribonucleotide) (5 ' -cytidylic acid, hereinafter referred to as 5 ' -CMP), a polar compound, and an ampholyte; the N-glycosidic bond of 5' -CMP is relatively stable and only the phosphate group is cleaved under severe hydrolysis conditions. The CDP and CTP can be generated through phosphorylation, and the main function is as a raw material for nucleic acid biosynthesis and is involved in activation of metabolites (such as choline, nuclear alcohol and the like). The molecular structure of 5' -CMP is as follows:

5' -CMP is insoluble in organic solvents such as benzene and ethanol, and the ratio of 98% ultraviolet absorbance: a250 nm/A260 nm is 0.41-0.49, A280 nm/A260 nm is 2.03-2.17, and the water content is 5.28%.

5' -CMP is an important biochemical substance in biological cells and participates in various physiological and biochemical reactions. In industry, 5' -cytidine is mainly used for manufacturing drugs such as citicoline, cytidine triphosphate, cytarabine, polyinosinic. In recent years, the composition can be added into milk powder together with other nucleotide to enhance the immunity of infants. In 1965, nucleotides were added to infant milk in Japan, and in Spain and the United states, the use of nucleotides was recommended in 1983 and 1989, respectively. The european union started specifying the level of nucleotide addition in infant food in 1991. In the early 90 s, the nucleotide-containing imported branded infant milk powder began to appear in the domestic market. Related patents in China also introduce the effects of adding nucleic acid or nucleotide into high-energy milk, being easily absorbed by human bodies, promoting blood circulation, improving brain functions, promoting metabolism, resisting fatigue, strengthening physique, improving immunity and the like. The 5' -CMP can be used for infant formula milk powder, medical intermediates, new-generation health feed additives and the like. The main production modes of 5' -CMP are: RNA (ribonucleic acid) degradation, chemical synthesis, enzymatic synthesis and biological fermentation. At present, the chemical method is mainly used for industrially synthesizing the 5' -CMP, and the method has the main defects of high raw material cost, more three wastes, equipment corrosion and no harm to the health of personnel and industrial production. At present, there are reports about enzyme catalysis and biological fermentation methods, for example, in patent CN 202111282072.1, 5' -CMP is extracted after microbial fermentation broth is subjected to high-temperature inactivation, acid adjustment, membrane filtration, concentration, decolorization and elution crystallization, but there are problems of low yield, complex operation and the like, the traditional crystallization method is freezing below 10 ℃ until crystallization is precipitated, although the method has low requirements on equipment, the phenomena of particle aggregation and impurity wrapping are serious due to easy outbreak nucleation, and finally the crystal has uneven particle size, small bulk density, low crystallinity and poor stability; in industrial production, a plurality of times of low-temperature alcohol precipitation crystallization is adopted, but the process flow is long, the operation is complicated, the solvent consumption is high, and the purity and the yield of the obtained product are low; in addition, 5' -cytidine monophosphate trihydrate (Acta Crystal. (1979). B35,2141-2144), which is an orthorhombic system and is obtained by evaporative crystallization in a water-rich isopropanol solution, has been reported, but has problems such as instability under storage conditions; at present, few reports on 5 '-CMP crystallization documents are reported in China, and the process optimization progresses slowly, so that how to develop a high-quality 5' -CMP crystal product suitable for industrial production and a preparation method thereof is urgent.

Disclosure of Invention

The purpose of the invention is as follows: the technical problem to be solved by the invention is to provide a high-quality 5 '-cytidylic acid monohydrate crystal and a preparation method thereof aiming at the defects of the prior art, wherein the prepared 5' -cytidylic acid monohydrate crystal has good fluidity, good stability, uniform particle size distribution and high yield, and the product can be stably stored for a long time.

To realize the above technologyThe invention discloses a 5' -cytidine monophosphate monohydrate crystal with good stability and high bulk density, and the molecular formula of the crystal is C₉H₁₄N₃O₈P·H₂O, belonging to monoclinic system, space group is P₂₁Cell parameter

90.126 DEG of beta, unit cell volume

The minimum number of asymmetric units Z in the unit cell is 2, and in one minimum asymmetric unit, one 5' -cytidine monophosphate and one crystal water are contained. The minimum asymmetric unit diagram and the structure diagram of the unit cell are basically shown in fig. 1 and fig. 2, the stacking diagram of the unit cell is basically shown in fig. 3, and the detailed data of the crystal structure, namely the bond angle information, is shown in fig. 8. During the determination of the crystal structure, diffraction data are collected at 298K.

The monohydrate crystal has a diffraction pattern obtained by performing diffraction analysis by using CuKa rays as characteristic X rays as shown in figure 4, and has characteristic absorption peaks at diffraction angles 2 theta +/-0.1, wherein the diffraction angles are 5.21 degrees, 10.36 degrees, 11.83 degrees, 15.55 degrees, 18.28 degrees, 18.98 degrees, 21.78 degrees, 23.62 degrees, 30.13 degrees and the like.

The crystalline powder of 5' -CMP described above is white or off-white in color.

The crystals form a crystalline powder having a bulk density greater than 0.20g/mL, preferably greater than 0.30g/mL, more preferably greater than 0.40 g/mL.

The crystals form a crystalline powder having a tap density greater than 0.23g/mL, preferably greater than 0.33g/mL, more preferably greater than 0.44 g/mL.

The crystals form a crystalline powder having a d50 of greater than 10 μm, preferably greater than 30 μm, more preferably greater than 50 μm, most preferably greater than 60 μm d 50.

The crystals form a crystalline powder having a d10 of greater than 3 μm, preferably greater than 6 μm, more preferably greater than 10 μm, most preferably greater than 15 μm d 10.

D50 and d10 are typical amounts used to indicate particle size distribution. D50 is stated to be a value for particle size such that 50 vol.% of the crystals have a size less than this. Said d10 is a value for the particle size, such that 10 vol.% of the crystals have a size below this value.

The bulk and tap densities are quantities related to the flow characteristics of the powder. In general, high bulk and tap density values are desired. In short, bulk density indicates the weight per volume unit of powder under predetermined conditions. In other words, how many grams of powder can be fed into a sample holder having a specific volume under predetermined conditions. For this reason, bulk density is expressed as weight per volume unit, usually in g/mL. Tap density also indicates the weight per volume unit of powder, in this case, in the case where the holder of the powder is subjected to beating or vibration under predetermined conditions. Tap density is expressed as weight per volume unit, usually in g/mL. More powder can be fed into the holder by beating or vibrating. Thus, for the same powder, the tap density is higher than the bulk density.

The powder with large bulk density and tap density has large specific gravity, can reflect that the crystal product is thick and solid and has texture, and has relatively good stability; in another aspect, a product with a high bulk density generally has better flowability of granules, is also convenient for storage and transportation, and is more convenient for efficient mixing with other components.

Specifically, the particle size distribution, bulk density, and tap density are measured as follows:

the particle size distribution of the samples from the mixer (including d10 and d50) was determined using a Malvern instruments particle size analyzer, dry method;

bulk density of the granules was determined according to USP method II (page 1914);

the tap density of the particles was determined by means of an economical tap density tester of FZS4-4 according to GB/T5162-2006. Specifically, the measurement conditions were: the vibration stroke of the compaction device is 3 +/-0.1 MM, and the vibration frequency is 250 +/-15 times per minute.

It is noted that the above crystalline powder may exhibit different apparent water contents (including free water and bound water) due to differences in preparation methods, or differences in drying methods, or differences in humidity of storage environments, and thus, the crystalline powder may have a water content of 4.5 to 6%.

Specifically, the method for measuring the water content of the crystal powder is a karl fischer method, and can be measured by an 870Titrino plus practical volumetric method karl fischer moisture meter available from wangtong, switzerland.

When the crystal is prepared into an aqueous solution with the mass fraction of 5%, the pH value of the system is 2-5, preferably 3-4.

The crystalline powder of the present invention means a powder having a certain degree of crystallinity, as opposed to being amorphous.

The crystal is a solid with definite diffraction pattern for X-ray, and its atoms or molecules are arranged repeatedly in space according to a certain regular period.

The invention further provides a preparation method of the 5' -cytidine monophosphate monohydrate crystal, which is characterized by comprising the following steps:

(1) placing the 5' -cytidine monophosphate aqueous solution in a crystallizer provided with a probe type ultrasonic device, adjusting the pH value to be 4.85-5.00, preferably adjusting the pH value by using sodium hydroxide, and stabilizing the pH value for 30-40 min at the temperature of 10-40 ℃;

(2) starting a probe type ultrasonic device, acting on the reaction system in the step (1) under the power of 20-50W, keeping for 20-40 min, adding a reactant until the pH value of the system is reduced to 3.9-4.0, continuing to perform ultrasonic treatment for 2-5 min if the system is turbid in the dripping process, closing an ultrasonic emitter, and growing crystals at the stirring speed of 180-200 rpm until more crystal particles appear in the system and do not increase;

(3) Continuously adding a reactant into the system obtained in the step (2), stopping dropwise adding the reactant when the pH value of the system is reduced to 0.5-2.0, simultaneously adding a elution agent with the volume 0.2 times that of the raw material liquid, and maintaining stirring in the dropwise adding process;

(4) stopping dripping the solventing-out agent, maintaining the temperature of the system between 25 and 40 ℃, and continuously stirring for 3 to 6 hours;

(5) and carrying out solid-liquid separation on the crystal mush, washing and drying by blowing to obtain the crystal mush.

In the step (1), the reaction concentration of the 5' -CMP is 25 g/L-200 g/L, preferably 100 g/L-150 g/L.

In the step (2), the nucleation is promoted by adopting an ultrasonic adding mode, and the ultrasonic device has probe type ultrasonic action and continuous ultrasonic (non-pulse type) action.

In the step (2), when the pH of the system is 3.9-4.0, the 5' -CMP mainly exists in a zwitterionic form.

In the step (2), the action of the ultrasonic intervention time needs to be stopped before a large amount of nucleation, wherein the cavitation effect of ultrasonic waves is utilized to enable solute molecules in the system in a supersaturated state to obtain extra energy, reduce the surface energy of aggregation among solute molecules, promote irregular solute molecule aggregates in an association state to overcome nucleation energy barriers, further perform regular adjustment, form crystal nuclei and grow to apparent crystal grains, but prevent the crystal from being broken due to overlong ultrasonic time, if the system is observed to be turbid in the process of dripping the reactant till the pH value is 3.9-4.0, the ultrasonic emitter is turned off after continuing ultrasonic treatment for 2-5 min.

In the step (2), the reactant needs to be slowly dripped or pumped, the high speed is not beneficial to the growth of crystals, and the low speed can reduce the efficiency of the crystallization process; preferably, the adding speed of the reactant is 0.2-0.4 mL/min, preferably 0.2 mL/min; it should be noted that if the system is scaled up or used in industrial production, the flow rate can be adjusted according to the actual situation with reference to the ratio.

In the step (3), the volume of the system needs to be considered for the dosage of the reactant, and the addition amount of the reactant is controlled, namely the pH end point of the system is controlled, so that the final pH value falls within the zwitterionic interval of the 5' -CMP ion distribution curve.

In the steps (3) and (4), the reactant is hydrochloric acid with the concentration of 1-3 mol/L, the induction period is prolonged when the hydrochloric acid concentration is too low, and the system is easy to generate explosive nucleation when the hydrochloric acid concentration is too high.

The solvent-out agent is absolute ethyl alcohol; the addition of the solventing-out agent is to further reduce the solubility of the solute in the system, separate out more solute and improve the crystallization yield; the added reactant has the function of gradually converting 5 '-CMP from an ionic state to a molecular state in a system to separate out 5' -CMP; there is no specific requirement for the addition rate of the elution reagent, and proper slow dropping is preferred.

In the step (4), after the pH value of the system is reduced to 0.5-2.0, the dripping of the elution agent and the reactant is stopped, the temperature of the system is kept unchanged, and the stirring is continued for 3-6 hours, so that the 5' -CMP crystal has an after-ripening process, and the particle distribution of the crystal is more uniform.

In the step (5), the solid-liquid separation mode of the crystal mush is suction filtration or centrifugation.

In the step (5), the washing is necessary, and may be performed by using a solvent such as ethanol, isopropanol, methanol, propanol, and the like, and preferably, the washing is performed by using ethanol with a volume fraction of 90%, which is advantageous for washing off impurities in the slurry.

The crystallization temperature of the 5' -cytidine monophosphate is 10-40 ℃.

In the step (5), the drying is drying by air blast drying at 40-50 ℃. It should be noted that too high a drying temperature tends to lose part of the crystal water of the product, thereby causing moisture absorption of the subsequent product, and too low a drying temperature tends to lower the drying efficiency. The drying time is determined according to the amount of a sample, for example, for a wet filter cake smaller than 1kg, the drying can be completed within 4-8 hours under the condition that the wet filter cake is spread to be smaller than 1cm in thickness. It is noted that the above crystalline powder may exhibit different apparent water contents (including free water and bound water) depending on the drying manner or the humidity of the storage environment, and thus, the resulting crystalline powder may have a water content of 4.00 to 6.50 wt%, preferably 4.80 to 5.25 wt%.

The method for measuring the water content of the crystalline powder is a Karl Fischer method, and can be measured by an 870Titrino plus practical volumetric Karl Fischer moisture measuring instrument of Switzerland.

Has the beneficial effects that: the crystallization method is simple and easy to control, and the obtained 5' -cytidylic acid monohydrate crystal has the advantages of good fluidity, good stability, uniform particle size distribution and high yield, and the product can be stably stored for a long time.

Drawings

FIG. 1 is a diagram of a ball and stick with the smallest asymmetric unit of 5' -cytidine monophosphate;

FIG. 2 is a diagram of the unit cell stacking of 5' -cytidine monophosphate along the b-axis;

FIG. 3 is a unit cell stacking diagram of 5' -cytidine monophosphate along the b-axis at 3X 3;

FIG. 4 is an x-ray powder diffraction pattern of 5' -cytidine monophosphate;

FIG. 5 is a graph of the infrared spectrum of 5' -cytidine monophosphate;

FIG. 6 is a PXRD pattern for humidity stability of 5' -cytidine monophosphate;

FIG. 7 is bond angle information for 5' -cytidine monophosphate;

FIG. 8 is a graph of particle size analysis of 5' -cytidine monophosphate;

FIG. 9 is a TG-DSC of 5' -cytidine monophosphate;

FIG. 10 is an electron micrograph of 5' -cytidine monophosphate, in which (a) no ultrasound was added (b) (c) ultrasound was added

FIG. 11 is a diagram showing the state of 5' -cytidine monophosphate trihydrate.

FIG. 12 is a graph showing the 5' -cytidine monophosphate ion distribution.

Detailed Description

The detection method and the detection instrument of the crystal structure of the 5' -CMP are as follows:

the single crystal X-ray diffraction measurement crystal structure and the analysis method are as follows: cutting the cultured 5' -CMP single crystal into about 0.22 × 0.24 × 0.26mm³The block with the size is obtained by a Bruk APEX-II CCD diffractometer Mo Kalpha radioactive source (a graphite monochromator,

) Irradiating the sample, collecting diffraction data, reducing the diffraction data by SAINT, performing structural analysis by SHELXTL software direct method, and analyzing the structure based on F²The full matrix least squares refinement of (a), all non-hydrogen atoms are refined by anisotropy. Final number ofAccording to the graph by Mercury or Diamond software.

Powder X-ray diffraction: about 0.100g of the ground sample was taken, and diffraction data was collected at room temperature by a powder X-ray diffractometer (Smartlab or Bruker D8 Advance, Japan) using a light source of Cu Ka ray with a scanning step of 0.02 DEG, a scanning voltage of 40kV, a current of 40mA, a scanning rate of 0.2s/0.02 DEG, a scanning range of 2 theta of 5 to 50 DEG, and data were plotted by JADE software processing.

Infrared spectrum: about 0.001g of the ground sample was subjected to background subtraction and data collection by Fourier Infrared Spectroscopy (ThermoFisher scientific, USA) at room temperature with a resolution of 4cm ^-1The collection range is 4000-500 cm^-1。

Thermogravimetric analysis and differential scanning calorimetry: about 0.05g of the ground sample is respectively put into a thermogravimetric analyzer and a differential scanning calorimeter sample tray, and temperature measurement is carried out from room temperature to 500 ℃, the atmosphere is nitrogen, and the heating rate is 20 ℃/min.

And (3) particle size analysis: about 0.1g of 5' -CMP crystal powder was subjected to particle size measurement by a particle size analyzer (Malvern instruments) at room temperature, and the dispersant was ethanol.

The invention is explained in detail below with reference to specific embodiments and the drawing.

Example 1

Preparing 50g of 5' -cytidylic acid amorphous solid powder into 1000mL of aqueous solution, adjusting the pH value to 5.0 by using 3mol/L sodium hydroxide, putting the aqueous solution into a crystallizer with a probe type ultrasonic emission device, starting stirring at the rotating speed of 200rpm, controlling the temperature to be 10 ℃ in the whole process, and stabilizing the system for 30 min; starting an ultrasonic emission device, setting the power to be 20W, simultaneously pumping hydrochloric acid with the concentration of 1.5mol/L at the flow rate of 0.2mL/min to observe turbidity and pH change in the system, stopping the flow of the hydrochloric acid when the pH is reduced to 4.0, continuously keeping the ultrasonic state for 5min, if the system is obviously turbid, closing the ultrasonic, and continuously stirring for 2h for growing crystals; and after 2h, continuously pumping 1.5mol/L hydrochloric acid at the dropping rate of 0.2mL/min, monitoring the pH change in the system process, stopping adding acid when the pH of the system is reduced to 2.0, simultaneously pumping absolute ethyl alcohol at the flow rate of 0.6mL/min, stopping dropping when the system is stable, continuously stirring for 6h, and discharging. And (2) carrying out suction filtration on the crystal mush, washing the crystal mush by using 60mL of ethanol with volume fraction of 90%, placing the crystal mush into a 40 ℃ air-blast dryer for drying for 6h to obtain white crystalline solid particles, wherein the measured crystal yield is 94.5%, the measured bulk density is 0.41g/mL, the flowability is good, the liquid chromatogram purity is 99.5%, the measured water content by the Karl Fischer method is 4.9 +/-0.5%, and the color, the purity and the flowability of the product do not obviously change after the product is stored at room temperature in a sealed manner for 3 months.

TABLE 15' -Cytidine acid monohydrate Single Crystal Structure data

Example 2

Preparing 150g of 5' -cytidylic acid amorphous solid powder into 1000mL of aqueous solution, adjusting the pH value to 5.2 by using sodium hydroxide, putting the aqueous solution into a crystallizer with a probe type ultrasonic emission device, starting stirring, controlling the temperature to be 25 ℃ in the whole process, and stabilizing the system for 30 min; starting an ultrasonic emission device, setting the power to be 50W, simultaneously pumping hydrochloric acid with the concentration of 1.5mol/L at the flow rate of 0.2mL/min to observe turbidity and pH change in the system, stopping the flow of the hydrochloric acid when the pH is reduced to 4.0, continuously keeping the ultrasonic state for 8min, if the system is obviously turbid, closing the ultrasonic, and continuously stirring for 2h for growing crystals; continuously pumping 1.5mol/L hydrochloric acid at the dropping speed of 0.2mL/min after 2h, monitoring the pH change of the system process, stopping adding acid when the pH of the system is reduced to 2.0, simultaneously pumping absolute ethyl alcohol at the flow speed of 0.6mL/min, stopping dropping when the system is stable, and continuously keeping stirring for 6 h; and (3) carrying out suction filtration on the crystal mush, washing the crystal mush by using 120mL of ethanol with the volume fraction of 90%, and then putting the crystal mush into a 40-DEG C oven for drying for 6h to obtain a 5' -cytidylic acid white crystal product, wherein the crystal appearance is shown as figure 10b and is in a more regular sheet crystal habit. The crystal yield was found to be 95.3%, the bulk density was found to be 0.43g/mL, the fluidity was good, the liquid chromatography purity was 99.5%, and the moisture content by the Karl Fischer method was found to be 4.9. + -. 0.5%. The corresponding PXRD pattern is shown in figure 4. The color, purity and fluidity of the product are not obviously changed after the product is hermetically stored for 3 months at room temperature.

Example 3

Preparing 200g of 5' -cytidylic acid amorphous solid powder into 1000mL of aqueous solution, adjusting the pH to 5.0 by using 3mol/L sodium hydroxide, putting the aqueous solution into a crystallizer with a probe type ultrasonic emission device, starting stirring at the rotating speed of 200rpm, controlling the temperature to be 35 ℃ in the whole process, and stabilizing the system for 30 min; starting an ultrasonic emission device, setting the power to be 30W, simultaneously pumping hydrochloric acid with the concentration of 1.5mol/L at the flow speed of 0.2mL/min to observe the turbidity and pH change in the system, stopping the flow of the hydrochloric acid when the pH is reduced to 4.0, continuously keeping the ultrasonic state for 15min, if the system is obviously turbid, turning off the ultrasonic, and continuously stirring for 2h for growing crystals; and continuously pumping 1.5mol/L hydrochloric acid at the dropping speed of 0.2mL/min after 2h, monitoring the pH change of the system process, stopping adding acid when the pH of the system is reduced to 2.0, simultaneously pumping 0.2 times of absolute ethyl alcohol by the volume of the feed liquid at the flow speed of 0.6mL/min, continuously stirring for 6h after the anti-solvent dropping is finished, and discharging the tank. Filtering the crystal slurry, washing with 90% ethanol 60mL, blowing and drying at 40 deg.C for 6h to obtain white crystalline solid particles with good crystal morphology (as shown in FIG. 10c), crystal yield of 96.5%, bulk density of 0.45g/mL, good fluidity, liquid chromatography purity of 99.5%, and water content of 4.9 + -0.5% by Karl Fischer method, corresponding crystal structure shown in FIGS. 1-3, crystal information data shown in Table 1 and 7, corresponding PXRD spectrogram shown in FIG. 4, corresponding infrared spectrogram shown in FIG. 5, and characteristic peak at 3560cm ^-1、3375cm^-1、3123cm^-1、2948cm^-1And the corresponding particle size distribution curve is shown in figure 8, the median particle size is 63.1 mu m, the color, purity and fluidity are not obviously changed after the room-temperature closed storage for 3 months, the crystal form is not changed, the TG-DSC thermogram is shown in figure 6, and the water loss weight ratio of the product is 5.20 percent.

Example 4

Preparing 200g of 5' -cytidylic acid amorphous solid powder into 1000mL of aqueous solution, adjusting the pH to 5.0 by using sodium hydroxide, putting the aqueous solution into a crystallizer with a probe type ultrasonic emission device, starting stirring, controlling the temperature to be 40 ℃ in the whole process, and stabilizing the system for 30 min; starting an ultrasonic emission device, setting the power to be 50W, simultaneously pumping hydrochloric acid with the concentration of 1.5mol/L at the flow rate of 0.2mL/min to observe turbidity and pH change in the system, stopping the flow of the hydrochloric acid when the pH is reduced to 4.0, continuously keeping the ultrasonic state for 10min, if the system is obviously turbid, closing the ultrasonic, and continuously stirring for 2h for growing crystals; continuously pumping 1.5mol/L hydrochloric acid at a dropping rate of 0.2mL/min after 2h, monitoring the pH change in the system process, stopping adding acid when the pH of the system is reduced to 2.0, simultaneously pumping absolute ethyl alcohol at a flow rate of 0.6mL/min, stopping dropping when the system is stable, and continuously stirring for 6 h; and (3) carrying out suction filtration on the crystal mush, washing the crystal mush by using 100mL of ethanol with the volume fraction of 90%, and then putting the crystal mush into a 40-DEG C oven for drying for 6h to obtain a 5' -cytidylic acid white crystal product. The crystal yield was found to be 95.1%, the bulk density was found to be 0.45g/mL, the fluidity was good, the liquid chromatography purity was 99.5%, and the moisture content by the Karl Fischer method was found to be 4.9. + -. 0.5%. The measured ion distribution curve is shown in fig. 12. After the solid is stored for 3 months in a sealed manner at room temperature, the color, the purity and the fluidity of the solid are not obviously changed.

Comparative example 1

The same as in example 3, except that ultrasonic treatment was not used. Specifically, 200g of 5' -cytidylic acid amorphous solid powder is prepared into 1000mL of aqueous solution, the pH value is adjusted to 5.5 by sodium hydroxide, the aqueous solution is placed into a crystallizer with a probe type ultrasonic emission device, stirring is started, and the temperature is controlled to be 40 ℃ in the whole process of circulating water bath; pumping hydrochloric acid with the concentration of 1.5mol/L at the flow rate of 0.2mL/min to observe turbidity and pH change in the system, stopping adding the hydrochloric acid when the pH value is reduced to 4.0, continuing stirring for 30min, keeping the ultrasonic emission device in a closed state, and after continuing stirring for 30min, ensuring that the system has no obvious change and no phase change occurs. Continuously pumping 1.5mol/L hydrochloric acid at the dropping speed of 0.2mL/min, monitoring the pH change in the system process, when the pH is 3.89, generating explosion crystal nuclei in the system, continuously feeding acid until the pH is 2.0, stopping dropping, pumping 0.2 times of absolute ethyl alcohol by the volume of the feed liquid at the flow speed of 0.6mL/min, continuously stirring for 6h, and discharging. And (3) carrying out suction filtration on the crystal mush, washing the crystal mush by using 60mL of ethanol with the volume fraction of 90%, drying the crystal mush in an oven at 40 ℃ for 6 hours, wherein the dried sample has a hardening phenomenon, poor crystal form (as shown in figure 10a) and low crystallinity, the measured water content is 5.5 +/-0.5%, and the product turns yellow after being stored in a closed manner at room temperature for 1 month.

Comparative example 2

The same as example 3, except for the ultrasonic pretreatment, that is, the ultrasonic treatment was carried out for 120min by probe type before the crystallization. Specifically, 200g of 5' -cytidylic acid amorphous solid powder is prepared into 1000mL of aqueous solution, the pH value is adjusted to 5.5 by using sodium hydroxide, the aqueous solution is put into a crystallizer with a probe type ultrasonic emission device, the ultrasound is started, the power is set to be 50W, the ultrasonic emission device is maintained for 120min, and the temperature of a circulating water bath is controlled to be 40 ℃; simultaneously pumping hydrochloric acid with the concentration of 1.5mol/L at the flow rate of 0.2mL/min to observe turbidity and pH change in the system, stopping adding acid when the pH of the system is reduced to 2.0, simultaneously pumping absolute ethyl alcohol at the flow rate of 0.6mL/min, stopping dropwise adding when the system is stable, continuously stirring for 6h, and observing that crystals in the system are agglomerated; and (3) carrying out suction filtration on the crystal mush, washing the crystal mush by using 100mL of ethanol with the volume fraction of 90%, putting the washed crystal mush into a 40-DEG C oven for drying for 6h to obtain a 5' -cytidylic acid crystal product, wherein the dried sample has a hardening phenomenon and a poor crystal form, and the product turns yellow after being hermetically stored at the room temperature of 5.5 +/-0.5% by measuring the water content.

Comparative example 3

The same as example 3, except that no ethanol is added dropwise after the pH is adjusted to 1.5-2.5. Specifically, 200g of 5' -cytidylic acid amorphous solid powder is prepared into 1000mL of aqueous solution, the pH value is adjusted to 5.5 by using sodium hydroxide, the aqueous solution is placed into a crystallizer with a probe type ultrasonic emission device, stirring is started, and the temperature is controlled to be 40 ℃ in the whole process; starting an ultrasonic emission device, setting the power to be 50W, simultaneously pumping hydrochloric acid with the concentration of 1.5mol/L at the flow rate of 0.2mL/min to observe turbidity and pH change in the system, stopping the flow of the hydrochloric acid when the pH is reduced to 4.0, continuously keeping the ultrasonic state for 10min, if the system is obviously turbid, closing the ultrasonic, and continuously stirring for 2h for growing crystals; continuously pumping 1.5mol/L hydrochloric acid at the dropping rate of 0.2mL/min after 2h, monitoring the pH change of the system process, stopping adding acid when the pH of the system is reduced to 2.0, and continuously stirring for 6 h; and (2) carrying out suction filtration on the crystal mush, washing the crystal mush by using 100mL of ethanol with the volume fraction of 90%, putting the washed crystal mush into a 40-DEG C oven for drying for 6h to obtain a 5' -cytidylic acid crystal product, wherein the dried sample has a caking phenomenon, the crystal form is poor, the measured water content is 5.5 +/-0.5%, and the color, the purity and the fluidity of the product are not obviously changed after the product is stored at room temperature in a sealed manner for 1 month.

Comparative example 4

The same as in example 3, except that the ultrasound intervention was performed at a different time. Specifically, 200g of 5' -cytidylic acid amorphous solid powder is prepared into 1000mL of aqueous solution, the pH value is adjusted to be 5.5 by sodium hydroxide, the aqueous solution is placed into a crystallizer with an ultrasonic emission device, and the temperature is controlled to be 40 ℃ in the whole process; pumping hydrochloric acid with the concentration of 1.5mol/L at the flow rate of 0.2mL/min to observe the turbidity in the system and monitor the pH change, if the system is turbid, starting an ultrasonic device and setting the power to be 50W, keeping the ultrasonic state for 20min, and observing the change of crystal nuclei in the system (microscopic examination); stopping ultrasonic treatment when a large amount of crystals appear in the system, and continuously stirring for 2 hours for crystal growing. After 2h, pumping 0.2 times of feed liquid volume of absolute ethyl alcohol at the flow rate of 1mL/min, simultaneously pumping 2% by mass of hydrochloric acid at the flow rate of 0.2mL/min, detecting the change of pH in the system, stopping feeding when the pH is reduced to 1.5, and continuously keeping stirring for about 4 h. And (3) carrying out suction filtration on the crystal mush, washing the crystal mush by using 100mL of ethanol with the volume fraction of 90%, and then putting the crystal mush into a 40-DEG C oven for drying for 8 hours to obtain a white powdery product of the 5' -cytidine monophosphate, wherein the water content is measured to be 5.5 +/-0.5%, the product is hardened after being dried, and the bulk density is low.

Comparative example 5

5 '-Cytidine monophosphate crystals were obtained according to the method for preparing 5' -Cytidine monophosphate trihydrate reported in the literature (Acta Crystal st. (1979). B35, 2141-Astro 2144). Specifically, 1.00g of monohydrate crystals are respectively put into 5mL of isopropanol solutions with the volume percentages of 1%, 5% and 10%, stirred for 12 hours and then placed in a 40 ℃ oven, and slowly evaporated until the solvent is completely volatilized, the crystal growth state is observed as shown in a figure 11, and the content of water measured by Karl Fischer is 13.80% +/-0.5%, which is close to the theoretical content of trihydrate; 5' -cytidine monophosphate obtained by evaporation and crystallization is placed for 24 hours at room temperature, and the crystal is found to be dehydrated; after the crystal is hermetically stored for 15 days at normal temperature and pressure, the crystal surface is yellow, moist and hardened and has poor fluidity.

Comparative example 6

According to a refining method of high-purity nucleotide in patent CN 108892699A, similarly, heating the obtained 5' -cytidine monophosphate solution to 60 ℃, preserving heat for 2 hours, then adding ethanol at a flow rate of 1mL/min, preserving heat for 30 minutes, slowly cooling to 40-45 ℃, preserving heat for 30 minutes, cooling to 30 ℃, preserving heat for 30 minutes, cooling to 20-25 ℃, preserving heat for 1 hour, and filtering. Leaching with 75% ethanol, 85% ethanol and 95% ethanol in volume fraction, drying under reduced pressure to obtain white powdery 5' -cytidine monophosphate crystal, drying to obtain crystals, wherein the crystals aggregate and agglomerate, and the crushed crystals have low fluidity and poor crystal form.

Comparative example 7

According to the 5 ' -cytidylic acid refining method in the patent CN 105348346A, similarly, 80g/L of 5 ' -cytidine monophosphate solution is prepared, hydrochloric acid is used for adjusting the pH value to be 2.5-3.0, ethanol which is 1.5 times of the volume of the original eluent is added to separate out crystals, and the 5 ' -cytidine monophosphate product is obtained by filtering, rinsing and drying, wherein the sample is adhered to the inner wall of a funnel during filtering and is not easy to wash, and the hardening phenomenon is generated after drying, so that the crystal form is poor.

The present invention provides the idea and method of 5' -cytidine monophosphate crystal and the preparation method thereof, and the above description is only the preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention. All the components not specified in this embodiment can be implemented by the prior art.

Claims

1. 5' -cytidine monophosphate monohydrate crystal is characterized in that the molecular formula is C₉H₁₄N₃O₈P·H₂O, belongs to the monoclinic system, and has a space group of P₂₁The unit cell parameters a (a) =4.858, b (a) =8.3267, c (a) =17.0704, β =90.126 °, unit cell volume V =690.515 a ³The minimum number of asymmetric units in the unit cell, Z =2, and in one minimum asymmetric unit, one 5' -cytidine monophosphate and oneOne crystal water.

2. The crystalline 5' -cytidine monophosphate monohydrate according to claim 1, wherein an X-ray powder diffraction pattern measured using Cu-ka radiation has characteristic absorption peaks at 5.21 °, 10.36 °, 11.83 °, 15.55 °, 18.28 °, 18.98 °, 21.78 °, 23.62 °, and 30.13 ° 2 Θ.

3. The crystalline 5' -cytidine monophosphate monohydrate crystals according to claim 1, wherein the crystalline powder formed by the crystals has a bulk density of not less than 0.20 g/mL and a d50 of not less than 10 μm.

4. A method for producing 5' -cytidine monophosphate monohydrate crystals as claimed in any one of claims 1 to 3, comprising the steps of:

(1) placing the 5' -cytidine monophosphate aqueous solution in a crystallizer provided with a probe type ultrasonic device, adjusting the pH value to be 4.85-5.00, and stabilizing the pH value for 30-40 min at the temperature of 10-40 ℃;

(2) starting a probe type ultrasonic device, acting on the reaction system in the step (1) under the power of 20-50W, keeping for 20-40 min, adding a reactant until the pH value of the system is reduced to 3.9-4.0, continuing to perform ultrasonic treatment for 2-5 min if the system is turbid in the dripping process, and then closing an ultrasonic emitter, and growing crystals at the stirring speed of 180-200 rpm until more crystal particles appear in the system and do not increase any more;

5. The method according to claim 4, wherein the concentration of the 5' -cytidylic acid solution in the step (1) is 25-200 g/L.

6. The method according to claim 4, wherein the crystallization temperature of the 5' -cytidine acid solution in the step (1) is 10 to 40 ℃.

7. The method according to claim 4, wherein in steps (2) and (3), the reactant is 1.5-3.0 mol/L hydrochloric acid, and the addition rate of the reactant is 0.2-0.4 mL/min.

8. The method according to claim 4, wherein the elution solvent is absolute ethanol.

9. The method according to claim 4, wherein the crystallization temperature of 5' -cytidine monophosphate is 10 to 40 ℃.

10. The method according to claim 4, wherein in the step (5), the air-blowing drying is performed at 40 to 50 ℃.